patents.google.com

CN107630003A - A kind of umbilical cord mesenchymal stem cells are divided into the inducing culture and abductive approach of neural-like cells - Google Patents

️Fri Jan 26 2018

A kind of umbilical cord mesenchymal stem cells are divided into the inducing culture and abductive approach of neural-like cells Download PDF

Info

Publication number

CN107630003A

CN107630003A CN201711081393.3A CN201711081393A CN107630003A CN 107630003 A CN107630003 A CN 107630003A CN 201711081393 A CN201711081393 A CN 201711081393A CN 107630003 A CN107630003 A CN 107630003A Authority

China

Prior art keywords

cells

culture

neural

umbilical cord

mesenchymal stem

Prior art date

2017-11-03

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Granted

Application number

CN201711081393.3A

Other languages

Chinese (zh)

Other versions

CN107630003B (en

Inventor

魏伟

嵐山芮

王雅茹

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Guangzhou Rui Platinum Health Management Consulting Co Ltd

GUANGZHOU TIANHE NUOYA BIOLOGICAL ENGINEERING Co Ltd

Original Assignee

Guangzhou Rui Platinum Health Management Consulting Co Ltd

GUANGZHOU TIANHE NUOYA BIOLOGICAL ENGINEERING Co Ltd

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2017-11-03

Filing date

2017-11-03

Publication date

2018-01-26

2017-11-03 Application filed by Guangzhou Rui Platinum Health Management Consulting Co Ltd, GUANGZHOU TIANHE NUOYA BIOLOGICAL ENGINEERING Co Ltd filed Critical Guangzhou Rui Platinum Health Management Consulting Co Ltd

2017-11-03 Priority to CN201711081393.3A priority Critical patent/CN107630003B/en

2018-01-26 Publication of CN107630003A publication Critical patent/CN107630003A/en

2021-01-19 Application granted granted Critical

2021-01-19 Publication of CN107630003B publication Critical patent/CN107630003B/en

Status Active legal-status Critical Current

2037-11-03 Anticipated expiration legal-status Critical

Landscapes

Micro-Organisms Or Cultivation Processes Thereof (AREA)
Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention belongs to biological technical field, and in particular to a kind of umbilical cord mesenchymal stem cells are divided into the inducing culture and abductive approach of neural-like cells.The invention provides inducing culture, the abductive approach that a kind of umbilical cord mesenchymal stem cells are divided into neural-like cells, the inducing culture includes piceatannol, minimal medium, and concentration of the piceatannol in inducing culture is 62.5~250 μm of ol/L；The abductive approach is that umbilical cord mesenchymal stem cells are obtained into neural-like cells with above-mentioned inducing culture culture, directed differentiation.Method induction time provided by the invention is short, and can induce the neural-like cells for producing neuron and neuroglia mixing, and a kind of new cell derived is provided for the injury repair of central nervous system.

Description

A kind of umbilical cord mesenchymal stem cells are divided into the inducing culture of neural-like cells and lured Guiding method

Technical field

The invention belongs to biological technical field, and in particular to a kind of umbilical cord mesenchymal stem cells are divided into neural-like cells Inducing culture and abductive approach.

Background technology

The serious health that threaten the mankind of the nervous system disease, Parkinson's as everyone knows and alzheimer ' Silent disease, and hemorrhagic cerebral apoplexy, trauma disorders and spinal cord injury etc. are due to that the nerve cell of differentiation and maturation can not divide Propagation is caused by make up damage and dead cell.At present, the method for treating the nervous system disease is mostly nerve autograft, But nerve autograft is there is limited source, therefore the cell for finding other sources is to carry out the nervous system disease cell transplantation With the key point for the treatment of.

