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CN109620844A - A kind of cell mixture and its preparation method and application being overexpressed Nurr1 - Google Patents

️Tue Apr 16 2019

A kind of cell mixture and its preparation method and application being overexpressed Nurr1 Download PDF

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Publication number

CN109620844A

CN109620844A CN201811529591.6A CN201811529591A CN109620844A CN 109620844 A CN109620844 A CN 109620844A CN 201811529591 A CN201811529591 A CN 201811529591A CN 109620844 A CN109620844 A CN 109620844A Authority

China

Prior art keywords

nurr1

cell

overexpressed

nscs

microglia

Prior art date

2018-12-14

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Pending

Application number

CN201811529591.6A

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Chinese (zh)

Inventor

邓兴力

钱源

陆地

李力燕

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First Affiliated Hospital of Kunming Medical University

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First Affiliated Hospital of Kunming Medical University

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2018-12-14

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2018-12-14

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2019-04-16

2018-12-14 Application filed by First Affiliated Hospital of Kunming Medical University filed Critical First Affiliated Hospital of Kunming Medical University

2018-12-14 Priority to CN201811529591.6A priority Critical patent/CN109620844A/en

2019-04-16 Publication of CN109620844A publication Critical patent/CN109620844A/en

Status Pending legal-status Critical Current

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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs

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Abstract

本发明涉及医疗技术领域，具体涉及一种过表达Nurr1的细胞混合液及其制备方法和应用。本发明的目的在于提供一种过表达Nurr1的细胞混合液及其制备方法和应用，所述细胞混合液是将过表达Nurr1基因的NSCs和过表达Nurr1基因的小胶质细胞按2:1的数量混合而得，细胞悬浮所用液体是无菌生理盐水，所得混合液可以用于治疗帕金森病。实验明证过表达Nurr1基因的NSCs和小胶质细胞联合移植PD大鼠后发现，PD大鼠相对行为表象更为正常，而且移植的NSCs在大鼠体内相对于现有的治疗手段存活时间更长与存活率更高。The invention relates to the field of medical technology, in particular to a cell mixture overexpressing Nurr1 and a preparation method and application thereof. The object of the present invention is to provide a cell mixture overexpressing Nurr1, a preparation method and application thereof, wherein the cell mixture is a ratio of 2:1 between NSCs overexpressing Nurr1 gene and microglia overexpressing Nurr1 gene. It is obtained by mixing the quantities, and the liquid used for cell suspension is sterile physiological saline, and the obtained mixed liquid can be used for the treatment of Parkinson's disease. Experiments proved that after the combined transplantation of NSCs overexpressing Nurr1 gene and microglia in PD rats, it was found that the relative behavioral appearance of PD rats was more normal, and the transplanted NSCs survived longer in the rat compared with the existing treatment methods. with a higher survival rate.

Description

A kind of cell mixture and its preparation method and application being overexpressed Nurr1

Technical field

The present invention relates to field of medical technology, and in particular to it is a kind of be overexpressed Nurr1 cell mixture and its preparation side Method and application.

Background technique

Neural stem cell (neural stem cell, NSC), which refers to, to be present in nervous system, is had and is divided into nerve First, astroglia and oligodendroglia potential can be carried out self more so as to generate a large amount of brain cell tissues Newly, and it is enough to provide the cell masses of a large amount of brain tissue cells.Neural stem cells transplantation is by neural stem cells transplantation to host It is interior, make neural stem cells system diseased region become to going, aggregation, and survive, be proliferated, being divided into neuron and/or colloid Cell, thus a kind of technology for promoting the part of host's missing function to restore.In recent years, neural stem cell research becomes treatment mind Hot spot through degenerative disease and central lesion.Neural stem cells transplantation holds out broad prospects in clinical application, Research to it is always hot spot in recent years.

Microglia (microglia) be in nerve fiber exclusive source in mesoblastic cell.This cell is in maincenter Distribution is less in nervous system.Multidigit is also shown in white matter near pericaryon or around thin vessels in grey matter It arrives.Function is equivalent to macrophage.

