CN106520676B - The method and its application of human amnion membrane are prepared from Human plactnta amnion - Google Patents

The present invention relates to the method and its application that human amnion membrane is prepared from Human plactnta amnion.Specifically, being related to the method for preparing human amnion membrane from placenta amnion, method includes the following steps: placenta basic balanced salt solution repeated flushing surface carries out disinfection to placenta；It is torn with surgical forceps and fetal membrane outer layer amnion is taken to set in glass dish, with basic balanced salt solution clean the surface crimson blood, shredded into, fritter；Film fritter is uniformly affixed on culture dish, and amnioic epithelium layer downward, after drying, adds complete medium, culture；Fluid infusion after 2 days continues to cultivate；To see that epithelial cell climbs out of, and removes tissue, changes liquid and continue to cultivate；The typical epithelial cell group in many places to appear when meromixis rate is up to 90%, carries out secondary culture；Harvest: collecting cell after being digested with pancreatin, count, freeze to get human amnion membrane.Further relate to obtained human amnion membrane with and application thereof.Advantage described in specification is presented in the method for the present invention.

The method and its application of human amnion membrane are prepared from Human plactnta amnion

Technical field

The invention belongs to stem-cell therapy technical field, the method for being related to preparing epithelial cell from Human plactnta amnion is also related to And the human amnion membrane and their therapeutical uses prepared by this method.

Background technique

Stem cell is the basis of regenerative medicine development, and effective application is to realize long-lived human health, anti-resisting cancer and senility, forever Protect the effective ways of the ultimate dreams such as younger state, constantly improve quality of life.In recent years, embryonic stem cell and adult stem cell In cell biology and regenerative medicine research field by attention.However, although embryonic stem cell has other cells can not The totipotency reached, but the factors such as immunological rejection after oncogenicity, allograft and unavoidable dispute of ethic It is greatly limited in clinical application, and content is less in the tissue for adult stem cell, lacks the table of specificity The characteristics of face indicates also becomes the yoke of its clinical application.In contrast, human amnion membrane (Human Amniotic Epithelial Cells, hAECs) also have embryonic stem cell characteristic, there are stronger differentiation capability and plasticity, and there is no wind Danger and ethics problem, and rich content are easy to obtain, and possess many embryonic stem cells and the advantages of adult stem cell does not have, To become now the hot spot of studies and clinical application both at home and abroad.

Human amnion membrane because its without oncogenicity, without immunological rejection, highly-safe, rich content, be easy obtain, Do not cause dispute of ethic and obtains the extensive favorable comment of medical field.So far, human amnion membrane has been applied in a variety of diseases In, it is significant in efficacy.What is wherein continued saying it with interest the most by researcher is it in autoimmune disease and nervous system injury disease The curative effect of sick aspect.Amniotic epithelial cells can break up neuroblast, secrete various neurotransmitters and neurotrophic factor comes Repair the nervous system disease that is impaired and degenerating, such as: senile dementia, Parkinson's disease, multiple sclerosis, cerebral hemorrhage, in brain Wind etc., this is not available for other stem cells and immunocyte.Meanwhile human amnion membrane is expressed extensively by paracrine Anti-inflammatory factors are composed, a variety of growth factors are expressed, targetedly to inhibit the abnormal inflammatory of autoimmunity disease.This is autoimmune Disease opens a kind of new targeted treatment method.For autoimmune disease patient, such as systemic loupus erythematosus, class Rheumatic arthritis etc. brings better treatment method.In addition to this, damage of the human amnion membrane for Non nervous system Wound property disease: such as diabetes, lung and the fibrosis lesion of liver, myocardial infarction, acute liver damage, radioactive damage salivary gland, mouth The diseases such as chamber jaw defect have certain curative effect.In the recent period, goal in research has been placed on premature ovarian failure by researcher both domestic and external In terms of corneal restoration, so, the prospect of human amnion membrane is immeasurable.

Current research discovery, human amnion membrane not only has embryonic stem cell characteristic, have stronger differentiation capability and Plasticity, while again without reference to the risk of ethics problem, and rich content, easy acquisition can fully meet stem cell clinic Application security, validity, the condition of quality controllability requirement, it has also become after embryonic stem cell and adult stem cell another The regenerative medicine of unique feature and the ideal seed cell of cell therapy clinical application.

Amnion is located at the chorial surface of fetus, is wrapped in amniotic fluid and fetus, provides the environment of embryo cutting, is tire The innermost layer tissue of disk is contacted between close embryonic development early stage product and parent and fetus with developmental fetus Carry out the vital tissue of mass exchange.Amnion is translucent film, and flexible, impassivity, blood vessel, lymphatic vessel are wrapped in sheep Water and fetus provide the environment of embryo cutting.Its thickness is only 0.02~0.5mm, by epithelial layer, basal layer and hypothallus (connective tissue layer) composition.

Human amnion membrane is the epithelial cell by placenta from the amnion tissue separation of fetus side, is had more Energy stemness has immortality and anti-inflammatory property, no oncogenicity and immunogenicity, and many years through numerous scientists both at home and abroad grind Study carefully demonstration, it has following characteristics:

Full tissue differentiation ability: amniotic epithelial cells and embryonic stem cell have same developmental tissue source, have multipotency Stemness has stronger differentiation capability and plasticity；It can break up to the Various Tissues cell type in triploblastica；Outer embryo can be divided into Neuron, astroglia, the Deiter's cells in layer source；Osteocyte, cartilage cell, the fat of mesoderma origin are thin Born of the same parents, cardiac muscle cell, myocyte；And liver cell, the pancreatic cell of endoderm origin；Amniotic epithelial cells have to three germinal layers The potential of differentiation, amniotic epithelial cells can express some labels of early stage stem cell, since this explanation amnion is formed in embryo Cell mass mesoderm growing early stage, it is possible to retain the poorly differentiated stem cell of prematurity, still retain more differentiation potentials；Currently, having more Show that human amnion membrane is easy to be induced to differentiate into nerve cell in vitro come more results of study, it can also be without body Outer induction is grafted directly to interior therapeutic neurotrosis and degenerative disease；Therefore, in treatment the nervous system disease and damage side Face, amniotic epithelial cells obviously have the potential of more differentiating into nerve cells；