Mescenchymal stem cell (mesenchymal stem cell, MSCs) be it is a kind of have height self-renewal capacity and The multipotential stem cell of multi-lineage potential, under given conditions can be thin to Gegenbaur's cell, nerve cell, adipocyte, cardiac muscle Born of the same parents, muscle cell, cartilage cell etc. break up.Mescenchymal stem cell is derived from the mesoblastic adult stem cell of mesoderm growing early stage, There is very big application value in terms of cell replacement therapy and organizational engineering.Mescenchymal stem cell can be in marrow, umbilical cord, skin Separate and obtain in the tissue such as skin, muscle, peripheral blood, wherein the mescenchymal stem cell in people's umbilical cord source and the mesenchyma in other sources Stem cell is compared, and the cost of umbilical cord is low compared with low immunogenicity and source does not have ethics dispute.

Chinese medicine is the quintessence of Chinese culture in China, has been approved by the world in the treatment that aspect of promoting health and curing diseases has mystery.Some Chinese medicines exist There is good advantage in terms for the treatment of the nervous system disease.There is presently no piceatannol so that mescenchymal stem cell directed differentiation For the report of neural-like cells.Chinese medical extract piceatannol is a kind of polyphenol substance found in grape, sugarcane etc., is had Stronger antioxidation, and there is certain nervous system protective effect.

The content of the invention

The shortcomings that in order to overcome prior art and deficiency, the invention provides umbilical cord mesenchymal stem cells to be divided into neural sample The inducing culture and abductive approach of cell.

In addition, present invention also offers a kind of piceatannol or above-mentioned inducing culture in inducing umbilical cord mesenchymal stem The application being divided into neural-like cells.

To achieve these goals, the invention provides following technical scheme：

A kind of umbilical cord mesenchymal stem cells are divided into the inducing culture of neural-like cells, and the inducing culture includes white Skin China fir alcohol, minimal medium.

As the further improvement to above-mentioned technical proposal, concentration of the piceatannol in inducing culture is 62.5 ~250 μm of ol/L.

As the further improvement to above-mentioned technical proposal, concentration of the piceatannol in inducing culture is 62.5 μm ol/L, 125 μm of ol/L or 250 μm of ol/L.

As the further improvement to above-mentioned technical proposal, the minimal medium is L-DMEM culture mediums, MEM culture mediums Or one kind in RPMI1640 culture mediums.

A kind of umbilical cord mesenchymal stem cells are divided into the abductive approach of neural-like cells, comprise the following steps：By between umbilical cord Mesenchymal stem cells obtain neural-like cells with inducing culture culture described above, directed differentiation.

As the further improvement to above-mentioned technical proposal, 0.5~6 hour time of the culture.

Described neural-like cells are neuron and neuroglial composite nerve like cell

As the further improvement to above-mentioned technical proposal, the umbilical cord mesenchymal stem cells are divided into neural-like cells Abductive approach, comprise the following steps：

(1) hUC-MSCs in mescenchymal stem cell medium culture is taken, mescenchymal stem cell culture medium is discarded, adds life Normal saline washing is managed, is digested with pancreatin digestive juice, adds culture medium to terminate digestion, centrifugation, supernatant discarding, adds mescenchymal stem cell training Support base and cell is resuspended；

(2) by cell reach through in six orifice plates of the poly-D-lysine coating containing sterilization cover glass, treat cell reach 60~ During 70% degrees of fusion, the culture medium containing piceatannol is changed into.

Further, step (1) the mescenchymal stem cell culture medium is that Fasgrow culture mediums or UltraCULTURE are trained Support base.

Further, the method for coating of step (2) six orifice plates of the poly-D-lysine coating containing sterilization cover glass is： In six orifice plates of cover glass are placed with, poly-D-lysine is added, and is put in incubator and cultivates, six holes are taken out from incubator Plate, poly-D-lysine is discarded, cleaned with pure water, dried, it is standby；

Further, the PL200 concentration is 100 μ g/ml, and dosage 1ml, described culture is 37 DEG C, 5%CO₂Training Support in case and cultivate 3h.

