CN103865956B

CN103865956B - Utilize the method that recombinant slow virus induced nerve stem cells breaks up to dopaminergic neuron

Info

Publication number: CN103865956B
Application number: CN201310229277.7A
Authority: CN
Inventors: 谭雪锋; 金国华; 秦建兵
Original assignee: Nantong University
Current assignee: Nanjing Elp regenerative medical science and Technology Co., Ltd.
Priority date: 2013-06-09
Filing date: 2013-06-09
Publication date: 2016-08-10
Anticipated expiration: 2033-06-09
Also published as: CN103865956A

Abstract

The present invention relates to a kind of biotechnology, a kind of method utilizing gene recombined virus induced nerve stem cells to break up to dopaminergic neuron: recombined lentivirus vector builds；The separation of neural stem cell, cultivate and identify；Recombinant virus transfection NSCs；The qualification of recombinant slow virus transfection NSCs；Recombinant virus transfection NSCs: take the digestion of the NSCs after passing on 3 times and be prepared as single cell suspension, be seeded in culture plate；Transfection TH+Brn4 gene recombinaton slow virus；Add slow virus and ploybrene, after rocking mixing, culture plate is put in incubator and cultivate, after 12 hours, change fresh division culture medium；Screen with puromycin, to obtain the cell of stable transfection simultaneously；It is an advantage of the current invention that: by building the slow virus carrier of genes of interest TH and Brn4, after transfection NSCs, allogenic gene can be expressed at cell inner stablity；A high proportion of TH positive dopaminergic neuron is produced after differentiation；Brn4 can promote the high expressed of neurotrophic factor, and has the function of inhibited apoptosis.

Description

Utilize the method that recombinant slow virus induced nerve stem cells breaks up to dopaminergic neuron

Technical field

The present invention relates to a kind of method utilizing recombinant slow virus induced nerve stem cells to break up to dopaminergic neuron.

Background technology

Parkinson disease (Parkinson ' s Disease, PD) it is a kind of common central nervous system degenerative disease, its base This pathological change is that specific substantia nigra of midbrain dopaminergic neuron reduces because of a variety of causes gradually row regression necrosis, quantity, causes Dopamine In Striatum neurotransmitter regulator is made to reduce, thus the range of motion impairment property such as generation is trembled, muscle rigidity.

Traditional Drug therapy and surgical intervention are only limitted to improve symptom, but can not stop PD, it is impossible to fundamentally solve Certainly problem.Owing to this pathological changes mainly invades substantia nigra of midbrain dopaminergic neuron, and the target area of substantia nigra dopaminergic neuron projection Striatum location clearly, so transplant in nigro-striatal system can the cell of Dopamine Secreted, recover the nerve of dopamine Loop is a Perfected process for etiological treatment.

In the last thirty years, the intracerebral transplantation research of PD achieves bigger progress, has many PD patients to receive in aborted fetus The transplanting of cerebral tissue, transplant the dopamine neuron in tissue can long-term surviving, and constantly discharge dopamine, make the fortune of patient Dynamic symptom is obviously improved.This shows that cell transplantation replacement therapy is treatment PD method more likely.But because of fetal transplatation thing The problems such as the repulsion of limited source, alloimmune and ethics, embryo brain tissue transplantation is very limited in actual applications.

Neural stem cell (Neural stem cells, NSCs) is one of current focus studied in the world, and it is not only present in In fetal brain tissue, also have been observed that in adult brain.NSCs not only has the ability of division growth and self renewal, also has Many differentiation potentials, can be divided into the polytype cells of nervous system such as neuron, astrocyte and oligodendrocyte, Including dopaminergic neuron.Therefore, NSCs treats the nervous system such as PD as the donorcells of cell transplantation replacement therapy and moves back Row disease has huge potentiality and superiority.But, research shows that the NSCs of In vitro culture is divided into the most mostly Glial cell, is seldom divided into neuron, the most specific dopaminergic neuron, and this transplants to clinical practice NSCs and controls Treat PD and cause great obstacle.Meanwhile, after current research shows independent NSCs transplanting, cell survival rate only has 5%-10%, And can not migration and differentiation be incorporated into the neural circuitry of host effectively, thus well release neurotransmitters can not play physiology Function.

It is divided into the method for dopamine neuron so inquiring into induction NSCs and promotes that the cell transplanted is survived, time longer in vivo Between Dopamine Secreted be problem in the urgent need to address in NSCs transplantation treatment PD research.

Tyrosine hydroxylase (Tyrosine Hydroxylase, TH) is the speed limit during intracellular tyrosine synthesis dopamine Enzyme, usually used as the specificity marker of dopaminergic neuron, its expression is that can NSCs be converted into dopaminergic neuron Key.Research finds, TH content and the linearly positive correlation of midbrain DOPAMINE CONTENT IN RABBIT, TH and TH mRNA in PD patient's brain in brain Level significantly lower than normal person, prompting supplements TH gene in brain can promote the biosynthesis of dopamine.By people or Mus TH gene Proceed to the internal of various kinds of cell as genes of interest and experiment in vitro proves, through the cell of TH genetic modification, there is Dopamine Secreted Ability.Therefore, manage to improve the TH activity of NSCs, promote that the generation of dopamine and release are that NSCs transplanting associating transgenic is controlled Treat the new method of PD.

Brn-4 is one of transcription factor POU protein family the 3rd class members.Have been well documented that, multiple hox genes, Such as HOX, POU, LIM, PAX etc., the transcription factor of coding in embryo development procedure to the differentiation of neurocyte, migration and certain The expression of a little tissue-specific genes plays important regulating and controlling effect.Wherein, POU hox genes is at development of central nervous system During play key player.According to whole amino acid sequence homologous degree in POU district, mammal POU albumen is divided into Class I～Class VI totally six class, Brn-4 and Brn-1, Brn-2, Tst-1 belong to the IIIth class, embryo Brn-1 in early days, Brn-2, Brn-4 i.e. occurs in nervous system, and widely distributed, points out these factors may participate in the most neural generation.Exist in the recent period In a report on " Nature ", three transcription factor Ascl 1, Brn-2, Mytl 1 are imported from adult rat afterbody Skin Cell, after one week, the experimental mouse Skin Cell that there are about 20% is converted into neurocyte, and these neurocytes are the most permissible Express neuroprotein, and synaptic contact can be formed with other neurocytes, show that POU protein family member Brn-2 is at nerve Developmental pivotal role.Visible, the IIIth class factor pair NSCs differentiation destiny of POU protein family may play conclusive work With.The research of Le Moine etc. also finds, blocks Adult Mammals black substance and projects to striatal dopaminergic nerve, can draw Brn-4 up-regulated in graining shape body.Show Brn-4 may with Adult Mammals go dopaminergic nerve domination after striatum Interior neuranagenesis is relevant with reparation.