No oncogenicity: compared with stem cell, amniotic epithelial cells lack Telomerase, do not surpass which dictates that it is expanded in vitro After 6 generations, there is limited external competence for added value, and produce it can in vivo unlike embryonic stem cell or other stem cells Raw teratoma or other tumours；Just because of this feature of amniotic epithelial cells, make it that can successfully avoid stem cell clinic from answering Oncogenicity problem, proliferation is limited and ensure that the safety of amniotic epithelial cells treatment without the characteristics of oncogenicity；

Without distribution type: amniotic epithelial cells do not express MHC- class Ⅱ antigens, have immune tolerance ability, and when transplanting is not necessarily to HLA or Gene Type Matching；

No repellency: amniotic epithelial cells lack major histocompatibility complex antigen, while also expression is with immune The HLA antigen of inhibitory activity, therefore immunogenicity will not be generated after transplanting；Since amnion tissue is derived from the product of fetus, It is exposed under mother's immune system monitoring, so that immunogenicity will not be generated after transplanting；In addition, human amnion membrane while table Up to immunosuppressive activity HLA-E ,-G antigen；It, can when making its clinical treatment just because of these features of amniotic epithelial cells Repeatedly infusion is without causing any immunological rejection；

Superpower neural restoration ability: amniotic epithelial cells have the function of stem cell paracrine, can secrete a variety of nerves Trophic factors (NGF, BDNF, NT3 etc.), neurotransmitter and growth factor, can promote the work of host's self neural stem cells function Jump and itself reparation, can promote nerve growth, repairing nerve damage；Nerve to occur morning is thought in biology of nerve growth research Phase, amnion tissue are directly contacted with neural epithelium, release neurotransmitters and neurotrophic factor into amniotic fluid, are sent out in nervous system It plays an important role during educating；Amniotic epithelial cells have the characteristic in terms of nerve cell biochemistry, can synthesize and secrete Dopamine (DA) expresses dopamine receptor D1 and D2 on cell membrane, also has dopamine transporter expression on plasma membrane；In addition, sheep Film epithelial cell energy secretory nerve trophic factors (such as BDNF, NT-3, NGF) and other neurotransmitters and quenched (such as catechol Amine, acetylcholine, norepinephrine, histamine, serotonin, urotensin 1, neurotensin and Somat Deng)；Amniotic epithelial cells can not only itself differentiation nerve fiber, and can secrete various neurotransmitters and neurotrophy because Son promotes the active of host's autologous stem cells function and itself repairs；

Unique immunoregulation capability: a variety of anti -inflammatory cytokines of amniotic epithelial cells energy secreting, expressing and GAP-associated protein GAP, energy The proliferation for inhibiting lymphocyte, can be used for cell therapy inflammatory, at fibroid and autoimmune disease；What it was secreted exempts from Epidemic disease inhibiting factor then has preferable effect to the immunological regulation of autoimmune disease and sub-health population；

Accelerate tissue repairing ability: the growth factor (EGF, TGF, IGF etc.) of amniotic epithelial cells secretion not only can promote Vascular endothelial reparation promotes the generation of the capillary of damage zone, increases collagen synthesis ability, and can activate body The autologous stem cells of interior dormant state are migrated to internal damage location, assemble and are converted, to accelerate angiogenic growth, accelerate tissue It repairs；

Ethics is accepted, is from a wealth of sources: amniotic epithelial cells derive from newborn's postpartum waste amnion, are placenta surface one The strippable biomembrane of layer, without embryonic stem cell application bring ethnics Problem；In addition, being easy a large amount of acquisition cells, easily It is controlled in quality, an amnion can at least obtain 1 × 108 amniotic epithelial cells, and patient's single therapy only needs 1 × 107 It is a, that is to say, that an amnion meets multiple patients and uses.

There are many reports about the preparation method for taking cell on amnion.Such as