As the further improvement to above-mentioned technical proposal, the umbilical cord mesenchymal stem cells are divided into neural-like cells Abductive approach, separation, culture, the amplification of umbilical cord mesenchymal stem cells are also included before the abductive approach step.

As the further improvement to above-mentioned technical proposal, the umbilical cord mesenchymal stem cells are divided into neural-like cells Abductive approach, immunohistochemical staining is also included after the abductive approach.

Technical scheme provided by the invention has the beneficial effect that：

(1) present invention uses the inducing culture culture umbilical cord mesenchymal stem cells containing piceatannol, makes its orientation point Turn to neural-like cells；

(2) abductive approach induction time provided by the invention is short, and induces generation neuron and neuroglia to mix

The neural-like cells of conjunction, a kind of new cell derived is provided for the injury repair of central nervous system.

Brief description of the drawings

Fig. 1 is Flow cytometry figures of the P4 for umbilical cord mesenchymal stem cells MSC surface markers；

Fig. 2 is umbilical cord mesenchymal stem cells NSE colored graphs (100 times and 200 times) after piceatannol induces；

Fig. 3 is umbilical cord mesenchymal stem cells GFAP colored graphs (100 times and 200 times) after piceatannol induces.

Embodiment

Embodiment 1

The present invention is a kind of embodiment for the inducing culture that umbilical cord mesenchymal stem cells are divided into neural-like cells, this reality Applying the example umbilical cord mesenchymal stem cells and being divided into the inducing cultures of neural-like cells includes piceatannol and minimal medium, Concentration of the piceatannol in inducing culture is 62.5 μm of ol/L, and the minimal medium is L-DMEM culture mediums.

The abductive approach that the present embodiment umbilical cord mesenchymal stem cells are divided into neural-like cells is specific as follows：

①

Separation, culture, the amplification of umbilical cord mesenchymal stem cells

Under aseptic condition, the umbilical cord 10cm of healthy fetus is taken, is put into contain and is soaked one minute in spirituous culture dish, then put Cleaned in the culture dish for entering to fill physiological saline and remove clot and residual alcohol；It is subsequently placed into next physiological saline of filling In culture dish, whole piece umbilical cord is cut into the segment of 3cm length, and extruded the blood remained in blood vessel with scissors and tweezers, so Umbilical cord scissors opening or cleaning is washed into inside along arteria umbilicalis afterwards.

The umbilical cord tissue for being cut into segment is put into last empty culture dish, torn umbilical cord with tissue clamps, is peeled off Two blood vessels, are torn and are distributed in circumvascular Wal Tong Shi glue, and the Wal Tong Shi glue separated is put into 50ml centrifuge tubes In, 3ml FasGrow culture mediums are added, Wal Tong Shi glue is cut into the fragment of 1mm sizes with scissors, adds 15ml FasGrow culture mediums, averagely it is put into 6 T25 blake bottle after mixing.

Blake bottle being taken out every 3 days after preparation and observing cell growth state under the microscope, the 3rd after preparation day is to each About 1ml culture mediums are added in blake bottle, are reentered into 37 DEG C, 5%CO in incubator₂Culture；The 7th day under the microscope after preparation Cell state is observed, then pours out the culture medium in blake bottle and tissue block, changes fresh culture, each blake bottle 3ml, it is reentered into 37 DEG C, 5%CO in incubator₂Culture.Gained cell is the primary mescenchymal stem cell of umbilical cord.

Blake bottle in incubator is taken out into observation cell growth condition, the cell in more than half blake bottle is all When reaching 80% density, represent that this collection of cell can pass on.In general 7 are needed when primary cell grows into 80% density~ 14 days.It it is later 3 days per cultivated days between instead of.