Nurr1 is Nur correlation factor, belongs to the superfamily of orphan nuclear receptor.The Nurr1 of the mankind is located on No. 2 chromosomes, There are 8 exons, there is extensive expression in central nervous system, generation with midbrain dopaminergic neuron is developed and deposited Work has substantial connection.

Parkinson's disease (Parkinson disease, PD) is the common Central nervous system degenerative disease of person in middle and old age, Clinical manifestation is a series of neurological symptoms such as static tremor, myotonia, bradykinesia and posture abnormal gait.Nowadays, PD has become in harm, the 2nd nervous system degeneration disease of senior health and fitness.PD illness rate reaches in the old man of over-65s 1%.With the aging of population, the disease incidence of PD will be further up.Therefore effective prevent and treat actively is found to arrange It applies and has been a hot spot of research.

Currently, the treatment method of PD mainly has traditional drug therapy and surgical intervention.Drug therapy includes that dopamine replaces Generation treatment, anticholinergic preparation, dopamine-receptor stimulant, monoamine oxidase inhibitors, catechol-oxygen position-transmethylase Inhibitor, glutamate receptor antagonists etc., these drugs can only mitigate symptom but cannot prevent progression of the disease, and prolonged application Can not only curative effect be caused to decline, there is also various complication such as on-off phenomenon, agent end phenomenon and dyskinesia etc..Surgery is controlled It treats using the damage of globus pallidus and thalamus as main method, since operation wound is big, is limited to using object, it is not a kind of preferably to control Treatment means.

The cellular transplantation therapy of PD starts from 20th century mid-term, people successively using different types of cell as donorcells, Experimental studies have found that, dopamine neuron can be alleviated into PD correlation to the rat model of corpus straitum transplantation treatment PD by many Symptom.Cell transplantation has been achieved for more progress in the research that PD is treated, and is applied to the clinical prospect that will have radiance. But existing research still suffers from many problems, such as optimizes transplantation Project, inhibits cell quantity and whether needs immune suppression Preparation, its survival, field planting etc. encounter difficulties after neural stem cells transplantation.

Summary of the invention

In view of the above shortcomings of the prior art, the purpose of the present invention is to provide a kind of mixing with cells for being overexpressed Nurr1 Liquid and its preparation method and application, the cell mixture are the NSCs and overexpression Nurr1 gene that will be overexpressed Nurr1 gene Microglia be mixed in a certain ratio and obtain, gained cell mixture can be used for treating Parkinson's disease.

In order to achieve the above object, the technical scheme is that

A kind of cell mixture being overexpressed Nurr1, including the NSCs and overexpression for being overexpressed Nurr1 gene The microglia of Nurr1 gene.

Further, in the cell mixture of above-mentioned overexpression Nurr1, the NSCs for being overexpressed Nurr1 gene and excessively table Quantity ratio up to the microglia of Nurr1 gene is 4~3:2~1.

Preferably, in the cell mixture of above-mentioned overexpression Nurr1, the NSCs for being overexpressed Nurr1 gene and excessively table Quantity ratio up to the microglia of Nurr1 gene is 2:1.

Further, the cell mixture of above-mentioned overexpression Nurr1, liquid component are sterile physiological saline.

Further, in the cell mixture of above-mentioned overexpression Nurr1, cell concentration therein is 4.5 × 10⁷~4.5 ×10⁹A/mL.

Preferably, in the cell mixture of above-mentioned overexpression Nurr1, cell concentration therein is 9 × 10⁷A/mL.

The present invention also provides the preparation methods of the cell mixture of above-mentioned overexpression Nurr1, comprising the following steps:

1. the separation and identification of primary microglia；

2. the separation and identification of primary NSCs；

3. being overexpressed the NSCs of Nurr1 gene and the building respectively of microglia；

4. the preparation of cell mixture.

Application of the cell mixture of above-mentioned overexpression Nurr1 in preparation prevention and treatment Parkinson's disease drug.