CN104818244A (Chinese Patent Application No. 201510263291.8, become a fine day) discloses a kind of amniotic epithelial cells The method of separation, culture, it is characterised in that this method is completed by the following steps: one, amnion tissue separation and disinfection: by sheep Film is removed from placenta, is then used normal saline flushing 2~3 times, then in the physiological saline containing 1%~3% cefoperazone Impregnate 3~10min；Two, amniotic epithelial cells separate: amnion is cut into 50~150cm²Bulk, then will cut after sheep The epithelium of film is pasted on up on sterile nitrocellulose membrane, 3~10mL physiological saline is added into culture dish A, then by amnion Epithelium posted in culture dish A down, stand 2~3min, obtain amniotic membrane patch；Amniotic membrane patch is placed in culture dish B, The pancreas enzyme -EDTA that 10~30ml mass concentration is 0.25% is added and digests 15~20min, then by culture dish after sealed membrane sealing It is put into constant-temperature table and digests, obtain digestive juice, add 2~5ml fetal calf serum and terminate enzyme reaction, then remove amniotic membrane patch The upper amniotic epithelial cells reunited, be adhered, then amniotic membrane patch is transferred in the culture dish C equipped with physiological saline, it cleans adherent Amniotic epithelial cells, obtain cleaning solution, collect digestive juice and cleaning solution, filtered through 100 mesh cell sieves, it is thin to obtain amnioic epithelium Born of the same parents' single cell suspension；Three, amniotic epithelial cells purifying culture: the amniotic epithelial cells single cell suspension that step 2 is obtained is 4 DEG C, 5~15min of pelleted by centrifugation of 1500rpm, abandon supernatant, the low sugar DMEM/F12 training containing epidermal growth factor be then added It supports base weight and hangs cell, 0.9~1.1 × 10 are pressed after mixing⁵/cm²Inoculum concentration be inoculated in culture bottle, then be placed in 37 DEG C, CO₂Body It is cultivated in the incubator that product concentration is 5%, after cell confluency reaches 85%, reject non-adherent cell, and rushed with physiological saline It washes 2 times, the pancreas enzyme -EDTA that mass concentration is 0.05% is then added and digests, 37 DEG C of 2~5min of digestion, reject digestive juice, then use Normal saline flushing 1~2 time, then be added mass concentration be 0.25% pancreas enzyme -EDTA, under the conditions of 37 DEG C digest 3~ 6min adds FBS and terminates enzyme reaction, collects digestive juice, and digestive juice is centrifuged under conditions of 4 DEG C, 1500rpm rotation concussion 5~15min abandons supernatant, DMEM/F12 culture medium is then added, cell is resuspended, it is slender for amniotic epithelial cells to obtain high-purity P0 Born of the same parents' suspension, that is, complete.It is believed that the invention amnion tissue can guarantee that amnioic epithelium face is smooth after amniotic membrane patch is made, while amnion Back side mucus is fitted closely with connective tissue and nitrocellulose membrane, promotes separating for pancreatin digestive efficiency and cell and tissue, short Time digestion collects whole amniotic epithelial cells, avoids the reduction of adherence rate, carries out twice after short time culture Pancreatin digestion, obtains high-purity amniotic epithelial cells.It is believed that the invention can be applied to basic research and clinical transplantation field.

CN104371971A (Chinese Patent Application No. 201310357351.3, consonance) discloses a kind of separation acquisition amnion The method of epithelial cell is incubated for this method comprises: collagenase solution is added in amnion tissue；Then pancreas egg is added in amnion tissue White enzyme solutions are digested；It terminates and digests and filter；Amniotic epithelial cells are obtained after supernatant is centrifuged.Operation of the present invention letter It is single, and the original activity of cell and growth conditions are able to maintain, improve the yield of amniotic epithelial cells.

CN104974980A (Chinese Patent Application No. 201510261707.2, Chongqing) discloses a kind of human amniotic membrane epithelium The separation method of cell, comprising the following steps: collected fresh human placenta is first put into and fills by the first step, acquisition transport placenta In the aseptic plastic bag of 0.9% mass concentration physiological saline, it is put into protecting box after sealing；It is 2 that protecting box, which is put into temperature, again DEG C~8 DEG C of transport case in, and cell separation chamber is transported in 12 hours；This process ensure that the fresh and sheep of placenta tissue The activity of film epithelial cell is separately cultured separation and provides quality assurance to be next；Second step tears and takes amnion, cell separation The fresh human placenta received is placed in disk by personnel, first uses normal saline flushing fresh human placenta surface, then from fresh human placenta On completely tear amnion；Third step, rinse amnion, first by the amnion torn be put into kidney shape disk be unfolded rinse, and with stop blooding The trace of blood on pincers removal amnion, then with physiological saline repeated flushing at least 5 times, until amnion is clean；4th step, cuts out amnion, The amnion rinsed well is first cut into circle, the more corresponding big 3~4cm of culture dish diameter of round diameter size, by circular sheep Film epithelium is covered on culture dish up, and circular amnion edge must be on culture dish edge；It operates in this way and is Ensure in next digestion process trypsase only independent contact amnioic epithelium face；5th step digests amnioic epithelium face, The trypsin solution of 0.25% mass concentration is added in the culture dish for being covered with amnion, is filled until trypsin solution is close Culture dish makes amnioic epithelium face come into full contact with trypsase simultaneously, other location contacts of amnion are less than trypsase；37 The static digestion of DEG C constant temperature 40~80 minutes；The length of digestion time because the thickness of Different Individual amnion, area etc. difference due to Variation；6th step terminates digestion, after digestion, first the trypsin solution in culture dish is sucked out with pipette and abandons it, then The EMDM-F12 culture solution for containing 10% volumetric concentration fetal calf serum is added with liquid-transfering gun, and blows and beats the sheep in culture dish with liquid-transfering gun Film epithelial surface enters the amniotic epithelial cells disengaging amnion by digestion above in cell culture fluid, by blowing repeatedly It beats until then the muddiness that cell culture fluid becomes is collected cell culture fluid and is placed in 50ml centrifuge tube；This step repeats 3~5 It is secondary, to separate down more amniotic epithelial cells；7th step, is collected by centrifugation cell, by the cell culture fluid being collected into from In scheming, with 300~500g centrifugal force 5~10 minutes；8th step, amniotic epithelial cells are resuspended and inoculated and cultured, centrifugation After discard supernatant liquid, be centrifuged bottom of the tube amniotic epithelial cells precipitating with 10mlEMDM-F12 culture solution be resuspended, take a small amount of suspension For cell count, survival rate test and flow cytometer detection, with 1.0~1.3 × 10⁵/cm²Density inoculating cell.It is believed that the invention The amniotic epithelial cells of the isolated high-purity of energy, and cell quantity is more, activity is high, is the research and clinic of amniotic epithelial cells Using laying the foundation.