When cell density length is to 80% or so, culture medium is discarded, adds pancreatin 0.5ml to disappear with normal saline flushing twice Change, blake bottle is horizontal, rock back and forth so that the whole bottom of bottle of pancreatin uniform fold；Digestion one minute, naked eyes visible cell is Sheet of disengaging bottom of bottle, you can the culture medium 2ml added in centrifuge tube terminates digestion.Cell suspension is moved into centrifuge tube, 500g Centrifuge 5min.Supernatant discarding, add culture medium and cell is resuspended, pour into T75 blake bottles in 37 DEG C, 5%CO₂Cultivated in incubator, P1 is designated as cell.

When cell propagation merges up to more than 80%, above succeeding generations are repeated, P2 generations is designated as, by that analogy, continues to pass In generation, carries out flow cytometer detection to P4 generations.

The results detailed in Fig. 1, P4 is 99.76%, CD105 positive thin for CD90 positive cell numbers in umbilical cord mesenchymal stem cells Born of the same parents' number is that 98.54%, CD73 positive cell numbers are that 97.34%, CD45 positive cell numbers are that 0.04%, CD34 positive cell numbers are 0.03%, it is mescenchymal stem cell.

②

Piceatannol inducing mesenchymal stem cell is divided into neural-like cells

Poly-D-lysine is coated with six orifice plates：The μ g/ml poly-D-lysine 1ml of concentration 100 are taken, add six holes for being placed with cover glass In plate, and it is put in 37 DEG C, 3h or so in 5%CO2 incubators, six orifice plates is taken out from incubator, discard poly-D-lysine, use is pure Water 2ml is cleaned 3 times, is dried, standby.

Growth period and the good P4 of growth conditions take the logarithm for hUC-MSCs, discards culture medium, adds normal saline flushing 3 It is secondary, digested with 1ml pancreatin digestive juice, add culture medium 5ml to terminate digestion, cell suspension is moved into centrifuge tube, 500g centrifugations 5min. Supernatant discarding, add culture medium and cell is resuspended to concentration 1 × 10⁵/ ml, cell is reached coated containing disappearing through poly-D-lysine In six orifice plates of malicious cover glass, per hole 3ml, when cell reaches 60-70% degrees of fusion, change into containing 62.5 μm of ol/L piceatannols L-DMEM nutrient solutions induction, every half an hour with inverted microscope observation cellular morphology change 1 time, induction six hours after terminate Induction.

③

Immunohistochemical staining

Culture medium when discarding above-mentioned induction 6h in six orifice plates, gently washed 2 times with 0.01M PBS, then with 4% poly Formaldehyde fixes 20min at room temperature；With PBS in room temperature washing 3 times, each 5min,；With the PBS rooms containing 0.5%Triton-X-100 Warm lucifuge rupture of membranes 15min；With PBS in room temperature washing 3 times, each 5min；3%H2O2 deionized waters incubation at room temperature 5min is added, Block endogenous peroxidase activity；PBS is washed 3 times, each 5min；The serum working solution of closing normal goats is added dropwise, 15min is incubated at room temperature, inclines, does not wash；NSE (1 in proportion is added dropwise:200)、GFAP(1:200)、NF-H(1:200) the one of dilution Anti-, 4 DEG C overnight；PBS is washed 3 times, each 5min；Biotinylated secondary antibody working solution is added dropwise, is incubated at room temperature 15min；PBS is washed 3 times, each 5min；The strepto- avidin working solution of horseradish enzyme mark is added dropwise, is incubated 15min at room temperature；PBS washings 3 times, every time 5min；The DAB nitrite ions colour developing 1min of Fresh, haematoxylin are redyed；Running water rinses repeatedly.With micro- sem observation, Take pictures.Piceatannol inducing mesenchymal stem cell is shown in Fig. 2, Fig. 3 for the immunohistochemical staining of neural-like cells, NSE and GFAP immunochemistry histochemical stainings show as brown color, and colour developing is positive.

As a result：Add the L-DMEM culture mediums induction containing 62.5 μm of ol/L piceatannols, the oriented neural-like cells shape of cell The transformation of state, but most only 20% mescenchymal stem cells have the transformation of neural-like cells form.