In early-stage Study, block to set up to the cholinergic nerve fibers of hippocampal projections from septal area by cutting rat fimbria-fornix and go Innervation Hippocampus model, is transplanted to NSCs in Hippocampus, and the NSCs of implantation is easier to survival in the Hippocampus of denervation, And it is divided into neuron more, concurrently there are and also go out under denervation damage stimulates in the endogenous NSCs of dentate gyrus Now obvious hypertrophy, migration and neuron differentiation.The above results shows, after denervation damage, and local microenvironment in Hippocampus Change into the survival of NSCs, regeneration and Differentiating Into Neurons and provide good condition.We further with in situ hybridization, The multiple method detections such as RT-PCR, immunohistochemistry and Western blot find, the transcript and expression level of Brn-4 is being gone The most substantially raise in the Hippocampus of innervation.The denervation Hippocampus extracting solution obtained adds in the Brn-4 antibody of excess With Brn-4 therein, then co-culture with NSCs, it was found that in antibody and after, denervation Hippocampus extracting solution promotes god The effect broken up through unit is obviously reduced.Utilize RNA perturbation technique to proceed in NSCs by the small RNA fragments for Brn-4, lower The level of intracellular Brn-4, result NSCs is divided into the ratio of neuron and is decreased obviously.On the contrary, the NSCs of process LAN Brn-4 The ratio being divided into neuron is significantly raised, and the Maturity of newborn neuron substantially increases.These results explanation Brn-4 exists NSCs Differentiating Into Neurons maturation process plays a very important role.Research also finds further, transfection Brn-4 gene After NSCs breaks up 7 days in vitro, Western blot detection finds that the expression of apoptosis-related protein Caspase-3 is significantly lower than right According to group, prompting Brn-4 is likely to be of the effect of inhibited apoptosis.The research of Shimazaki etc. finds, source of requiring mental skill in vitro Property nerve growth factor (Brain-derived neurotrophic factor, BDNF) and insulin-like growth factor-i (Insulinlike growth factor-1, IGF-1) stimulation derives from mice embryonic striatal NSCs atomization, Brn-4 expresses and raises rapidly, and Brn-4 albumen is mainly expressed in the neuron of new life；Application Antisense OligodeoxynucleotideTechnique Technique resistance The function of disconnected Brn-4, NSCs is divided into the ratio of mature neuron and then declines.Illustrate that Brn-4 may pass through some neurotrophys The mediation of the factor regulates and controls the neuronotropic differentiation of NSCs and maturation thereof.But, Brn-4 promotes that NSCs divides to mature neuron Change and maintain the molecular mechanism of Cell survival at home, the most do not study.If can be effective by TH and Brn-4 gene Ground induction NSCs to ripe dopaminergic neuron differentiation, and maintained by Brn-4 transplant after the survival of cell, be allowed to long Dopamine Secreted temporally, pathology and the biochemistry of correcting PD are abnormal, improve clinical symptoms, and this will move for polygene combined NSCs Plant treatment PD and new method and experimental basis are provided.

Summary of the invention

It is an object of the invention to overcome weak point of the prior art, it is provided that one utilizes recombinant slow virus induced nerve stem cells Method to dopaminergic neuron differentiation.

In order to realize the purpose of the present invention, we will adopt the following technical scheme that and be practiced:

The method that recombinant slow virus induced nerve stem cells breaks up to dopaminergic neuron, described method is utilized to include following reality Execute step:

One, pLV5-TH+Brn4 recombined lentivirus vector builds:

Requirement according to pLV5-EF1a-GFP Lentiviral and the cDNA function total length of GenBank rat TH and Brn4 Sequence is respectively the gene of NM_012740 and NM_017252, synthesis genes of interest and build pLV5-TH, pLV5-Brn4 and PLV5-TH+Brn4 Lentiviral, carries out order-checking and identifies and titre qualification after packaging；

Two, neural stem cell separation, cultivate and identify:

The cultural method of neural stem cell: aseptically, is organized as raw material with the SD tire Mus ventral mesencephalan of pregnant 14d, uses Suction pipe is blown and beaten repeatedly until being cell suspension, and through 200 mesh nylon net filters, collects the cell suspension after filtering, train with basis Support after base cleans 3 times and be resuspended in NSCs culture fluid, after cell counting and vigor are observed, connect by 1 × 105/ml cell density Plant in the Tissue Culture Flask containing 10ml NSCs culture fluid, be placed in the CO2 incubator of 5%, 37 DEG C cultivation, can after 3 days Form, in seeing culture bottle, the NSCs ball suspended, after 7 days, carry out Secondary Culture；

The separation method of neural stem cell: after neural stem cell cultivates 3 days in culture bottle, collects NSCs ball with low-speed centrifugal, Digest with Accutase enzyme after cleaning 3 times, terminate digestion with the basic culture solution containing 10%FBS after about 10min, pass through The mode of machinery piping and druming makes it be dispersed into single cell suspension, continues to cultivate after carrying out cell counting in culture bottle；

The authentication method of neural stem cell: take P3 and add the BrdU of final concentration of 5 μm ol/L for NSCs when passing in culture fluid It is marked, makes BrdU be incorporated in neural stem cell DNA, after 7 days, carry out BrdU Immunofluorescence test；Separately take P3 generation with After NSCs sub-clone ball Nestin Immunofluorescence test prove its embryo source property；P10 is inoculated in for NSCs ball and puts in advance Enter to scribble in 24 well culture plates of poly-D-lysine coverslip, add the DMEM/F12 differentiation culture liquid containing 10%FBS and break up, MAP-2, GFAP and CNP Immunofluorescence test is carried out respectively to prove many differentiation potentials of NSCs after 2 weeks；

Four, the qualification of recombinant slow virus transfection NSCs, including:

Transfection efficiency detection is that the NSCs ball after transfecting is seeded to be pre-coated with in 24 orifice plates of poly-D-lysine dome sheet, adds DMEM/F12 differentiation culture liquid containing 2%FBS, differentiation culture terminates after 1 week cultivating, sucks culture fluid, add 4% poly first Aldehyde fixes 30min, dyes with Hoechst33342 after PBS, observed result under fluorescence microscope；

The detection of TUNEL method apoptosis is by after each group of NSCs differentiation culture 2 weeks, sucks the culture fluid in each hole, uses 0.01M PBS 3 times, then fixes 20min with under the 0.1PBS room temperature containing 4% paraformaldehyde, sucks paraformaldehyde, and PBS is clear Washing 3 times, carry out apoptosis detection by Roche company's T UNEL apoptosis detection kit by operating instruction, last PBS develops a film After 3 times, under the conditions of lucifuge, adding Hoechst33342, the wet box of room temperature hatches 30min.PBS develops a film after 3 times, neutral sweet Sheet for oil seal；Just put fluorescence microscopy Microscopic observation TUNEL, Hoechst positive cell core and take the photograph sheet；Soft by Stata10.0 statistics Each group of data are carried out comparing between variance analysis and group by part；

In high performance liquid chromatography detection differentiation culture liquid, dopamine (Dopamine, DA) content is to take NSCs differentiation training by each group Nutrient solution 1ml, adds 0.1mol/L and crosses chloric acid acidifying culture fluid, and 14000rpm is centrifuged 15min, takes after supernatant filters by Wu The method row high-performance liquid chromatogram determination of the report such as Yan Qiong；

It is characterized in that: the method is implemented step and also included:

Three, recombinant virus transfection NSCs:

The transfection method of described recombinant virus: take P3 and be prepared as single cell suspension for NSCs digestion, be seeded to after cell counting In 24 well culture plates, 4 × 104 cells of every hole inoculation, add 500 μ l basic culture solution suspension cells；Cell is divided into 4 groups Transfect, often organize 6 holes: Mock group transfects pLV5-GFP slow virus negative control；TH group transfection pLV5-TH recombinant lentiviral is sick Poison；Brn4 group transfection pLV5-Brn4 recombinant slow virus；TH+Brn4 group transfection pLV5-TH+Brn4 recombinant slow virus；Every hole Add slow virus and 5 μ g/ml ploybrene that 20 μ l 1 × 108TU/ml infection multiplicities are 50, after jiggling mixing Culture plate is put in incubator and cultivate, after 12 hours, change fresh division culture medium；After infecting 72 hours, show being inverted fluorescence Micro-Microscopic observation luciferase expression situation, adds 1 μ g/ml puromycin in the medium simultaneously and screens, to obtain stable turning The cell of dye；The NSCs ball formed each group after 7 days carries out Secondary Culture；

Four, the qualification of recombinant slow virus transfection NSCs.