The utility model has the advantages that

The present invention is experimentally confirmed: dopamine can be improved in the Nurr1 for being overexpressed the NSCs expression secretion of Nurr1 gene The expression quantity of correlation factor TH and DAT can be used for neural restoration so that NSCs be promoted to be divided into dopaminergic neuron；Cross table Neurotrophic factor BDNF and the expression quantity of GDNF can be improved in Nurr1 up to the microglia expression secretion of Nurr1 gene, To promote the growth of NSCs around small colloid, it can be used for neural restoration；Highly expressed Nurr1 makes proinflammatory thin in microglia The expression quantity of intracellular cytokine significantly reduces, and alleviates inflammatory reaction, good growing environment can be provided for NSCs.

The cell mixture provided by the invention for being overexpressed Nurr1 is implanted into the corpus straitum of PD rat model, and and its Its unicellular or non-overexpressing cell mixed liquor transplantation group is compared, as a result, it has been found that, overexpression Nurr1 provided by the present invention Cell mixture the expression quantity of DA correlation factor can be improved in PD rat striatum, to promote neural stem cell to DOPA Amine neuron cell differentiation；Long-term engraftment experiment discovery, the NSCs for the overexpression Nurr1 gene transplanted and microglia exist Can exist steadily in the long term in PD rat striatum, solve death rate height etc. after existing cell transplantation therapy NSCs is transplanted and ask Topic.

The cell mixture provided by the present invention for being overexpressed Nurr1 can be applied to treatment PD, and can solve existing thin Many problems present in born of the same parents' implantation technique have application prospect.

Detailed description of the invention

In each figure, " * " " △ " " # " " ≠ " label is otherness of the NNSC+NMG group respectively compared with other groups.

Fig. 1 is microglia immunofluorescence dyeing CD11b/c positive findings figure；

Fig. 2 is NSCs immunofluorescence dyeing cellular identification positive findings: Fig. 2A: Nestin is positive；Fig. 2 B:GFAP is positive； Fig. 2 C:Tuj1；

Fig. 3 is the result that NSCs and microglia are overexpressed Nurr1: Fig. 3 A:NSCs slow-virus transfection positive findings；Figure The Nurr1 being overexpressed in 3B:Western blot detection NSCs；Fig. 3 C: microglia slow-virus transfection positive findings；Figure The Nurr1 being overexpressed in 3D:Westernblot detection microglia；Compared with the control group, P < 0.001 * P < 0.01, * * *.

Fig. 4 is to be overexpressed Nurr1 to the influence result of NSCs and microglia secretion: Fig. 4 A-D:Western Blot and RT-PCR detection is overexpressed NSCs TH and the DAT expression quantity of Nurr1；Scheme E, F:ELISA detection and is overexpressed Nurr1's Microglia neurotrophic factor and proinflammatory factor expression quantity；Compared with the control group, P < 0.01 * P < 0.01, * *；***P< 0.001。

Fig. 5 is the PD rat speed variation statistical result after cell transplantation:

In figure, " * △ # ≠ " label is otherness of the NNSC+NMG group compared with other groups, the meaning respectively indicated It is: *, compared with sham-operation group, P < 0.01；△, compared with NNSC+MG group, P < 0.05；#, compared with NSC group, P < 0.05；≠, Compared with NNSC group, P < 0.05.

Fig. 6 is the expression quantity of Nurr1, TH, DAT, Pitx3 in PD rat striatum: Fig. 6 A, the detection of B:RT-PCR method The mrna expression amount of Nurr1, TH, DAT, Pitx3；Fig. 6 C, D:Western blot detect Nurr1, TH, DAT, Pitx3 albumen Amount；*, compared with sham-operation group, P < 0.05；△, compared with NSC group, P < 0.05；#, compared with NNSC group, P < 0.05；≠, with NNSC+MG group is compared, P < 0.05.

Fig. 7 is after cell transplantation 12 weeks in body fluorescence immunoassay TH positive cell quantity statistical result: △, with NSCs group phase Than P < 0.05；#, compared with NNSC group, P < 0.05；≠, compared with NNSC+MG group, P < 0.05.

Fig. 8 is after cell transplantation 12 weeks in body fluorescence immunoassay Iba1 positive cell quantity statistical result: △, with NSC group phase Than P < 0.05；#, compared with NNSC group, P < 0.05；≠, compared with NNSC+MG group, P < 0.05.

