CN102899285A - Method for differentiation of embryonic stem cells into nerve cells through in vitro induction - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to a method for differentiation of embryonic stem cells into nerve cells through in vitro induction, especially to a method for induction of Olig2<+>-GFP<+>-mES nerve cell differentiation through a purine derivative Purmorphamine, and uses thereof. According to the present invention, an embryoid body mediated nerve induction method is adopted, Olig2-GFP<+>-mES is adopted as a cell model, a purine derivative Purmorphamine and all-trans retinoic acid are combined to carry out directed induction on the Olig2-GFP<+>-mES cells to obtain spinal motor neurons and oligodendrocyte progenitor cells through differentiation; experiment results show that the Purmorphamine can be used as a substitute of SHH, can effectively induce Olig2<+>-GFP<+>-mES differentiation to obtain high purity and function spinal motor neurons and oligodendroglial cells, and can cause expression changes of related genes; and transplant experiment results show that the induced nerve cells can promote partial function and morphology restoration after rat spinal cord injury, and have effects of spinal cord injury regeneration promotion and function reconstruction.

Description

A kind of external evoked ES cell differentiation becomes the neurocyte method

Technical field

The invention belongs to biomedical sector, relate to the method that neurocyte is induced, being specifically related to a kind of external evoked ES cell differentiation becomes the neurocyte method, relates in particular to purine derivative (Purmorphamine) and induces Olig2 ⁺-GFP ⁺Method of the neurocyte differentiation of-mES and uses thereof.

Background technology

Spinal injury (Spinal cord injury, SCI) is one of clinical common traumatic illness.The generation of SCI comes from traffic accident injury, Falling Injury or motion wound etc. mostly, and is in recent years in rising trend.SCI brings very big burden for patient, family and society in case formation usually causes serious behavioral function obstacle.In recent years, regenerative medicine brings new hope to the treatment of Spinal injury.Embryonic stem cell (ESC) has very strong propagation and differentiation capability with it, can be induced in large quantities differentiation to become the advantages such as neurone, oligodendrocyte and astroglia cell, becomes the ideal source of regenerative medicine transplantation donor.But owing to have the teratomatous danger of formation in the embryonic stem cell directly transplanting body, therefore it is general intensive through precursor cell or neurocyte at Differentiation Induction in vitro first to use transplantation of differentiated embryonic stem cells for treatment Spinal injury, and then myeloid tissue is entered in transplanting, particularly, at present the process of clinical implementation transplantation of differentiated embryonic stem cells for treatment Spinal injury comprises following two portions: the one, and external evoked ES cell differentiation becomes the neurocyte that purity height, quantity are many, function is arranged; The 2nd, be transplanted to that cell in the spinal cord is transportable, propagation and form good integration with host tissue, thus the performance function.

Current, in the Differentiation Induction in vitro method of embryonic stem cell, also exist the problems such as the cell purity of inducing differentiation is low, quantity is few.The process of Neural Differentiation is subjected to the regulation and control of many A signal pathways, and its middle pitch Weihe acid (Sonic hedgehog, SHH) signal path plays an important role.Purine derivative Purmorphamine is micromolecular compound, has been proved to be combined by the Smoothened in the SHH signal path, activates the SHH signal path, is considered to the surrogate of SHH, plays an important role in growth.

Known at present, the Olig gene is alkaline whorl one spiral (basic helix-loop-helix, bHLH) a member of transcription factor family comprises two kinds of Olig1 and Olig2, is considered at first oligodendrocyte or the specific expressed gene of its precursor cell and gains the name; Olig1 participates in the growth of oligodendrocyte specifically, and Olig2 just expresses when oligodendrocyte not yet occurs in the time of 8.5 days as far back as the embryo.There are some researches show that Olig2 is positioned at 2/3 zone of veutro in the spinal cord fetal development, the early stage generation that can promote motor neuron, suppress V2 district relay cell under the inducing of SHH (a kind of factor that is produced by notochord in the embryo development procedure); Stage promotes oligodendrocyte, suppresses the generation of astroglia cell, discloses the synergy of Olig2 and other transcription factor and jointly regulates occurring in sequence of neurone and oligodendrocyte.

At present, there is not yet the relevant Purmorphamine of application and substitute SHH, the inducing embryo stem cell differentiation, dynamoneure and oligodendrocyte that acquisition purity is high, quantity is many, have function, and Purmorphamine substitutes SHH, can cause the variation of genes involved in SHH signal path, the back of the body-abdomen axle path, motor neuron and the oligodendrocyte growth, induce simultaneously the oligodendrocyte precursor cells of differentiation to be transplanted in the spinal cord injury tissue, can integrate with the host, and then move, break up the report of bringing into play the effect of repairing damage.

Summary of the invention

The purpose of this invention is to provide a kind of method of inducing neurocyte, being specifically related to a kind of external evoked ES cell differentiation becomes the neurocyte method, relates in particular to purine derivative (Purmorphamine) and induces Olig2 ⁺-GFP ⁺The method of the neurocyte differentiation of-mES.

Further purpose of the present invention provides the purposes of described method in the preparation of preparation reparation Spinal injury.

The present invention adopts the nerve-inducing method of embryoid body mediation, with Olig2-GFP ⁺-mES is as cell model, utilize this clone in the nerve-inducing atomization, to express oligodendrocyte transcription factor 2 (Oligodendrocyte transcription factor 2, Olig2), also can conveniently observe green fluorescent protein (Green Fluorescent Protein, GFP) expression, thereby it is the method for dynamoneure and oligodendrocyte precursor cells in conjunction with the all-trans-retinoic acid Induction of committed differentiation of lower concentration that Purmorphamine is provided.