The recombinant slow virus induced nerve stem cells that utilizes described in utilization obtains one transfection to the method that dopaminergic neuron breaks up The neural stem cell of pLV5-TH+Brn4 recombinant slow virus: be by stablizing of obtaining in the enforcement step 3 described in described method The NSCs of transfection pLV5-TH+Brn4 virus makes single cell suspension, with basic culture solution, cell is made into 2 × 106/ml, adds Enter in 1.5ml cell cryopreservation tube, be simultaneously introduced 0.4ml calf serum and 0.1ml dimethyl sulfoxide, seal after mixing, put 4 DEG C 1 hour ,-20 DEG C 2 hours, be then directly placed in-80 DEG C of ultra cold storage freezers preservation obtain.

The qualification of described recombinant slow virus transfection NSCs also includes: Western Blot detection, Immunofluorescence inspection Survey:

Described Western Blot detection is that the NSCs ball after transfecting each group is seeded in 24 orifice plates break up, and separately sets As a control group, differentiation culture terminates after 2 weeks cultivating the NSCs of one group of untransfected, by the requirement of RIPA lysate description, Extract the albumen of cell in each group, with 10%SDS-PAGE vertical electrophoresis, albumen is separated, the half-dried transferring system of Bio-Rad Transferring film 15V, 50min, 5% defatted milk powder is separately added into following one and resists after closing: little mouse-anti β-actin antibody, little mouse-anti TH Antibody, rabbit anti-Brn4 antibody, rabbit anti-GDNF antibody, rabbit anti-GFR α-1 antibody, rabbit anti-ret antibody, incubated at room 2h, 4 DEG C overnight, and the goat anti-mouse igg and the goat anti-rabbit igg two that are separately added into horseradish peroxidase combination next day resist, incubated at room 4h, is then exposed, develops and with gathered image, is averaged Western blot result light by image analysis software Density analysis, and carry out comparing between variance analysis and group to each group of data by the method for statistics；

Described Immunofluorescence detection is to terminate after each group of NSCs breaks up 2 weeks cultivating, to negative control group and each restructuring In virus transfection group, cell carries out Immunofluorescence test, is summarized as follows: used one anti-is respectively as follows: little mouse-anti TH, big mouse-anti DAT； Two resist and are: be combined with the mountain sheep anti-mouse igg of Alexa Fluor 568；One resists in 4 DEG C of overnight incubation, two anti-incubated at room temperature 2h, carries out nuclear targeting 30min with Hoechst33342 subsequently, all carries out routine with 0.01M PBS between middle each step Develop a film, finally with 20% glycerol buffer mounting, and under FV10i Intelligent laser Laser Scanning Confocal Microscope, observe TH's or DAT Expression, and calculate GFP/TH and GFP/DAT double-labeled cell and account for the percentage ratio of Hoechst labeled cell, and use statistical method Carry out comparing between variance analysis and group to each group of data；

The chromatographic condition of DA content in described high performance liquid chromatography detection differentiation culture liquid: chromatographic column is 4.6 × 250mm, The Inertsil ODS-SP type octadecylsilane chemically bonded silica post of granularity 5 μm, column temperature 40 DEG C, flowing is phosphate mutually Buffer and the mixed liquor of methanol, flow velocity is 1.0ml/min, and detector is SPD-20A type UV-detector, detects wavelength 280 nm；Qualitative analysis: compare qualitative with standard substance retention time with the retention time of sample peak；Quantitative analysis: first with DA standard Product draw standard curve, carry out sample determination the most successively, with quantified by external standard method, represent content with peak area.

Described phosphate buffer is by potassium dihydrogen phosphate 6.742g, dipotassium hydrogen phosphate 0.0114g, adds water and is dissolved to 1000 Ml, with phosphorus acid for adjusting pH value to 4.2, and obtains with 0.12 μm cellulose ester membrane filtration.

Described flowing is the mixed liquor of phosphate buffer and methanol mutually, and its ratio is 97:3.

Described NSCs culture fluid is made up of DMEM/F12,2%B27,20ng/mlEGF, 20ng/ml bFGF.

The infection multiplicity of described slow virus is 50.

Beneficial effect

One, our cell of isolated from Mus embryo mesencephalic tissue has constantly propagation and self-renewal capacity, and has differentiation For many differentiation potentials of neuron, astrocyte and oligodendrocyte, it it is the stem cell of central nervous system；

Two, successfully building the slow virus carrier with genes of interest TH and Brn4, after transfection NSCs, allogenic gene can be at cell Interior stable expression, obtains rat NSCs-TH+Brn4 cell strain；NSCs-TH+Brn4 can produce higher proportion after breaking up in vitro TH positive dopaminergic neuron, and these neurons are in form, phenotype and the most ripe.Brn4 can promote Neurotrophic factor GDNF and the high expressed of receptor GFR α-1 and Ret thereof, the most also have the function of inhibited apoptosis, this A bit for differentiation, the maturations of dopamine neuron and maintain its survival to play an important role.

Accompanying drawing explanation

Fig. 1 is structure and the slow virus packaging test result figure of slow virus carrier；

Fig. 2 is the cultivation of NSCs, separation and qualification result figure；

Fig. 3 is the Efficiency testing result figure of recombinant virus transfection NSCs；

Fig. 4 is Western blot testing result figure；

Fig. 5 is Immunofluorescence test result figure；

Fig. 6 is apoptosis testing result figure.

Detailed description of the invention

In conjunction with accompanying drawing, the present invention is described further:

Part I experiment in vitro

The preparation of the neural stem cell of transfection recombinant slow virus and the qualification of this neural stem cell

Experiment material

1.1, laboratory animal

Pregnant 14d Sprague-Dawley (SD) rat, cleaning grade, Nantong University's Experimental Animal Center provide, production licence Number: SCXK (Soviet Union) 2002-0019.