Fig. 9 is 5 months after transplanting NNSCs+NMG operation immunofluorescence experiment results: blue-fluorescence indicates that TH is positive thin Born of the same parents, green (GFP) and red (RFP) fluorescence indicate slow virus.

Specific embodiment

In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with implementation of the invention Example, technical scheme in the embodiment of the invention is clearly and completely described.Based on the embodiments of the present invention, this field Those of ordinary skill's every other embodiment obtained without making creative work, belongs to protection of the present invention Range.

About embodiment coherent detection albumen introduction:

Pitx3:Pituitary homeobox 3, the homologous frame 3 of pituitary, by the albumen of PITX3 genetic transcription.Pitx3 Specifically expressing plays key effect in brain DA neuron, for the differentiation and maturation of brain dopaminergic neuron, is considered It is transcription factor necessary to midbrain dopaminergic neuron specific development.

TH:Tyrosine hydroxylase, tyrosine hydroxylase are responsible for catalytic amino acid l-tyrosine and are changed into two The enzyme of hydroxy phenylalanine (DOPA).DOPA is a precursor of dopamine, therefore TH promotes dopamine to generate.

DAT:dopamine transporter, Dopamine Transporter are located at central dopaminergic neuronal tip, are one Kind memebrane protein, physiological action are that the dopamine (DA) that physiological effect has been played in synaptic cleft is taken in presynaptic membrane again, In case utilizing again, while the transmitting of the information between nerve cell is terminated, existing research shows that the DAT of Early Parkinson's disease patient Level is substantially reduced.

BDNF:brain derived neurotrophic factor, brain-derived neurotrophic factor are closed in intracerebral At a kind of protein, it is distributed widely in central nervous system, during development of central nervous system, to neuron Survival, differentiation, growth and development play an important role.Brain-derived neurotrophic factor can prevent the death of neuronal damage wound, improve mind Pathological state, promotion through member are damaged the biological effects such as neuron regeneration and differentiation, and are also mature maincenter and surrounding The neuron of nervous system is survived and normal physiological function institute is required.

GDNF, glialcellline-derived neurotrophic factor, glia cell line-derived neurotrophy because Son was purified and was named first from the culture solution of rat glioma cells system B49 by Lin etc. in 1993.At present more Discovery GDNF expression, has the function of Target- derived neurotrophic factors in the culture of kind nerve cell and neural relevant cell.

Iba1: it is specific expressed in macrophage/microglia, and raised during these cell activations, Iba1 is macrophage/microglia specific actin crosslinking protein necessary to the film of M-CSF induction wrinkles.

A kind of embodiment one: preparation for the cell mixture being overexpressed Nurr1

The in vitro culture and identification of 1 primary microglia

Take newborn 24-48 hours of SD suckling mouse brain cortex for microglia in vitro culture, using containing 10% tire ox The DMEM/F12 complete medium culture mixing primary glia 10d of serum, multi ANN is presented in cell, after purification at cell In quiescent condition, in forms such as shuttle shape, branch-likes.Form is compared with quiescent condition without significant change after PBS processing 3h；LPS(10μg/ Ml, Sigma) processing 3h, cell process shortens or disappearance, cell space become larger, are rounded, it is seen that pseudopodium, like amoebiform, in activation shape State.Through immunofluorescent staining after microglia purifying, show that CD11b/c is positive, positive findings are as shown in Figure 1.

The in vitro culture and identification of 2 primary neural stem cell

Take the SD tire mouse Ventral Midbrain tissue of pregnant 12.5-14.5d for primary Culture of neural stem cells, culture solution forms: DMEM/F12,100U/ml mycillin of serum-free, 2%B27 (Gibco, USA), 20ng/mLbFGF (Peprotech, USA), 20ng/mL epidermal growth factor (Peprotech, USA).Culture environment: 37 DEG C, 5%CO₂.It can be formed after culture 5-7d The nerve ball of 150-200 μm of size of diameter shows that nestin (Nestin) is positive, such as Fig. 2A institute through immunofluorescent staining Show.The nerve ball of culture to 7d is further broken up into culture, under differentiation culture medium effect, nerve ball is gradually broken up, Sphere surrounding growth goes out protrusion, and most of nerve ball differentiation shows colloid through immunofluorescent staining after differentiation culture to 7d Fibrillary acidic protein (GFAP) positive findings, such as Fig. 2 B, tubulin (Tuj1) positive findings, such as Fig. 2 C.