CN104371970A (Chinese Patent Application No. 201310355824.6, consonance) discloses a kind of for cultivating people sheep The culture medium of film epithelial cell, it is characterised in that the culture medium by DMEM, fetal calf serum, Sodium Pyruvate, L-Glutamine, EGF, Beta -mercaptoethanol and nonessential amino acid composition；Wherein the content of DMEM is 10g/1000ml；Wherein the volume of fetal calf serum contains Amount is 5~15%；Wherein the content of Sodium Pyruvate is 0.0550~0.2200g/1000ml；Wherein the content of L-Glutamine is 0.0731~0.2193g/1000ml；Wherein the content of beta -mercaptoethanol is 1.9287~5.7861g/1000ml；Wherein EGF Content is 5~15ng/ml；Wherein nonessential amino acid is l-Alanine, L- asparagine, L-Aspartic acid L, L- paddy ammonia It is formed in acid, glycine L, L-PROLINE and L-- serine；The content of nonessential amino acid is 0.5~2mM.It is believed that the invention Culture medium be suitable for human amnion membrane culture, be particularly suitable for the separation of primary human amnion membrane and purified Culture in journey, while the probability triggered an immune response during Transplanting Human can be reduced.

CN103642751A (Chinese Patent Application No. 201310650384.7, with pool) discloses one kind and makes from people's amnion The method for taking stem cell refers specifically to separate epithelial cell and mescenchymal stem cell method in a kind of amnion, is related to cell separation skill Art field.It protects liquid to be acquired placenta by homemade sterile, efficient, stable placenta, utilizes digestion method and creep plate method Combination, amniotic epithelial cells and amnion mesenchymal stem cell are separated, the amniotic epithelial cells of high-purity are respectively obtained And amnion mesenchymal stem cell.The present invention solves the cell after people's amnion acquisition pollution rate height, acquisition exists in the prior art Poor activity, the problem of directly affecting subsequent isolation and culture of cell.Be to from people's amnion separate stem cell method into Row improvement and innovation, form a set of standardized separation scheme, can be more stable, make full use of the stem cell resource of amnion, Pollution-free, purity is high, cell quantity is more, while keeping cell activity, ensure that the primary characteristic of stem cell.

CN105420179A (Chinese Patent Application No. 201510963676.5, Si Tanmu) discloses a kind of from umbilical cord and tire The method that argali membrane tissue extracts epithelial cell and mescenchymal stem cell simultaneously, it is characterised in that: steps are as follows: (1) umbilical cord and tire Argali film materials and inoculation: 1. high-temperature sterilization surgical instrument and big glass dish, while disinfection by ultraviolet light superclean bench；2. preparing Culture medium: the composition of complete medium is: DMEM/F12 adds the fetal calf serum of complete medium total volume 5%, complete medium The mixing additive of the insulin of total volume 1%, siderophillin and selenium, final concentration of 20 nanogram/milli in complete medium Final concentration of 20 ngs/ml of basic fibroblast growth factor in the epidermal growth factor and complete medium risen, It is sterile filtered, 4 DEG C of preservations；The volume ratio of DMEM:F12 is 1:1 in the DMEM/F12；The mixing additive and add in mixing Add 5 mcg/ml of final concentration of insulin in object, 10 mcg/ml of siderophillin and 5 ngs/ml of sodium selenite； 3. strictly screening donor, blood testing excludes infectious disease；4. transporting umbilical cord and placenta: mechanical stripping placenta amnion in low temperature ice box Later, umbilical cord and placenta amnion are respectively placed in big glass dish, following steps all aseptically carry out, and umbilical cord is cut into Placenta amnion is cut into the square of about 5X5cm long by the segment of 1cm long, with the penicillin of phosphate buffered saline+total volume 1%+total The streptomysin cleansing tissue of volume 1% twice, the blood of wash clean umbilical cord；5. umbilical cord and placenta amnion tissue are placed on 4 DEG C In phosphate buffered saline, cell survival is influenced to prevent tissue from overheating when machinery shreds, is divided using tissue cutting machine It is not broken into the fragment of 1-3mm, using low speed disrupting tissue, while keeping cell activity, obtains broken umbilical cord and amnion group It knits；6. broken umbilical cord and amnion tissue are transferred to respectively in T175 Tissue Culture Flask, and tiled with tissue scraper In culture bottle bottom, be carefully added into a small amount of complete medium, make culture solution can cover broken umbilical cord and amnion tissue block and As for it is floated, it is placed in 37 DEG C, cultivates in saturated humidity 5%CO2 incubator；(2) mescenchymal stem cell and amnioic epithelium Cell is expanded and is passed in culture bottle: 1. being observed visible cell in incubation and gradually is vacillated out from tissue block, goes forward side by side one Step realizes adherent growth and proliferation, at the 3-4 days of culture, when cell growth merges in blocks, uses sterile phosphate buffered saline The tissue for washing away remaining, is all changed to fresh complete medium and continues to cultivate；2. hereafter every 4 days change a culture solution until cell 90-100% culture bottle floor space is covered with, pancreatin had digestive transfer culture is just carried out；3. pancreatin had digestive transfer culture: abandoning culture solution, washed with PBS Cell 2 times, every T175 is digested 10 minutes with the pancreas enzyme -EDTA that 10 milliliters of mass percents are 0.25% at 37 DEG C, outstanding to cell 1 milliliter of fetal calf serum is added to neutralize pancreatin reaction in each T175 after floating, and 1000 revs/min are centrifuged 10 minutes, is resuspended in completely In culture medium, in the passage of 1:4 cell number ratio into new T175 culture bottle；(3) the mirror of mescenchymal stem cell and amniotic epithelial cells It is fixed: when 1. the third generation is passed in pancreatin digestion, with the purity of flow cytometry culture cell: the marker of amniotic epithelial cells It include: keratin CK19, CD29 and CD34；The marker of placenta and umbilical cord mesenchymal stem cells includes: CD73, CD90, and CD105；2. flow cytometer verifying cell purity reaches 90% or more, chemical examination excludes common infectious disease, and excludes bacterium, props up Mycoplasma contamination is the mixture complied with standard to get epithelial cell and mescenchymal stem cell.It is believed that the method cost of the invention It is low, simple and quick, umbilical cord and placenta amnion are shredded into minimum fragment using tissue cutting machine, are extremely conducive to directly be inoculated with Cell is vacillated to outside tissue after to culture bottle；Without using any protease reagent, so that cost be greatly reduced, system is reduced The standby time, so that short time mass cell is prepared into reality；This method uses addition growth factor and other nutrients Cell culture medium saves serum.