1.2 experimental apparatus

(1) SPD-20A type UV-detector (Shimadzu, Japan)；

(2) CO2 incubator (Jouan company of France)；

(3) superclean bench (SW-CJ-1F, SuZhou Antai Air Tech Co., Ltd.)；

(4) freezing centrifuge (Beckman company of Germany)；

(5) Leica DMR is just putting fluorescence microscope (Leica company of Germany)；

(6) Leica DMIRB inverted fluorescence microscope (Leica company of Germany)；

(7) ultra cold storage freezer (Nuaire company of the U.S.)；

(8) SDS-PAGE electrophoresis tank and membrane-transferring device (Bio-Rad company)；

(9) LeicaQwin image analysis system (Leica company)；

(10) Molecular Imager ChemiDoc XRS System gathers image (Bio-Rad company)；

(11) FV10i Intelligent laser Laser Scanning Confocal Microscope (Olympus company)；

(12) TUNEL apoptosis detection kit (Roche company)；

(13) Tissue Culture Flask (BD company)；

(14) 200 mesh nylon wires (BD company)；

(15) cell cryopreservation tube (BD company)；

(16) cellulose adipose membrane (BD company)；

(17) Inertsil ODS-SP type octadecylsilane chemically bonded silica post (Shimadzu company)；

(18) Quantity One image analysis software (Bio-Rad company).

1.3 experiment reagent

1.3.1 slow virus builds reagent

(1) pLV5-EF1a-GFP slow virus (Genepharma company)；

(2) restriction endonuclease BamHI (MBI)；

(3) restriction endonuclease NotI (MBI)；

(4) T4DNA Ligase (NEB company)；

(5) DNA purification kit (Axygen company)；

(6) DH5 α competent cell (TransGen Biotech company)；

(7) ammonia benzyl antibiotic solution (Invitrogen company)；

(8) packaging plasmid Packaging Mix (Genepharma company)；

(9) 293FT cell (Invitrogen company)；

(10) DMEM+10%FBS (Invitrogen company)；

(11) Lipofectamine 2000 (Invitrogen company)；

(12) Opti-MEM culture fluid (Invitrogen company)；

(13) 0.05%Trypsin (Invitrogen company).

1.3.2 cell cultivates main agents

(1) DMEM culture medium (Dulbecco ' s Modified EagleMedium, Cat.No.12100-038, Gibco company)；

(2) F12 culture medium (F12Nutrient Mixture, Cat.No.21700-026, Gibco company)；

(3) serum-free culture additive B 27 (B27Supplement, Cat.No.17504-044, Gibco company)；

(4) hyclone (Fetal Bovine Serum, FBS, Cat.No.16140-087, Gibco company)；

(5) epidermal growth factor (Epidermal Growth Factor, EGF, Cat.No.E4127, Sigma company)；

(6) basic fibroblast growth factor (Fibroblast Growth Factor-Basic, bFGF, Cat.No.F0291, Sigma Company)；

(7) cell dissociation buffer Accutase (Sigma company)；

(8) BrdU (5-Bromo-2-deoxyUridine, sigma company)；

(9) RIPA lysate (the green skies, P0013B)；

(10) dimethyl sulfoxide (dimethyl sulfoxide, sigma company).

(11) puromycin (puromycin, sigma company).

1.3.3 main antibody

(1) little mouse-anti TH (1:200, Millipore company)；

(2) big mouse-anti DAT (1:5000, Millipore company)；

(3) the mountain sheep anti-mouse igg (1:500, Invitrogen company) of Alexa Fluor 568 it is combined with；

(4) little mouse-anti β-actin antibody (1:2000, Sigma company)；

(5) little mouse-anti TH antibody (1:200, Millipore company)；

(6) rabbit anti-Brn4 antibody (1:1000, Santa Cruz company)；

(7) rabbit anti-GDNF antibody (1:200, Abcam company)；

(8) rabbit anti-GFR α-1 antibody (1:500, Abcam company)；

(9) rabbit anti-ret antibody (1:5000, Abcam company)；

(10) goat anti-mouse igg (1:1000, Pierce company) that horseradish peroxidase combines；

(11) goat anti-rabbit igg (1:1000, Pierce company)；

(12) Hoechst33342 (1:2000, sigma company).

1.4 experimental technique

One, recombined lentivirus vector builds

(1) genes of interest obtains

1) design of primers

Requirement according to Genepharma company pLV5-EF1a-GFP Lentiviral and GenBank rat The cDNA function full length sequence design synthetic pcr primer thing of TH (NM_012740) and Brn4 (NM_017252), upstream is drawn Thing 5 ' end adds BamHI site sequence, and downstream primer 5 ' end adds NotI site sequence, primer sequence TH-1F, TH-1R, Brn4-1F, Brn4-1R, TH+Brn4, TH+Brn4 such as sequence table:

2) amplifying target genes fragment and glue reclaim

(1) PCR system is as follows:

(2) PCR condition is as follows:

95℃ 3min

(94 DEG C of 30sec, 58 DEG C of 30sec, 68 DEG C of 30sec) circulate 30 times

68℃ 10min；

(3) agarose gel electrophoresis, and use DNA purification kit to reclaim genes of interest fragment.

3) structure and the order-checking of recombiant plasmid is identified

(1) by BamHI and NotI enzyme action genes of interest fragment, 37 DEG C of enzyme action 30min, enzyme action system used is:

(2) with BamHI and NotI enzyme action pLV5-EF1a-GFP plasmid, enzyme action system is:

Digestion products carries out after (3) 37 DEG C of enzyme action 30min the parallel glue of electrophoresis reclaim；

(4) connecting genes of interest fragment and 16 DEG C of carrier connects 2h, linked system is:

(5) take 5 μ l and connect product conversion DH5 α competent cell；

(6) picking positive colony, and sequence verification.

(2) packaging of slow virus and the mensuration of virus titer

With the slow virus carrier pLV5-EF1a-GFP built and packaging plasmid (pGag/Pol, pRev, pVSV-G) cotransfection 293T cell also carries out slow virus packaging, collects virus stock solution used, and ultracentrifugation concentrates, and measures titre.

1) slow virus packaging

(1) take cell state good, be in the 293T cell of exponential phase, after cell counting, according to each 10cm's Culture dish 6 × 10⁶Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；

Remove culture fluid before transfection in (2) the 2nd days, change 5ml Opti-MEM culture fluid；

(3) take 9 μ g Packaging Mix and 3 μ g slow virus plasmids add in 37 DEG C of preheating 1.5ml Opti-MEM, gently Mixing；

(4) taking 36 μ l lipofectamine 2000 to add in 1.5ml Opti-MEM, mix gently, room temperature places 5min；

(5) mixing plasmid solution (3) and lipofectamine 2000 diluent (4), put room temperature 20min；

(6) 3ml plasmid liposome complex (5) is carefully added in Tissue Culture Dish, mixes gently, 37 DEG C, 5% CO₂Incubator in hatch 4_~After 6h, change complete culture solution DMEM+10%FBS；

(7) collecting cells and supernatant after 48h, 3000rpm is centrifuged 10min, removes cell and fragment, and by 0.45 μm Filter filters；

(8) by pLV5-TH, pLV5-Brn4 or pLV5-TH+Brn4 virus stock solution used ultracentrifugation 2h under 50000g, go Except supernatant, being resuspended in 2ml DMEM culture fluid, subpackage 200 μ l often manages, and is positioned over-80 DEG C and saves backup.