3NSCs and microglia are overexpressed the building of Nurr1 cell model

CDNA will be overexpressed to be inserted into the multiple cloning sites in pCDH (Inbavio company, Chinese Shanghai), opened in CMV Mover regulation is lower to express Nurr1.PCDH-copGFP-Nurr1 and pCDH-copRFP-Nurr1 slow virus carrier is purchased from Inbavio.The sky skeleton carrier (pCDH-copGFP and pCDH-copRFP) is used as negative control.Slow virus carrier is transfected into In 293 cell of HEK (Invitrogen, USA), the supernatant containing virion is harvested after culture 72 hours.By virus titer It is adjusted to 3.1 × 10⁹A particle/mL.Carry the recombined lentivirus vector (pCDH-copGFP-Nurr1) and RFP (pCDH- of GFP CopRFP-Nurr1 NSCs and microglia) are transfected respectively, transfect 72 hours detection transfection results.To overexpression Nurr1 base The NSCs of cause and microglia carry out fluorescent marker and Western blot detection, as a result as shown in Figure 3.

Detect 72 hours nerve balls and mesoglia after green fluorescent protein and red fluorescent protein transfect pCDH Cell is respectively pCDH-GFP-Nurr1 and pCDH-RFP-Nurr1 (Fig. 3 A and 3C).Western blot analysis determines that Nurr1 exists Stablize expression in NSCs and microglia.The result shows that Nurr1 transfection group is compared with empty vectors control group, the egg of Nurr1 White matter expression quantity rises, and the two has significant difference, P < 0.05 (Fig. 3 B and 3D).

The preparation of 4 cell mixtures

It will be resuspended after the resulting NSCs for being overexpressed Nurr1 of step 3 and microglia centrifugation, in sterile saline In, cell quantity ratio: NSCs: microglia=2:1, total cell concentration is 9 × 10 after mixing with cells⁷A/mL.

Embodiment one illustrates the separation of NSCs and microglia and qualification process, is overexpressed Nurr1 cell line and builds Vertical process and cell mixture preparation process, while positive findings reference being provided.Easy to operate, experimenter is easy to implement.

Embodiment two: derived from effect of the NSCs and Nurr1 of microglia in dopamine neuron differentiation

Nurr1 has been proved to play a key effect in dopamine neuron differentiation, by the NSCs of the Nurr1 of overexpression Transplantation treatment is carried out with after microglia mixing, is conducive to improve the body dis environment after neuronal cell moves into vivo, reduce The death rate.

Experimental procedure is as follows:

Nerve ball (neural stem cell generation) is randomly divided into two groups by 1 further checks that Nurr1 is overexpressed the shadow to NSCs It rings: empty carrier group (being transfected with empty carrier) and Nurr1 group (transfection pCDH-GFP-Nurr1's).

2 by these cell culture in 1%FBS culture medium, without bFGF in culture medium.After differentiation culture 7 days, use The expression of Westernblot and RT-PCR technology detection TH and DAT.As a result as shown in figures 4 a-d, the overexpression of Nurr1 can draw Play significantly improving for TH and DAT expression quantity, P < 0.01.

3 in order to study effect of the Nurr1 in microglial activation, and microglia is divided into following four groups:

Control group is handled with PBS；

LPS group is handled with LPS (100ng/mL)；

Nurr1 group is handled for 48 hours after transfecting Nurr1 with PBS；

LPS-Nurr1 group is handled for 48 hours after transfecting Nurr1 with LPS (100ng/mL).

4 collect culture medium, and ELISA method detection correlation factor changes after processing 24 hours.With significantly high expression The expression of the neurotrophic factor BDNF and GDNF of the microglia of Nurr1 increases (Fig. 4 E).On the contrary, high in microglia The Nurr1 of expression causes the expression quantity of proinflammatory cytokine to significantly reduce, such as tumor necrosis factor-alpha (TNF-α) and (leucocyte - 1 β of interleukin (IL-1 β) (Fig. 4 F).