However, this field is still expected to have new method to prepare human amnion membrane, and expect that this method is in Reveal one or more kinds of beneficial technical effects.

Summary of the invention

The purpose of the present invention is to provide a kind of methods for preparing human amnion membrane from placenta amnion, expect this method Show one or more kinds of beneficial technical effects.

First aspect present invention provides a kind of method for preparing human amnion membrane from placenta amnion, and this method includes Following steps:

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, use basic balanced salt solution repeated flushing Surface carries out disinfection processing to placenta；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, after drying 30-60min, is slowly added Complete medium did not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 6 days or so, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), removes tissue, changes Liquid (i.e. above-mentioned complete medium) continues to cultivate；

(5) the typical epithelial cell group (10d or so) in many places to appear is in paving stone sample close-packed arrays, meromixis rate When up to 90%, carrying out secondary culture, (picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, cell count Instrument meter number, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: with cell is collected after the digestion of pancreatin digestive juice, counting, freeze thin to get people's amnioic epithelium in P1 generation Born of the same parents carry out secondary culture to it when necessary.

The method of any embodiment according to a first aspect of the present invention, wherein including strepto- in the basis balanced salt solution Element and two kinds of antibiotic of penicillin.

The method of any embodiment according to a first aspect of the present invention, wherein the basis balanced salt solution is by as follows What mode was prepared: by NaCl, 0.35g of Na2HPO4.12H2O, 8g of KH2PO4,0.132g of KCl, 0.06g of 0.4g The D-Glucose of NaHCO3,1.0g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

The method of any embodiment according to a first aspect of the present invention, wherein complete medium described in step (3) include: DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor (EGF).In one embodiment, the complete training Support also added in base 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) thiamine mononitrate and 0.05~ 0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.It has been unexpectedly discovered that adding into culture medium After adding both micro thiamine mononitrate and maltose, the yield of human amnion membrane can be explicitly improved, without the use of it The two or while only increasing one of them can not obtain this effect.

The method of any embodiment according to a first aspect of the present invention, wherein in step (4), continue culture to it can be seen that Apparent oval epithelial cell climbs out of, and cultivates about 5~7 days, normally about 6 days at this time, and be also shown part mesenchyma at this time The heteroproteose cells such as stem cell, fibroblast.

There is the typical epithelium in many places wherein in step (5) in the method for any embodiment according to a first aspect of the present invention It is culture about 8~12 days, normally about 10 days or so when cell mass.

The method of any embodiment according to a first aspect of the present invention, wherein in step (5), secondary culture is according to following behaviour What work carried out: picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, and cell counter counts, by 3000/ cm²It is seeded in culture bottle, continues to cultivate)；

The method of any embodiment according to a first aspect of the present invention, wherein in step (5), pancreatin digestive juice is to include 0.25% trypsase and 0.02% EDTA digestive juice.

Further, second aspect of the present invention provides a kind of human amnion membrane, is first party through the invention Face the method is prepared.

Human amnion membrane according to a second aspect of the present invention is to be prepared by a method comprising the following steps to obtain :

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, use basic balanced salt solution repeated flushing Surface carries out disinfection processing to placenta；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, after drying 30-60min, is slowly added Complete medium did not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 6 days or so, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), removes tissue, changes Liquid (i.e. above-mentioned complete medium) continues to cultivate；

(5) the typical epithelial cell group (10d or so) in many places to appear is in paving stone sample close-packed arrays, meromixis rate When up to 90%, carrying out secondary culture, (picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, cell count Instrument meter number, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: with cell is collected after the digestion of pancreatin digestive juice, counting, freeze thin to get people's amnioic epithelium in P1 generation Born of the same parents carry out secondary culture to it when necessary.

The human amnion membrane of any embodiment according to a second aspect of the present invention, wherein the basis balanced salt solution In include two kinds of antibiotic of streptomysin and penicillin.

The human amnion membrane of any embodiment according to a second aspect of the present invention, wherein the basis balanced salt solution It prepares and obtains in the following way: by Na2HPO4.12H2O, 8g of KH2PO4,0.132g of KCl, 0.06g of 0.4g The D-Glucose of NaHCO3,1.0g of NaCl, 0.35g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at The solution of 1L.

The human amnion membrane of any embodiment according to a second aspect of the present invention, wherein described in step (3) completely Culture medium includes: DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor (EGF).In one embodiment, institute It also added 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) nitric acid sulphur in the complete medium stated Amine and 0.05~0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.

The human amnion membrane of any embodiment according to a second aspect of the present invention continues to cultivate wherein in step (4) Extremely it can be seen that apparent oval epithelial cell climbs out of, about 5~7 days, normally about 6 days are cultivated at this time, and be also shown at this time The heteroproteose cells such as part mescenchymal stem cell, fibroblast.

There are many places wherein in step (5) in the human amnion membrane of any embodiment according to a second aspect of the present invention It is culture about 8~12 days, normally about 10 days or so when typical epithelial cell group.