2) slow virus activity titers measures

(1) preparation of slow virus dilution culture medium: addition 2%FBS in DMEM, then add the Polybrene of 8 μ g/ml, After mix homogeneously standby.

(2) slow virus liquid dilution: slow virus stock solution, with after 10 times of dilution proportion of slow virus dilution culture medium, respectively takes 100ul Infect the HEK293 cell in 96 orifice plates, specifically comprise the following steps that

1. No. 1 diluent: 5 μ l slow virus liquid+245 μ l viral dilution culture medium, containing slow virus liquid 2 × 10 in every 100 μ l^-3ml；

2. No. 2 diluents: 25 μ l 1 diluent+225 μ l viral dilution culture medium, containing slow virus liquid 2 × 10 in 100 μ l^-4ml；

3. No. 3 diluents: 25 μ l 2 diluent+225 μ l viral dilution culture medium, containing slow virus liquid 2 × 10 in 100 μ l^-5ml；

4. No. 4 diluents: 25 μ l 3 diluent+225 μ l viral dilution culture medium, containing slow virus liquid 2 × 10 in 100 μ l^-6ml；

5. No. 5 diluents: 25 μ l 4 diluent+225 μ l viral dilution culture medium, containing slow virus liquid 2 × 10 in 100 μ l^-7ml；

……

(3) carefully suck the culture medium in 96 orifice plates, mix each pipe slow virus diluent gently, respectively take 100 μ l and add every hole In cell, each dilution factor two repetition.Put into overnight incubation in the cell culture incubator of 37 DEG C；

(4) slow virus titre is calculated: after 72h, fluorecyte quantity in each hole of fluorescence microscopy Microscopic observation, virus titer is The cell number average expressing fluorescence in each hole amasss divided by the slow virus liquid contained in every hole.

Two, neural stem cell separation, cultivate and identify

Aseptically, it is organized as former with SD tire Mus (the Nantong University's Experimental Animal Center provides) ventral mesencephalan of pregnant 14d Material, repeatedly blows and beats with suction pipe until being cell suspension, and through 200 mesh nylon net filters, collects the cell suspension after filtering, It is resuspended in NSCs culture fluid (by DMEM/F12,2%B27,20ng/mlEGF, 20ng/ml after cleaning 3 times with basal medium BFGF etc. form), after cell counting and vigor are observed, it is inoculated in containing 10ml NSCs by 1 × 105/ml cell density In the Tissue Culture Flask of culture fluid, it is placed in the CO2 incubator of 5%, 37 DEG C cultivation.Formed outstanding after 3 days in visible culture bottle Floating NSCs ball, carries out Secondary Culture after 7 days.Low-speed centrifugal collects NSCs ball, carries out with Accutase enzyme after cleaning 3 times Digestion, terminates digestion with the basic culture solution containing 10%FBS after about 10min, makes it be dispersed into list by the way of machinery is blown and beaten Cell suspension, continues to cultivate in culture bottle after carrying out cell counting.Take P3, for NSCs, culture fluid adds when passing on BrdU (final concentration of 5 μm ol/L) are marked, and make BrdU be incorporated in neural stem cell DNA, carry out BrdU immunity after 7 days Fluoroscopic examination.Separately take P3 and prove its embryo source property for later NSCs sub-clone ball Nestin Immunofluorescence test.By P10 It is inoculated in for NSCs ball in 24 well culture plates being pre-filled with scribbling poly-D-lysine coverslip, adds containing 10%FBS's DMEM/F12 differentiation culture liquid breaks up, and carries out MAP-2, GFAP and CNP Immunofluorescence test respectively with card after 2 weeks Many differentiation potentials of bright NSCs.

Three, recombinant virus transfection NSCs

Take P3 and be prepared as single cell suspension for NSCs digestion, be seeded to after cell counting in 24 well culture plates, every hole inoculation 4 × 104 cells, add 500 μ l basic culture solution suspension cells.Cell is divided into 4 groups and transfects, and often organizes 6 holes: Mock Group transfection pLV5-GFP slow virus negative control；TH group transfection pLV5-TH recombinant slow virus；Brn4 group transfection pLV5-Brn4 Recombinant slow virus；TH+Brn4 group transfection pLV5-TH+Brn4 recombinant slow virus.It is slow that every hole adds 20 μ l 1 × 108TU/ml Virus (MOI=50) and 5 μ g/ml ploybrene, put culture plate after jiggling mixing in incubator and cultivate, 12 hours The fresh division culture medium of rear replacing.After infecting 72 hours, under inverted fluorescence microscope, observe luciferase expression situation, simultaneously in training Foster base adds 1 μ g/ml puromycin screen, to obtain the cell of stable transfection.To each group of NSCs formed after 7 days Ball carries out Secondary Culture.The NSCs of stable transfection pLV5-TH+Brn4 virus is made single cell suspension, uses basic culture solution Cell is made into 2 × 106/ml, adds in 1.5ml cell cryopreservation tube, be simultaneously introduced 0.4ml calf serum and 0.1ml dimethyl Sulfoxide, after mixing seal, put 4 DEG C 1 hour ,-20 DEG C 2 hours, be then directly placed in-80 DEG C of ultra cold storage freezers preservation.

Four, transfection efficiency detection

Due to reporter gene GFP can respectively with genes of interest TH and Brn4 amalgamation and expression, therefore can by observe green fluorescence Come whether testing goal gene TH and Brn4 expresses in NSCs.NSCs ball after transfection is seeded to be pre-coated with poly rely In 24 orifice plates of propylhomoserin dome sheet, adding the DMEM/F12 differentiation culture liquid containing 2%FBS, differentiation culture terminates training after 1 week Support, suck culture fluid, add 4% paraformaldehyde and fix 30min, with Hoechst33342 (1:2000, sigma after PBS Company) dyeing, observed result under fluorescence microscope.Every coverslip randomly selects 5 visuals field, absorbs the same visual field respectively The cell photo that interior GFP and Hochest is positive, imports photo LeicaQwin image analysis system and carries out overlap processing, meter Number fluorescencepositive cell number and total cell number, both ratio is transfection efficiency.

Five, Western Blot detection

NSCs ball after transfecting each group is seeded in 24 orifice plates break up, and separately sets the NSCs of one group of untransfected as comparison Group, differentiation culture terminates after 2 weeks cultivating, and by the requirement of RIPA lysate (the green skies, P0013B) description, extracts each The albumen of cell in group, the method reported according to this seminar, with 10%SDS-PAGE vertical electrophoresis, albumen is separated, Bio-Rad half-dried transferring system transferring film 15V, 50min, 5% defatted milk powder is separately added into following one and resists after closing: little mouse-anti β -actin antibody (1:2000, Sigma company), little mouse-anti TH antibody (1:200, Millipore company), the anti-Brn4 of rabbit Antibody (1:1000, Santa Cruz company), rabbit anti-GDNF antibody (1:200, Abcam company), rabbit anti-GFR α-1 antibody (1:500, Abcam company), rabbit anti-ret antibody (1:5000, Abcam company), incubated at room 2h, 4 DEG C are overnight.Next day is separately added into Goat anti-mouse igg (1:1000, Pierce) that horseradish peroxidase (HRP) combines and goat anti-rabbit igg (1:1000, Pierce) Two resist, incubated at room 4h, are then exposed, develop and gather with Molecular Imager ChemiDoc XRS System Image (Bio-Rad company), enters Western blot result by Quantity One image analysis software (Bio-Rad company) Row average optical is analyzed, and carries out comparing between variance analysis and group to each group of data with Stata10.0 statistical software.