Embodiment two is by comparing dopamine the correlation factor TH's and DAT of overexpression and the non-NSCs for being overexpressed Nurr1 Expression quantity compares, show be overexpressed Nurr1 gene NSCs expression secretion Nurr1 can be improved dopamine correlation factor TH and DAT can be used for neural restoration so that NSCs be promoted to be divided into dopaminergic neuron；By comparing overexpression and non-overexpression The neurotrophic factor BDNF of the microglia of Nurr1 and the expression quantity of GDNF compare, and show to be overexpressed the small of Nurr1 gene Neurotrophic factor BDNF and the expression quantity of GDNF can be improved in the Nurr1 of spongiocyte expression secretion, to promote small colloid The growth of surrounding NSCs, can be used for neural restoration；By comparing overexpression and the non-microglia for being overexpressed Nurr1 in inflammation Under the conditions of proinflammatory cytokine expression quantity, show that highly expressed Nurr1 in microglia makes the expression of proinflammatory cytokine Amount significantly reduces, and alleviates inflammatory reaction, good growing environment can be provided for NSCs.

The experimental studies results of embodiment two are that the NSCs for being overexpressed Nurr1 and microglia cell mixture are being treated Application in the nervous system degenerative diseases such as Parkinson provides the experimental basis of cellular level.

Embodiment three: it is overexpressed application of the cell mixture of Nurr1 in P of Rats D model

The foundation of 1PD rat model

P of Rats D model is established by nigro-striatal access locating injection 6-OHDA, referring to professor Paxinos chief editor's " rat brain stereotaxic atlas " determines injection position: first point: 0.7mm before bregma, middle line right 3.0mm, Subdural space 4.5mm； Second point: 0.2mm after bregma, middle line right 2.6mm, Subdural space 6.0mm.Tip position is adjusted, carries out cranial drill using dental burr Hole, with 10 μ L micro syringes, two predetermined target spots of intracerebral are injected separately into 6-OHDA, every 4 μ L to the right.Postoperative intraperitoneal injection Penicillin is anti-infective, and continuous one week.After postoperative 2 weeks, intraperitoneal injection APO (apomorphine) induces circling behavior.Inject 10min Afterwards, manual count SD rat rotating cycle, records 30min altogether.Mean speed >=7 turn/min person be defined as PD the positive, continuous 3 weeks Positive rat model thinks modeling success.

The transplanting of 2PD rat cell mixed liquor

The above-mentioned rat for being defined as the PD positive is randomly divided into 5 groups, every group of cell is suspended in sterile PBS, and total cell Concentration is certain:

(1) sham-operation group (n=6)；

(2) NSCs transplantation group (n=6)；

(3) it is overexpressed NSCs transplantation group (the NNSC group of Nurr1；N=6)；

(4) cell mixture transplantation group (the NNSC+MG group of the NSCs of Nurr1 and the microglia of untransfected are overexpressed； N=6)；

(5) (embodiment one is made the cell mixture transplantation group of the NSCs of overexpression Nurr1 and microglia, NNSC+ NMG group；N=6).

PD rat model intraperitoneal injection of anesthesia yellow Jackets (50mg/kg) are simultaneously fixed in three-dimensional locating frame.It uses No. 22 syringe needles, by 5 μ L cell mixtures or single cell suspension (about 4.5 × 10⁵) it is injected into (A/P=+ in bilateral corpus straitum 0.5mm；L=+3.0mm；V=-5.0mm).Needle is left in place 5 minutes after per injection.Sham-operation rat is injected isometric Sterile saline.It sews up the incision, intramuscular injection 100KU penicillin is to prevent to infect.3 weeks after the transfer, 6 weeks, 9 weeks and 12 weeks It carries out performance testing: apomorphine (Sigma) is injected with the dose subcutaneous of 0.25mg/kg, the rotation of rat in monitoring 30 minutes Number.