The human amnion membrane of any embodiment according to a second aspect of the present invention, wherein in step (5), secondary culture Be to carry out according to following operation: picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, cell count instrument meter Number, by 3000/cm²It is seeded in culture bottle, continues to cultivate)；

The human amnion membrane of any embodiment according to a second aspect of the present invention, wherein in step (5), pancreatin digestion Liquid be include 0.25% trypsase and the digestive juice of 0.02% EDTA.

Further, third aspect present invention provides people's amnioic epithelium described in second aspect of the present invention any embodiment Cell is preparing the purposes in the drug as cellular therapeutic agent.

The purposes of any embodiment according to a third aspect of the present invention, wherein the human amnion membrane is by including What the method for following steps was prepared:

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, use basic balanced salt solution repeated flushing Surface carries out disinfection processing to placenta；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, after drying 30-60min, is slowly added Complete medium did not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 6 days or so, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), removes tissue, changes Liquid (i.e. above-mentioned complete medium) continues to cultivate；

(5) the typical epithelial cell group (10d or so) in many places to appear is in paving stone sample close-packed arrays, meromixis rate When up to 90%, carrying out secondary culture, (picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, cell count Instrument meter number, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: with cell is collected after the digestion of pancreatin digestive juice, counting, freeze thin to get people's amnioic epithelium in P1 generation Born of the same parents carry out secondary culture to it when necessary.

The purposes of any embodiment according to a third aspect of the present invention, wherein including strepto- in the basis balanced salt solution Element and two kinds of antibiotic of penicillin.

The purposes of any embodiment according to a third aspect of the present invention, wherein the basis balanced salt solution is by as follows What mode was prepared: by NaCl, 0.35g of Na2HPO4.12H2O, 8g of KH2PO4,0.132g of KCl, 0.06g of 0.4g The D-Glucose of NaHCO3,1.0g, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at 1L solution.

The purposes of any embodiment according to a third aspect of the present invention, wherein complete medium described in step (3) include: DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor (EGF).In one embodiment, the complete training Support also added in base 0.01~0.05% (such as 0.01~0.04%, such as 0.01~0.03%) thiamine mononitrate and 0.05~ 0.1% (such as 0.06~0.1%, such as 0.07~0.1%) maltose.

The purposes of any embodiment according to a third aspect of the present invention, wherein in step (4), continue culture to it can be seen that Apparent oval epithelial cell climbs out of, and cultivates about 5~7 days, normally about 6 days at this time, and be also shown part mesenchyma at this time The heteroproteose cells such as stem cell, fibroblast.

There is the typical epithelium in many places wherein in step (5) in the purposes of any embodiment according to a third aspect of the present invention It is culture about 8~12 days, normally about 10 days or so when cell mass.

The purposes of any embodiment according to a third aspect of the present invention, wherein in step (5), secondary culture is according to following behaviour What work carried out: picking amniotic epithelial cells dispel in centrifuge tube under stereomicroscope mirror, and cell counter counts, by 3000/ cm²It is seeded in culture bottle, continues to cultivate)；

The purposes of any embodiment according to a third aspect of the present invention, wherein in step (5), pancreatin digestive juice is to include 0.25% trypsase and 0.02% EDTA digestive juice.

Any technical characteristic possessed by any embodiment of either side or the either side of the present invention is equally applicable Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below The description of step.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and Phrase has that well known to a person skilled in the art general senses, nonetheless, the present invention remain desirable at this to these terms and Phrase is described in more detail and explains, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention Subject to the meaning stated.

It normally says, about 600 square centimeters of the area of a placenta amnion, amnion stem cell of the present invention is the placenta that has drawn from On amnion.

It is added to EGF (epidermal growth factor) in complete medium of the present invention, it is thin amnioic epithelium can to have been more efficiently facilitated The proliferation of born of the same parents.During adhere-wall culture, fibroblast or mescenchymal stem cell are inevitably grown, the present invention, which takes, to be chosen The method of clone efficiently improves the purity of amniotic epithelial cells, and the passage growth of more conducively amniotic epithelial cells.The present invention The whole flow process of method is easy to operate, and controllably, P1 generation can harvest a large amount of cells, provides possibility for Clinical application and research.

CK19 is the characteristic feature biomarker of epithelial cell marker.With discovery, income earner's amnion of the present invention Epithelial cell is detected through immunocytochemistry and immunofluorescence dyeing, is as a result shown, the primary and high table of 1-3 subtituted culturing cell Up to epithelium mark CK19, but do not express vimentin, it was demonstrated that the separated cell of the present invention is hAECs.In addition, for the present invention The flow cytometer detection of acquisition is shown: CD9, CD73, CD90, Nanog are the positive, and CD34, CD45, HLA-DR are negative；Induction differentiation knot Fruit shows it: being the positive at cartilage, at rouge, skeletonization.Furthermore, it has been found that by using the method for the present invention, every 400 squares lis Rice amnion available 10⁸A above human amnion membrane.

Detailed description of the invention

Fig. 1 shows human amnion membrane micrograph (200X) obtained by the present invention.

Specific embodiment

The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.

Material used in following experiments of the present invention for example wherein used in reagent be it is known in the art, can be with It is prepared using known method or is directly bought from market.

Embodiment 1: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 45min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor), did not had group It knits；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 6 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 10d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.

Embodiment 2: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 30min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor), did not had group It knits；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 7 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 8d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.

Embodiment 3: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 60min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor), did not had group It knits；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 5 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 12d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.