Six, Immunofluorescence detection

Each group NSCs terminates cultivating after breaking up 2 weeks, by the report of this seminar, transfects negative control group and each recombinant virus In group, cell carries out Immunofluorescence test, is summarized as follows: used one anti-be respectively as follows: little mouse-anti TH (1:200, Millipore are public Department), big mouse-anti DAT (1:5000, Millipore company)；Two resist and are: be combined with the mountain sheep anti mouse of Alexa Fluor 568 IgG (1:500, Invitrogen company)；One resists in 4 DEG C of overnight incubation, and two anti-incubated at room temperature 2h use Hoechst33342 subsequently Carry out nuclear targeting 30min, all carry out routine with 0.01M PBS between middle each step and develop a film, finally delay with 20% glycerol Rush fluid-tight sheet, and under FV10i Intelligent laser Laser Scanning Confocal Microscope (Olympus company), observe the expression of TH or DAT Situation, and calculate GFP/TH and GFP/DAT double-labeled cell and account for the percentage ratio of Hoechst labeled cell (total cellular score), use Each group of data are carried out comparing between variance analysis and group by Stata10.0 statistical software.

Seven, TUNEL method apoptosis detection

Each group NSCs differentiation culture, after 2 weeks, sucks the culture fluid in each hole, with 0.01M PBS 3 times, then with containing 20min is fixed under the 0.1PBS room temperature of 4% paraformaldehyde；Suck paraformaldehyde, PBS 3 times, use Roche company TUNEL apoptosis detection kit carries out apoptosis detection by operating instruction.Last PBS develops a film after 3 times, in lucifuge condition Under, adding Hoechst33342 (1:2000), the wet box of room temperature hatches 30min.PBS develops a film after 3 times, neutral glycerine mounting. Observe TUNEL, Hoechst positive cell core and take the photograph sheet under just putting fluorescence microscope (Leica DMR, Germany).Use Stata10.0 Each group of data are carried out comparing between variance analysis and group by statistical software.

Eight, DA content in high performance liquid chromatography (HPLC) detection differentiation culture liquid

Each group takes NSCs differentiation culture liquid 1ml, adds 0.1mol/L and crosses chloric acid acidifying culture fluid, and 14000rpm is centrifuged 15min, Take the method row high-performance liquid chromatogram determination reported after supernatant filters by Wu Yanqiong etc..Chromatographic condition: chromatographic column is Inertsil ODS-SP type octadecylsilane chemically bonded silica post (4.6 × 250mm, granularity 5 μm), column temperature 40 DEG C；Flowing is mutually (potassium dihydrogen phosphate 6.742g, dipotassium hydrogen phosphate 0.0114g add water and are dissolved to 1000ml, regulate with phosphoric acid phosphate buffer PH value to 4.2, and with 0.12 μm cellulose ester membrane filtration) with the mixed liquor (97:3) of methanol；Flow velocity is 1.0ml/min； Detector is SPD-20A type UV-detector, detects wavelength 280nm.Qualitative analysis: with retention time and the mark of sample peak The comparison of quasi-product retention time is qualitative.Quantitative analysis: draw standard curve first with DA standard substance, depend on the most under the same conditions Secondary carry out sample determination, with quantified by external standard method, represent content with peak area.

1.5 experimental result

The structure of slow virus carrier and slow virus packaging

As shown in Figure 1A, expand TH and Brn4 full length gene using Midbrain In The Rat mRNA as template, PCR, with PLV5-EF1a-GFP Lentiviral is attached, and converts DH5 α competent cell, picking positive colony warp Sequence verification, slow virus carrier pLV5-TH, pLV5-Brn4 and pLV5-TH+Brn4 successfully construct.With the slow virus built Carrier and packaging plasmid cotransfection 293T cell packaging virus, collect virus stock solution used, and ultracentrifugation concentrates, and measures titre.Slowly After viral vector and packaging plasmid transfection 293T cell 24h, under fluorescence microscope, see the green fluorescence of virus, such as Figure 1B Shown in, collect virus stock solution used after 48h, ultracentrifugation concentrates, and gained slow virus is cooked gradient dilution postoperative infection HEK293 cell, In 5. number dilution fluid apertures (liquid 2 × 10-7ml Han slow virus in 100ul), observe that the cell expressing green fluorescence is respectively 16 Individual and 24, virus titer is to express fluorecyte number average in each hole to amass divided by the slow virus liquid contained in every hole, therefore Virus titer is: (16+24)/2=20TU, 20TU/ (2 × 10-7) ml=1.0 × 108TU/ml.

The cultivation of NSCs, separate and identify

As in figure 2 it is shown, NSCs of the present invention is cultivating initial 1-2 days, it is seen that there are many small circular dead at the bottom of culture bottle Cell and fragment thereof and the most adherent colloid like cell, suspend in culture fluid the most adherent unicellular and small cell cluster.From 3rd day starts, it is seen that occurring in the cell mass of suspension that cell space is relatively big, endochylema color is relatively deep, not becoming of karyon/endochylema large percentage Ripe cell, it is seen that many cells divide.As shown in Figure 2 A, the cell number in cell mass increases sharply subsequently, and the 4th The cell ball containing dozens of cell is i.e. formed about it.As shown in Figure 2 B, hereafter the cell number in cell ball continues to increase, Cell ball continues to increase, and increases the most inconspicuous to about 1 week cell ball, and each neural ball is containing about hundreds of cells, composition nerve The cell size of ball is basically identical, and form rule, without enation.Cell proliferation growing state after passing on is similar with primary, The neural ball i.e. forming constant magnitude in about about 1 week.As shown in Figure 2 C, BrdU labelling and Immunofluorescence test show and are newly formed Cell clone ball in most cells karyon present strong green fluorescence, positive in BrdU immunoreation, show that these are cloned Cell is by the continuous division growth of the cell inoculated.As shown in Figure 2 D, Nestin Immunofluorescence test display cell clone Cell in ball all presents strong red fluorescence, show neural stem cell all in Nestin antigen positive, for embryo source sexual cell.As Shown in Fig. 2 E, NSCs ball there are about the MAP-2 of 100～150 fluorescence that take on a red color around visible each neural ball after breaking up 2 weeks Positive neuron, cell body is less, rounded, oval or triangle, and karyon is obvious, and not colored, projection is few and elongated, Based on double projections；As shown in Figure 2 F, the GFAP astrocyte number in green fluorescence is most, each neural ball Around there are about 500～800, cell is flat, cell space is relatively big, form is irregular, and circular karyon is obvious, stretches out many from cell space Branched projection；As shown in Figure 2 G, the CNP positive oligodendrocytes of the fluorescence that takes on a red color is minimum, around each neural ball Only 30～50, Chang Chengqun is distributed.These cell body are less, send many short and small and elongated projections from cell space.Above Result shows that the neurocyte of our separation and Culture has embryo source property, division growth ability and many differentiation potentials, is nervous system Stem cell.