After 3Westernblot and RT-PCR detection transplanting in corpus straitum DA (dopamine) correlation factor variation

Each group PD rat striatum body tissue is extracted respectively after transplanting 12w, is respectively used to Western blot and RT-PCR inspection The variation that Nurr1, DAT, Pitx3 and TH are expressed between survey each group.

After performing the operation 12 weeks, rat striatum body tissue total protein is extracted with RIPA lysate.Antibody used in Westernblot point Not are as follows: rabbit Nurr1 antibody (1:200；Abcam), multi-clone rabbit TH antibody (1:200；Abcam), multi-clone rabbit Pitx3 antibody (1:200；Abcam), multi-clone rabbit DAT antibody (1:200；Abcam), Monoclonal mouse β-actin antibody (1:2000； Santa)。

Total serum IgE (every group of n is extracted from tissue using TRIzol- common reagent (Tiangen Biotech, BeiJing, China) =4), and complementary DNA (cDNA) is synthesized.The amplimer of Nurr1, TH, Pitx3, DAT and internal reference GAPDH are as follows:

The primer sequence of Nurr1 gene:

F:5 '-AAGCCACCTTGCTTGTACCAAA-3 ',

R:5 '-CTTGTAGTAAACCGACCCGCTG-3 '；

The primer sequence of TH gene:

F:5 '-CAGGGCTGCTGTCTTCCTAC-3 ',

R:5 '-GGGCTGTCCAGTACGTCAAT-3 '；

The primer sequence of DAT gene:

F:5 '-TTGCAGCTGGCACATCTATC-3 ',

R:5 '-ATGCTGACCACGACCACATA-3 '；

The primer sequence of Pitx3 gene:

F:5 '-CTTAGTCCCTGCCAGTACGC-3 ',

R:5 '-GTGAGCCAAGGGTGAATTG-3 '.

RT-PCR reaction condition is as follows: 94 DEG C, 3 minutes, and 35 circulations；94 DEG C, 45 seconds；58 DEG C, 45 seconds；72 DEG C, 1 point Clock；Finally extend 72 DEG C, 10 minutes.

After the transplanting of 4 Immunofluorescence tests in corpus straitum TH and Iba1 variation

Cell transplantation 12 weeks after operation extracts NNSC+NMG combined transplantation group PD rat cerebral tissue, after being fabricated to frozen section Under the microscope, the microglia transplanted into carries RFP red fluorescence to fluorescence microscopy, and transplants the NSCs to enter and then carry GFP green fluorescence.12 weeks rats of each group post-transplantation extraction brain tissue is made into frozen section, each group is observed by immunofluorescence The quantity of TH positive cell and Iba1 positive cell in PD rat striatum body.In addition, by independent 3 NNSC+NMG cell mixtures Transplantation group PD rat feeding observes TH positive cell respectively and Iba1 is positive thin to taking brain to make frozen section after postoperative 5 months The quantity of born of the same parents.

5 experimental results

(1) PD rat model behavior observation

According to the behavior observation to the PD rat after cell transplantation as a result, the NSCs of Nurr1 overexpression and microglia Mixed liquor significantly improves the circling behavior of PD rat.Fig. 5 shows 3 weeks, 6 weeks, 9 weeks and 12 weeks each group PD rats pair after transplanting The circling behavior that APO (apomorphine) causes is as a result, the PD rat of the experimental group of the four celliferous cell liquid of transplanting all occurs Circling behavior, but sham-operation group do not rotate behavior.NSCs group reaches a plateau in the 6th week rotating cycle, Later period is risen；Rotating cycle is reduced NNSCs+NMG group at any time, the rotating cycle of each time of measuring compared with other groups all With significant difference.When transplanting 12 weeks, NNSCs+NMG group is compared with sham-operation group, P < 0.01；With NNSC+MG group, NSC Group, NNSC group are compared, and P value is respectively less than 0.05.

(2) to the detection of DA correlation factor expression

With the brain section tissue of fluorescence microscope NNSC+NMG group PD rat, there is red and green fluorescence label PD rat is to transfect successfully and transplant successful rat model.