Method through the embodiment of the present invention 1~3 prepares the human amnion membrane in P1 generation, for different placental samples, puts down It says, every available (1.73~2.21) × 10 of 400 square centimeters of amnions⁸A human amnion membrane.But through this hair The human amnion membrane that the method for bright embodiment 4~6 prepares P1 generation fifty-fifty says that every 400 is flat for different placental samples Available (7.46~9.38) × 10 of square centimetre of amnion⁸A human amnion membrane is higher than Examples 1 to 3 method significantly. The present invention has been found that both thiamine mononitrate and maltose in complete medium in the complementary testing referring to embodiment 4~6 It only adds one such, then prepares the human amnion membrane in P1 generation at this time, for different placental samples, fifty-fifty say, often Available (1.32~1.97) × 10 of 400 square centimeters of amnions⁸A human amnion membrane, this shows thiamine mononitrate and wheat One of bud sugar is appended in complete medium the yield that can not effectively improve human amnion membrane.In addition, ginseng of the present invention Examine other existing technical literatures, such as the method preparation P1 generation in the patent document quoted of the present invention is (with P1 on behalf of comparing Parameter, more comparativity) human amnion membrane when, on average every available people's amnion of 400 square centimeters of amnions Chrotoplast is less than 1.6 × 10⁸It is a.

Embodiment 4: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 45min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor, 0.02% Thiamine mononitrate, 0.085% maltose), do not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 6 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 10d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.Fig. 1 is shown Human amnion membrane micrograph obtained by the present embodiment.

Embodiment 5: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 30min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor, 0.01% Thiamine mononitrate, 0.1% maltose), do not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 7 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 8d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.

Embodiment 6: human amnion membrane is prepared from placenta amnion

(1) placenta is taken out from Placenta samples collecting cassette, be placed in ceramic whiteware disk, using basic balanced salt solution (by 0.4g KCl, 0.06g KH2PO4,0.132g Na2HPO4.12H2O, 8g NaCl, 0.35g NaHCO3,1.0g D- grape Sugar, the streptomysin of 0.10g, 0.06g penicillin with water dissolve and constant volume at the solution of 1L to get) repeated flushing surface, to tire Disk carries out disinfection processing；

(2) it is slowly torn with surgical forceps and fetal membrane outer layer amnion is taken to be placed in 150mm glass dish, it is anti-using basic balanced salt solution Multiple clean the surface crimson blood shreds into the amnion tissue fritter that diameter is 5mm；

(3) amnion fritter is uniformly affixed on 100mm culture dish, and amnioic epithelium layer downward, dries 30-60min (this test After 60min), it is slowly added complete medium (DMEM basal medium, 10%FBS, 10mg/L epidermal growth factor, 0.03% Thiamine mononitrate, 0.07% maltose), do not had tissue；It is cultivated in 37 DEG C, 5%CO2 incubator；

Fluid infusion (supplementing above-mentioned complete medium), continues to cultivate after (4) 2 days；To it can be seen that on apparent oval Chrotoplast climbs out of (normally about 5~7 days, this experiment 5 days, be also shown the heteroproteose cells such as part mescenchymal stem cell, fibroblast), Tissue is removed, liquid (i.e. above-mentioned complete medium) is changed and continues to cultivate；

(5) the typical epithelial cell group in many places to appear (normally about 8~12 days, this experiment 12d), it is close in paving stone sample Arrangement, when meromixis rate is up to 90%, carrying out secondary culture, (picking amniotic epithelial cells are in centrifuge tube under stereomicroscope mirror In, it dispels, cell counter counts, by 3000/cm²It is seeded in culture bottle, continues to cultivate using complete medium)；

(6) it harvests: being digested with pancreatin digestive juice (digestive juice comprising 0.25% trypsase and 0.02% EDTA) After collect cell, count, freeze to get the human amnion membrane in P1 generation, secondary culture carried out to it when necessary.

Test example 1: the verifying of amniotic epithelial cells:

(1), flow cytometer

CD45 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by CD45's Control reagent shakes up, and 20ul is added into Quality Control pipe, then CD45 reagent is shaken up, and the CD45 of 20ul is added into sample cell Then sample concussion is shaken up, 600ul sample is respectively added in Xiang Liangzhi streaming pipe, then mixes, is placed in 4 DEG C or so and keeps away by reagent Light is incubated for 30 minutes, and 1 milliliter of PBS is then separately added into Quality Control pipe and sample cell, is centrifuged 5 points for 1500 revs/min after mixing Clock, after discard supernatant, be added after the PBS of 240ul the upper machine that mixes, detection positive Jie Guo≤2% item be it is qualified, provide cell surface Mark analyte detection return on qualification.

HLA-DR detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by HLA-DR Control reagent shake up, 20ul is added into Quality Control pipe, then HLA-DR reagent is shaken up, is added into sample cell Then sample concussion is shaken up and 600ul sample is respectively added into two streaming pipes, then mixed, be placed in 4 by 20ulHLA-DR reagent DEG C or so be protected from light incubation 30 minutes, be then separately added into 1 milliliter of PBS in Quality Control pipe and sample cell, after mixing 1500 revs/min from The heart 5 minutes, after discard supernatant, the upper machine that mixes is added after the PBS of 240ul, detection positive Jie Guo≤2% item is qualification, is provided carefully Cellular surface marks analyte detection return on qualification.

SSEA-4 detection: prepare two streaming pipes, number, wherein it is sample cell that one, which is Quality Control Guan Yizhi, by SSEA-4 Control reagent shake up, 20ul is added into Quality Control pipe, then SSEA-4 reagent is shaken up, is added into sample cell Then sample concussion is shaken up and 600ul sample is respectively added into two streaming pipes, then mixed, be placed in 4 by 20ulSSEA-4 reagent DEG C or so be protected from light incubation 30 minutes, be then separately added into 1 milliliter of PBS in Quality Control pipe and sample cell, after mixing 1500 revs/min from The heart 5 minutes, after discard supernatant, the upper machine that mixes is added after the PBS of 240ul, detection positive Jie Guo≤80% item is qualification, is provided carefully Cellular surface marks analyte detection return on qualification.