The Efficiency testing of recombinant virus transfection NSCs

As it is shown on figure 3, have no obvious cell death under inverted microscope after NSCs infection virus, cell viability is preferable, refractive power Property very strong and visible cell multiple fission.After transfection 36h, under fluorescence microscope, in visible cell, green fluorescence occurs, wherein It is more weak that matched group fluorescence is relatively strong, other respectively organizes fluorescence, illustrates that the recombinant virus transfection efficiency importing genes of interest decreases.With Time lengthening fluorescence and gradually increase enhancing, after transfection, 72h fluorecyte quantity and fluorescence intensity reach peak, wherein TH, Brn4, And there is no notable difference between TH+Brn4 tri-groups, cell images after Hoechst33342 redyes under fluorescence microscope, warp Leica Qwin graphical analysis show that four groups of transfection efficiencies are similar, respectively 89.12%, 92.36%, 91.81%.Recombinant virus exists In NSCs, expression time is longer, and after passage is cultivated, green fluorescence the most substantially weakens.After passing on 3 times we will stablize turn The frozen conservation of NSCs of dye pLV5-TH+Brn4 virus, obtains rat NSCs-TH+Brn4 cell strain.

Four, Western blot testing result

As shown in Figure 4 A, Western Blot result show each group molecular weight be at 62kD and 39kD seen from more weak endogenous Property TH and Brn4 positive band.After recombinant slow virus transfects, the relative expression of TH albumen in TH group and TH+Brn4 group Measure relatively matched group and Mock group substantially increases, and in Brn4 group and TH+Brn4 group, the relative expression quantity of Brn4 albumen substantially increases Height, as shown in Figure 4 B, protein band average optical density value has significant difference (* P < 0.01), illustrate exogenous TH and Brn4 gene the most successfully imports in NSCs and can specifically strengthen the expression of intracellular corresponding albumen.Meanwhile, NSCs is respectively organized In also detect that faint endogenous GDNF and receptor GFR α-1, the expression of Ret albumen, its molecular weight be respectively 24kD, 50kD、124kD.After slow-virus transfection, the phase of GDNF, GFR α-1 and Ret albumen in Brn4 group and TH+Brn4 group Substantially increasing expression compared with other each group, protein band average optical density value has significant difference (* P < 0.01), prompting In NSCs, the process LAN of Brn4 albumen can induce the expression of GDNF, GFR α-1 and Ret albumen to increase.

Five, Immunofluorescence test result

As it is shown in figure 5, TH Immunofluorescence test result display differentiation culture is after 2 weeks, at matched group, Mock group and Brn4 In group, TH positive neuron is little, and statistical result shows that TH positive cell differentiation rate is respectively 3.05%, 2.97%, 4.59%, Without significant difference between three groups.As it can be seen from figure 5k, TH positive neuron showed increased, cell in TH group and TH+Brn4 group Differentiation rate respectively reaches 54.5%, 65.71%, there is significant difference between two groups, and as illustrated in fig. 6e in TH+Brn4 group TH positive neuron form is the most ripe, and cell body is relatively big, and projection is elongated abundant, and local is woven into netted.Such as Fig. 5 L institute Showing, DAT Immunofluorescence test result shows, DAT positive neurons seldom detected in matched group, Mock group and Brn4 group Unit, DAT positive cell differentiation rate is respectively 1.41%, 1.64%, 1.58%；DAT positive neuron showed increased in TH group, DAT positive cell differentiation rate is 16.12%, and in TH+Brn4 group, DAT positive neuron is most, and DAT positive cell divides Rate reaches 32.28%, and between two groups, difference has statistical significance.After illustrating to transfect slow virus pLV5-TH, it is possible to promote NSCs breaks up to dopaminergic neuron, and transfects pLV5-Brn4 and NSCs can not be induced to dopaminergic nerve positive for TH Unit's differentiation, but simultaneously transfection TH+Brn4 gene then to promote that NSCs is divided into form the most ripe, and express DAT's Ripe dopaminergic neuron.

Six, apoptosis testing result

As shown in Figure 6, TUNEL method detection apoptotic nucleus display red fluorescence, wherein Brn4 group and TH+Brn4 group TUNEL Positive apoptotic cells quantity is considerably less than other each group.Each group apoptotic cell percentage rate Analysis of variance and two-by-two comparative result show, Brn4 group and TH+Brn4 group TUNEL positive percentage well below with matched group, and Mock group, TH group are with right There was no significant difference according between group.Prompting Brn4 albumen high expressed energy inhibited apoptosis, improves the survival rate of cell.

Seven, high performance liquid chromatography testing result

Analyzing through high performance liquid chromatography detection, in the culture fluid of matched group, Mock group and Brn4 group, DOPAMINE CONTENT IN RABBIT is the lowest, TH group DOPAMINE CONTENT IN RABBIT slightly raises, and TH+Brn4 group DOPAMINE CONTENT IN RABBIT is the highest, reaches 6.78 ± 1.58 μ g/ml.Prompting Can promote after Brn4 and TH gene co-transfection that NSCs differentiation and development is the dopaminergic neuron that function is ripe.Each group data warp Variance analysis and two-by-two comparative result show, there was no significant difference between matched group, Mock group and Brn4 group, between remaining each group All there is significant difference (table 1).

Table-1HPLC detect DOPAMINE CONTENT IN RABBIT result in each component culture fluid (N=6)

* P < 0.01, compared with matched group, #P < 0.01, compared with TH group.

Claims

1. utilize the method that recombinant slow virus induced nerve stem cells breaks up to dopaminergic neuron, described method to include implementing as follows step:

One, pLV5-TH+Brn4 recombined lentivirus vector builds:

Requirement according to pLV5-EF1a-GFP Lentiviral and the cDNA function full length sequence of GenBank rat TH and Brn4 are respectively the gene of NM_012740 and NM_017252, build pLV5-TH, pLV5-Brn4 and pLV5-TH+Brn4 Lentiviral, carry out order-checking after packaging and identify and titre qualification；

Two, neural stem cell separation, cultivate and identify:

The cultural method of neural stem cell: aseptically, it is organized as raw material with the SD tire Mus ventral mesencephalan of pregnant 14d, repeatedly blow and beat until being cell suspension with suction pipe, and through 200 mesh nylon net filters, collect the cell suspension after filtering, it is resuspended in NSCs culture fluid after cleaning 3 times with basal medium, after cell counting and vigor are observed, by 1 × 10⁵/ ml cell density is inoculated in the Tissue Culture Flask containing 10ml NSCs culture fluid, is placed in the CO2 incubator of 5%, 37 DEG C cultivation, forms, in visible culture bottle, the NSCs ball suspended, carry out Secondary Culture after 7 days after 3 days；

The separation method of neural stem cell: after neural stem cell cultivates 3 days in culture bottle, NSCs ball is collected with low-speed centrifugal, digest with Accutase enzyme after cleaning 3 times, digestion is terminated with the basic culture solution containing 10%FBS after 10min, make it be dispersed into single cell suspension by the way of machinery is blown and beaten, after carrying out cell counting, culture bottle continues to cultivate；