When 62.5 μm of ol/L piceatannol induces 1h, about 10% cell body shrinks rounded or oval, shading Property become strong, cell edges obscure, and fraction cell process has branch, but branch is shorter, and have a small amount of cell detachment bottle wall, but not Completely disengage floating；When inducing 2h, cell quantity showed increased that cell space shrinks, ratio is up to 55% or so, and cell arm It is still shorter, and with more cell detachment；After inducing 4h, cell refractivity enhancing, cell edges become clear, cell arm still compared with It is short；When inducing 5h, the cellular morphology of 62.5 μm of ol/L piceatannol induction is with inducing cellular morphology during 4h without too big difference； After inducing 6h, cell arm is elongated, but most cell detachments, only surplus about 20% adherent neural-like cells.

Embodiment 2

The present invention is a kind of embodiment for the inducing culture that umbilical cord mesenchymal stem cells are divided into neural-like cells, this reality Applying the example umbilical cord mesenchymal stem cells and being divided into the inducing cultures of neural-like cells includes piceatannol and minimal medium, Concentration of the piceatannol in inducing culture is 125 μm of ol/L, and the minimal medium is L-DMEM culture mediums.

The present embodiment umbilical cord mesenchymal stem cells are divided into abductive approach concrete operations and the phase of embodiment 1 of neural-like cells Together, difference is that the concentration of inducing culture middle white skin China fir alcohol used is 125 μm of ol/L.

As a result：Add the L-DMEM culture mediums induction containing 125 μm of ol/L piceatannols, it is only necessary to inducing mesenchymal stem cell 2h When can obtain 90% neural-like cells.

When 125 μm of ol/L piceatannol induces 0.5h, about 45% cell body is shrunk to circular or ellipse, refractivity Become strong, projection occurs in fraction cell, but branch is very short；When inducing 1h, the ratios of neural-like cells is up to 65% or so, cell Projection becomes more and branch is elongated, and cell further shrinks, and adjacent cell arm is connected with each other intertexture and reticulated, and has a small amount of cell Depart from bottle wall, but do not completely disengage floating.When inducing 2h, cell process arm extension, and projection bifurcated is obvious, in the visual field Be connected with each other it can be seen that large stretch of neural-like cells, between each cell process in netted, neural-like cells ratio up to 90% or so, and Floating cells quantity is slightly aobvious than before to be increased；When inducing 4h, it is seen that the about 5% all cell detachments of nerve；When inducing 6h, about 20% Neural-like cells come off bottle wall.

Embodiment 3

The present invention is a kind of embodiment for the inducing culture that umbilical cord mesenchymal stem cells are divided into neural-like cells, this reality Applying the example umbilical cord mesenchymal stem cells and being divided into the inducing cultures of neural-like cells includes piceatannol and minimal medium, Concentration of the piceatannol in inducing culture is 250 μm of ol/L, and the minimal medium is L-DMEM culture mediums.

As a result：Add the L-DMEM culture mediums induction containing 250 μm of ol/L piceatannols, it is only necessary to inducing mesenchymal stem cell 2h When can obtain 90% neural-like cells.

After 250 μm of ol/L piceatannol induction 0.5h, the cell space of about 60% cell is shrunk to circular or ellipse, and stretches Go out shorter projection, refractivity becomes strong；After inducing 1h, cell process becomes more and length increases, and the ratio of neural-like cells is reachable 75% or so, cell further shrinks, and flanking cell projection is connected with each other intertexture and reticulated；During 2h, cell process is more, branch It is longer, and projection bifurcated is obvious, visible big sheet neural-like cells in the visual field, it is in net to be connected with each other between each cell process Shape, neural-like cells ratio is up to 90% or so；Visible about 5% neural-like cells floating during 4h；During 6h, cast-off cells ratio is about For 10%, neural-like cells ratio is up to 85%.