(2), (expression quantity of detection Nanog, Oct4, Kcf4 and SOX2 gene, primer is by professional gene for quantitative fluorescent PCR Company's synthesis) the following are operating procedures:

1. the extracting of sample RNA

1. be put on ice after taking out cell sample from -70 DEG C of refrigerators, after be transferred to the net platform of behaviour, it is fast in sample cell tube 1 milliliter of Trizol reagent is added in speed.This sample cell is designated as No. 1 pipe.It is anti-with 1 milliliter of liquid-transfering gun after cell in No. 1 pipe melts Multiple piping and druming draws 1 milliliter of cell sample and is transferred to No. 2 completely in the EP centrifuge tube without RNA enzyme until do not see any insoluble matter, Finish writing number corresponding with sample.

2. two-phase laminated flow: the sample being uniformly mixed being placed on palm centrifuge centrifugation 3s, makes the Trizol reagent of pipe lid Into in pipe.0.2 milliliter of chloroform is added in the sample of every 1 milliliter of TRIZOL reagent cracking, covers tightly pipe lid.By what is covered tightly No. 2 EP pipes acutely shake 15s manually, stand 5 minutes at 15 to 30 DEG C.12000rpm is centrifuged 15 minutes at 4 DEG C.It is mixed after centrifugation Liquid is classified into the red phenol chloroform phase of lower layer, middle layer and colourless aqueous phase upper layer.RNA is all distributed in water phase.Water The volume on phase upper layer is about the 60% of the TRIZOL reagent being added when being homogenized.

3. RNA precipitate: carefully taking out the sample being centrifuged from centrifuge, put on the super-clean bench.No. 2 pipes are tilted Aqueous top layer is transferred to No. 3 completely in the centrifuge tube without RNA enzyme, is sure not to encounter tube wall and is drawn onto middle layer by 45° angle.It is added Isometric isopropanol mixing covers tightly pipe lid, finishes writing corresponding number to precipitate RNA therein.It gently overturns 5 times, is allowed to It mixes.It is stood after ten minutes after mixing at 15 to 30 DEG C, 12000rpm is centrifuged 10 minutes at 4 DEG C.It is invisible before centrifugation at this time RNA precipitate gelatinous precipitate block will be formed in bottom of the tube and side wall.

4. RNA is cleaned: removing the supernatant in No. 3 pipes, be added at least 1 in the sample of every 1 milliliter of TRIZOL reagent cracking 75% ethyl alcohol (75% ethyl alcohol is prepared with DEPC water) of milliliter, cleans RNA precipitate.Pipe lid is covered tightly, overturns for several times repeatedly, makes to precipitate It floats.It is stored at room temperature 5 minutes.Then 7500rpm at No. 34 DEG C of pipe is centrifuged 5 minutes.

5. RNA is dry: carefully sucking all ethanol solutions in No. 3 pipes as far as possible, be sure not to siphon away precipitating.Blow in machine, wind It is 5-10 minutes or so dry.Then make RNA precipitate 5-10 minutes dry in air at room temperature.

6. dissolving RNA precipitate: suitable no RNase water is added, stands 5 minutes.Water-bath 8 divides in 58 DEG C of water-bath Clock.The RNA dissolved is taken out from water-bath, is shaken 10 seconds in vortex oscillator.The RNA solution of No. 3 pipes obtained saves It is stand-by in -80 DEG C.

2.RNA quality testing (ultraviolet absorption method measurement)

First spectrophotometer is returned to zero with the TE solution of dilution.Then a small amount of RNA solution TE dilution (1:100) is taken Afterwards, its absorption value at spectrophotometer 260nm and 280nm is read, the concentration and purity of RNA solution are measured.

1. concentration mensuration

Readings is 1 40ug RNA/ milliliters of expression under A260.Sample RNA concentration (ug/ milliliters) calculation formula are as follows: A260 × Extension rate × 40ug/ milliliters.Specific calculating is as follows:

RNA is dissolved in 40ul DEPC water, is taken 5ul, 1:100 to be diluted in the TE of 495ul, is measured A260=0.21

Concentration=0.21 × 100 RNA × 40ug/ milliliters=840ug/ milliliters or 0.84ug/ microlitres

After taking 5ul to be used to measure, remaining sample RNA is 35ul, remaining RNA total amount are as follows:

35ul × 0.84ug/ul=29.4ug

2. purity detecting

The ratio of two numerical value of RNA solution A260 and A280 is RNA purity, the A260/A280 of up-to-standard RNA solution Ratio range should be 1.8 to 2.1.

The human amnion membrane as obtained by the test method of this test example 1 test embodiment of the present invention 1~6, as a result shows Show that each parameter of above-mentioned test is consistent with human amnion membrane characteristic feature.For example, through flow cytometer detection, Examples 1 to 6 institute Human amnion membrane surface marker SSEA-4 percentage is obtained 94% or more.In addition, adopting with method known in this field Test CK19, CD9 of 1~6 gained human amnion membrane of the embodiment of the present invention, CD73, CD90, Nanog, CD34, CD45, HLA-DR, these markers meet with human amnion membrane as the result is shown.In particular, for example, CK19, CD9, CD73, CD90, Nanog are positive, and CD34, CD45, HLA-DR are negative.In addition, 1~6 income earner's amnion of the embodiment of the present invention Epithelial cell induction differentiated result show its at cartilage, at rouge, skeletonization be the positive.