The authentication method of neural stem cell: take P3 adds final concentration of 5 μm ol/L for NSCs when passing on BrdU in culture fluid and be marked, make BrdU be incorporated in neural stem cell DNA, carry out BrdU Immunofluorescence test after 7 days；Separately take P3 and prove its embryo source property for later NSCs sub-clone ball Nestin Immunofluorescence test；P10 is inoculated in for NSCs ball in 24 well culture plates being pre-filled with scribbling poly-D-lysine coverslip, add the DMEM/F12 differentiation culture liquid containing 10%FBS to break up, carry out MAP-2, GFAP and CNP Immunofluorescence test after 2 weeks respectively to prove many differentiation potentials of NSCs；

Three, recombinant virus transfection NSCs:

The transfection method of described recombinant virus: take P3 and be prepared as single cell suspension for NSCs digestion, be seeded to after cell counting in 24 well culture plates, every hole inoculation 4 × 10⁴Individual cell, adds 500 μ l basic culture solution suspension cells；Cell is divided into 4 groups and transfects, and often organizes 6 holes: Mock group transfects pLV5-GFP slow virus negative control；TH group transfection pLV5-TH recombinant slow virus；Brn4 group transfection pLV5-Brn4 recombinant slow virus；TH+Brn4 group transfection pLV5-TH+Brn4 recombinant slow virus；Every hole adds 20 μ l 1 × 10⁸The slow virus of TU/ml and 5 μ g/ml ploybrene, put culture plate after jiggling mixing in incubator and cultivate, change fresh division culture medium after 12 hours；After infecting 72 hours, under inverted fluorescence microscope, observe luciferase expression situation, add 1 μ g/ml puromycin in the medium simultaneously and screen, to obtain the cell of stable transfection；The NSCs ball formed each group after 7 days carries out Secondary Culture；

Four, the qualification of recombinant slow virus transfection NSCs, including:

Transfection efficiency detection is that the NSCs ball after transfecting is seeded to be pre-coated with in 24 orifice plates of poly-D-lysine dome sheet, add the DMEM/F12 differentiation culture liquid containing 2%FBS, differentiation culture terminates after 1 week cultivating, suck culture fluid, add 4% paraformaldehyde and fix 30min, dye with Hoechst33342 after PBS, observed result under fluorescence microscope；

The detection of TUNEL method apoptosis is by after each group of NSCs differentiation culture 2 weeks, sucks the culture fluid in each hole, with 0.01M PBS 3 times, then 20min is fixed with under the 0.1PBS room temperature containing 4% paraformaldehyde, suck paraformaldehyde, PBS 3 times, carry out apoptosis detection by TUNEL apoptosis detection kit by operating instruction, last PBS develops a film after 3 times, under the conditions of lucifuge, adding Hoechst33342, the wet box of room temperature hatches 30min, PBS develops a film after 3 times, neutral glycerine mounting；Just put fluorescence microscopy Microscopic observation TUNEL, Hoechst positive cell core and take the photograph sheet；Carry out comparing between variance analysis and group to each group of data by statistical method；

In high performance liquid chromatography detection differentiation culture liquid, DA content is to take NSCs differentiation culture liquid 1ml by each group, add 0.1mol/L and cross chloric acid acidifying culture fluid, 14000rpm is centrifuged 15min, take row high-performance liquid chromatogram determination after supernatant filters, chromatographic condition: chromatographic column is Inertsil ODS-SP type octadecylsilane chemically bonded silica post, column temperature 40 DEG C；Flowing is the mixed liquor of phosphate buffer and methanol mutually, and phosphate buffer is 97:3 with the mixed liquor ratio of methanol；Flow velocity is 1.0ml/min；Detector is SPD-20A type UV-detector, detects wavelength 280nm.

2. utilize the method utilizing recombinant slow virus induced nerve stem cells to break up to dopaminergic neuron described in claim 1 to obtain a kind of neural stem cell transfecting pLV5-TH+Brn4 recombinant slow virus, it is characterized in that: be that the NSCs of the stable transfection pLV5-TH+Brn4 obtained in the enforcement step 3 described in claim 1 virus is made single cell suspension, with basic culture solution, cell be made into 2 × 10⁶/ ml, adds in 1.5ml cell cryopreservation tube, is simultaneously introduced 0.4ml calf serum and 0.1ml dimethyl sulfoxide, seals, put 4 DEG C 1 hour after mixing ,-20 DEG C 2 hours, be then directly placed in-80 DEG C of ultra cold storage freezers preservation and obtain.

The method utilizing recombinant slow virus induced nerve stem cells to break up to dopaminergic neuron the most according to claim 1, it is characterized in that: the qualification of described recombinant slow virus transfection NSCs also includes: Western Blot detection, Immunofluorescence detection

nullDescribed Western Blot detection is that the NSCs ball after transfecting each group is seeded in 24 orifice plates break up，Separately set the NSCs of one group of untransfected as a control group，Differentiation culture terminates after 2 weeks cultivating，Requirement by RIPA lysate description，Extract the albumen of cell in each group，With 10%SDS-PAGE vertical electrophoresis, albumen is separated，Bio-Rad half-dried transferring system transferring film 15V，50min，5% defatted milk powder is separately added into following one and resists after closing: little mouse-anti β-actin antibody、Little mouse-anti TH antibody、Rabbit anti-Brn4 antibody、Rabbit anti-GDNF antibody、Rabbit anti-GFR α-1 antibody、Rabbit anti-ret antibody，Incubated at room 2h，4 DEG C overnight，The goat anti-mouse igg and the goat anti-rabbit igg two that are separately added into horseradish peroxidase combination next day resist，Incubated at room 4h，Then it is exposed、Develop and use gathered image，Western blot result is averaged photodensitometry by image analysis software，And carry out comparing between variance analysis and group to each group of data by the method for statistics；

Described Immunofluorescence detection is to terminate after each group of NSCs breaks up 2 weeks cultivating, and cell in negative control group and each recombinant virus transfection group is carried out Immunofluorescence test, is summarized as follows: used one anti-is respectively as follows: little mouse-anti TH, big mouse-anti DAT；Two resist and are: be combined with the mountain sheep anti-mouse igg of Alexa Fluor 568；One resists in 4 DEG C of overnight incubation, two anti-incubated at room temperature 2h, nuclear targeting 30min is carried out subsequently with Hoechst33342, all carry out routine with 0.01M PBS between middle each step to develop a film, finally with 20% glycerol buffer mounting, and under FV10i Intelligent laser Laser Scanning Confocal Microscope, observe the expression of TH or DAT, and calculate GFP/TH and GFP/DAT double-labeled cell and account for the percentage ratio of Hoechst labeled cell, and carry out comparing between variance analysis and group to each group of data by statistical method.

The method utilizing recombinant slow virus induced nerve stem cells to break up to dopaminergic neuron the most according to claim 1, it is characterised in that: described NSCs culture fluid is made up of DMEM/F12,2%B27,20ng/mlEGF, 20ng/ml bFGF.

The method utilizing recombinant slow virus induced nerve stem cells to break up to dopaminergic neuron the most according to claim 1, it is characterised in that: the infection multiplicity of described slow virus is 50.