Claims (10)

1. a kind of umbilical cord mesenchymal stem cells are divided into the inducing culture of neural-like cells, it is characterised in that：The induction training Supporting base includes piceatannol and minimal medium.

2. umbilical cord mesenchymal stem cells according to claim 1 are divided into the inducing culture of neural-like cells, its feature It is, concentration of the piceatannol in inducing culture is 62.5~250 μm of ol/L.

3. umbilical cord mesenchymal stem cells according to claim 1 are divided into the inducing culture of neural-like cells, its feature It is, concentration of the piceatannol in inducing culture is 62.5 μm of ol/L, 125 μm of ol/L or 250 μm of ol/L.

4. umbilical cord mesenchymal stem cells according to claim 1 are divided into the inducing culture of neural-like cells, its feature It is, the minimal medium is one kind in L-DMEM culture mediums, MEM culture mediums or RPMI1640 culture mediums.

5. piceatannol or the inducing culture as described in any one of Claims 1 to 4 divide in inducing umbilical cord mesenchymal stem Turn to the application in neural-like cells.

6. a kind of umbilical cord mesenchymal stem cells are divided into the abductive approach of neural-like cells, it is characterised in that comprise the following steps： By inducing culture culture of the umbilical cord mesenchymal stem cells described in any one of Claims 1 to 4, directed differentiation obtains neural sample Cell.

7. umbilical cord mesenchymal stem cells according to claim 6 are divided into the abductive approach of neural-like cells, its feature exists In the time of the inducing culture culture is 0.5~6 hour.

8. umbilical cord mesenchymal stem cells according to claim 6 are divided into the abductive approach of neural-like cells, its feature exists In the abductive approach comprises the following steps：

(1) hUC-MSCs in mescenchymal stem cell medium culture is taken, mescenchymal stem cell culture medium is discarded, adds physiology salt Water rinses, and is digested with pancreatin digestive juice, adds culture medium to terminate digestion, centrifugation, supernatant discarding, adds mescenchymal stem cell culture medium Cell is resuspended；

(2) cell is gone to through in six orifice plates of the poly-D-lysine coating containing sterilization cover glass, treating that cell reaches 60~70% During degrees of fusion, using the inducing culture culture described in any one of Claims 1 to 4.

9. umbilical cord mesenchymal stem cells according to claim 8 are divided into the abductive approach of neural-like cells, its feature exists In in the step (2), the method for coating of six orifice plates of the poly-D-lysine coating containing sterilization cover glass is：It is being placed with lid glass In six orifice plates of piece, poly-D-lysine is added, and is put in incubator and cultivates, six orifice plates are taken out from incubator, discard poly Lysine, cleaned, dried with pure water, it is standby.

10. the umbilical cord mesenchymal stem cells according to any one of claim 6~9 are divided into the induction side of neural-like cells Method, it is characterised in that also include step (1a) before the step (1)：Separation, culture, the amplification of umbilical cord mesenchymal stem cells.

CN201711081393.3A 2017-11-03 2017-11-03 Induction medium and induction method for differentiating umbilical cord mesenchymal stem cells into neural-like cells Active CN107630003B (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
CN201711081393.3A CN107630003B (en)	2017-11-03	2017-11-03	Induction medium and induction method for differentiating umbilical cord mesenchymal stem cells into neural-like cells

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
CN201711081393.3A CN107630003B (en)	2017-11-03	2017-11-03	Induction medium and induction method for differentiating umbilical cord mesenchymal stem cells into neural-like cells

Publications (2)

Publication Number	Publication Date
CN107630003A true CN107630003A (en)	2018-01-26
CN107630003B CN107630003B (en)	2021-01-19

Family

ID=61108030

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
CN201711081393.3A Active CN107630003B (en)	2017-11-03	2017-11-03	Induction medium and induction method for differentiating umbilical cord mesenchymal stem cells into neural-like cells