The PD rat of experiment is put to death, using RT-PCR and Western blot analyze measurement Nurr1, TH, DAT and As shown in figures 6 c and 6d, RT-PCR result is as schemed for the mRNA and protein expression level of Pitx3, Western blot testing result Shown in 6A and 6B.By testing result it is found that the expression quantity of TH, DAT and the Pitx3 of NNSCs+NMG group more than sham-operation group and its His transplantation group, and there is significant difference, P value is respectively less than 0.05.

12 weeks after immunofluorescence dyeing display transplanting, the transplanting brain of TH positive cell number PD rat in NNSCs+NMG group In with other group compare significant increase, Fig. 8 be TH positive cell number statistical result；On the contrary, Iba1 positive cell number reduces, table The microglia quantity of bright NNSCs+NMG group is reduced after the transfer.

5 months progress immunofluorescence experiments after NNSCs+NMG performs the operation are transplanted, further to study the effect of Long-term cell transplanting Fruit.As shown in figure 9, GFP and RFP from slow virus can be still observed under fluorescence microscope；It is visible in striatal section TH positive cell.The result shows that the microglia cell of Nurr1 gene overexpression and the nerve cord of Nurr1 gene overexpression are thin The co-transplantation of born of the same parents ensures that the neural stem cell transplanted in vitro is stabilized in the severe corpus straitum environment of PD rat brain And it plays a role.

Embodiment three is by being implanted into PD rat model for the cell mixture of the overexpression Nurr1 provided of the invention In corpus straitum, and it is compared with other unicellular or non-overexpressing cell mixed liquor transplantation group, as a result, it has been found that, the present invention is mentioned The cell mixture of the overexpression Nurr1 of confession can improve the expression quantity of DA correlation factor in PD rat striatum, to promote Neural stem cell is broken up to dopamine neuronal cell；Long-term engraftment experiment discovery, the overexpression Nurr1 gene transplanted NSCs and microglia can exist steadily in the long term in PD rat striatum, solve existing cell transplantation therapy NSCs The problems such as death rate is high after transplanting.

Embodiment three proves that the cell mixture provided by the present invention for being overexpressed Nurr1 can be applied by zoopery It in treatment PD, and can solve many problems present in existing cell transplant techniques, there is application prospect.

Place is not described in detail by the present invention, is the well-known technique of those skilled in the art of the present technique.Finally, it is stated that the above reality It applies example to be merely to illustrate explanation technical solution of the present invention rather than limit, modify to technical solution of the present invention or equivalent Replacement, without departing from the objective and range of technical solution of the present invention, is intended to be within the scope of the claims of the invention.

Claims (8)

1. a kind of cell mixture for being overexpressed Nurr1, it is characterised in that: including being overexpressed the NSCs of Nurr1 gene and crossing table Up to the microglia of Nurr1 gene.

2. the cell mixture according to claim 1 for being overexpressed Nurr1, it is characterised in that: the overexpression Nurr1 base The NSCs of cause and the quantity ratio for being overexpressed the microglia of Nurr1 gene are 4~3:2~1.

3. the cell mixture according to claim 1 for being overexpressed Nurr1, it is characterised in that: the overexpression Nurr1 base The NSCs of cause and the quantity ratio for being overexpressed the microglia of Nurr1 gene are 2:1.

4. the cell mixture according to claim 1 for being overexpressed Nurr1, it is characterised in that: the cell mixture Liquid component is sterile physiological saline.

5. the cell mixture according to claim 1 for being overexpressed Nurr1, it is characterised in that: the cell mixture Cell concentration is 4.5 × 10⁷~4.5 × 10⁹A/mL.

6. the cell mixture according to claim 1 for being overexpressed Nurr1, it is characterised in that: the cell mixture Cell concentration is 9 × 10⁷A/mL.

7. the preparation method of the cell mixture described in claim 1 for being overexpressed Nurr1, it is characterised in that: including following step It is rapid:

1. the separation and identification of primary microglia；

2. the separation and identification of primary NSCs；

3. being overexpressed the NSCs of Nurr1 gene and the building respectively of microglia；

4. the preparation of cell mixture.

8. application of the cell mixture described in claim 1 for being overexpressed Nurr1 in preparation prevention and treatment Parkinson's disease drug.

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