Purpose of the present invention is achieved through the following technical solutions:

One, sets up Olig2 ⁺-GFP ⁺-mES cell model

Change GFP albumen at the Olig2 of mouse embryo stem cell gene and set up Olig2 ⁺-GFP ⁺-mES cell model.Adopt the serum-free culture mode, Purmorphamine with different concns induces embryoid body (EBs), and observing it by methods such as cell cultures, immunocytochemistry, FACs and RT-PCR is the effect of dynamoneure and oligodendrocyte to Oriented Differentiation of Embryonic Stem Cell.Experimental result shows that Purmorphamine can substitute SHH fully and promote the embryonic stem cell differentiating into nerve cells; Promote the expression of Olig2 gene; Thereby promote to break up to dynamoneure behind EB8D, differentiation rate surpasses 50%, causes the expression variation with the dynamoneure genes involved; To the oligodendrocyte differentiation, differentiation rate surpasses 80% behind EB12D in promotion, causes the expression variation with oligodendrocyte genes involved, the back of the body-abdomen axle path and SHH signal path genes involved.

Two, set up effect after the Transplanted cells that rat spinal cord collision injury model validation obtains

Use NYU-Impactor II and set up rat spinal cord collision injury model, Purmorphamine is induced differentiation Olig2 ⁺-GFP ⁺The Olig2 that-mES obtains ⁺-GFP ⁺-OPC is transplanted in the spinal cord injury tissue, observe the recovery whether transplanted cells promotes spinal function by methods such as immunohistochemistry, transmission electron microscope, Western blot, study of behaviour and Electrophysiologies, confirm the Transplanted cells role in Spinal injury is recovered that obtains.

Experimental result of the present invention shows that Purmorphamine can induce Olig2 effectively as the surrogate of SHH ⁺-GFP ⁺-mES is divided into high purity, dynamoneure and the oligodendrocyte of function is arranged, and causes the change of genes involved; Transplant the Olig2 that induces ⁺-GFP ⁺-OPC can promote the part of function and morphology after the Spinal Cord Injury in Rats to recover, thereby promotes the effect of regeneration and reconstruction after the Spinal injury, for the clinical application Treatment of spinal cord injury with cellular transplantation provides certain experimental basis.

Among the present invention, described Olig2 ⁺-GFP ⁺-mES is the method for utilizing genetic engineering, by the tsiklomitsin inducible expression, makes at the Olig2 locus insertion GFP of mouse embryo stem cell (Mouse embryonic stem cell, mESC); It can judge appearance and the screening of the neurocyte of inducing intuitively in the nerve-inducing atomization, when also being convenient to Transplanted cells, determine the migration of transplanted cells in host, Differentiation and proliferation etc. by the expression of observing GFP.

Among the present invention, described Olig2 ⁺-GFP ⁺-OPC induces differentiation Olig2 by Purmorphamine ⁺-GFP ⁺-mES makes.

External evoked ES cell differentiation of the present invention becomes the neurocyte method, is the nerve-inducing method that adopts the embryoid body mediation, and namely comprehensive RA revulsion and five-step approach make Olig2 ⁺-GFP ⁺The method of the myelopetal motor neuron of-mES and oligodendrocyte differentiation, it may further comprise the steps:

1. with Olig2 ⁺-GFP ⁺-mES puts into the embryonic stem cell substratum suspension culture 2d that contains leukaemia inhibitory factor (LIF, 20u/ml);

2. put into and contain all-trans-retinoic acid RA (0.5 μ M) and the Neural Differentiation substratum of different concns Purmorphaine is cultivated 4d;

3. put into the Neural Differentiation substratum that contains bFGF (10ng/ml) and cultivate respectively 2d and 6d, adopt cell sorting (FACs) the screening Olig2 of fluorescence-activation ⁺-GFP ⁺-positive cell;

4. continue to cultivate at neurone substratum and oligodendrocyte substratum respectively, induce to generate dynamoneure and oligodendrocyte.

The step of the inventive method 4. in, the described cultivation in the neurone substratum is adherent culture 7～14d; Described is adherent culture 14d in the oligodendrocyte culture medium culturing.

In the method for the present invention, Purmorphamine is by affecting the related gene expression in SHH signal path and the back of the body-abdomen axle path, and inducing the Olig2+-GFP+-mES directed differentiation by the genes involved that raises motor neuron and oligodendrocyte respectively is dynamoneure and oligodendrocyte;

Can integrate with the host after inducing the Olig2+-GFP+-OPC of differentiation to be transplanted to rat spinal cord collision injury model, in host, continue propagation and differentiation, differentiation becomes the oligodendrocyte of pulpefaction sheath, promote the recovery of neurofilament protein and myelin protein, promote the recovery of Remyelination and neural function, significantly improve the SCI rat more after.

The method of neurocyte differentiation of the present invention has the following advantages:

1. purine derivative Purmorphamine substitutes SHH, and inducing the Olig2+-GFP+-mES directed differentiation is dynamoneure, induces differentiation rate to surpass 50%;

2. purine derivative Purmorphamine substitutes SHH, and inducing the Olig2+-GFP+-mES directed differentiation is oligodendrocyte, induces differentiation rate to surpass 80%;

3. purine derivative Purmorphamine causes the expression of motor neuron and oligodendrocyte genes involved in Olig2+-GFP+-mES nerve-inducing atomization;

4. purine derivative Purmorphamine causes the expression of genes involved in SHH signal path and the back of the body-abdomen axle path in Olig2+-GFP+-mES nerve-inducing atomization;

5. use the NYU spinal cord instrument of causing injury, successfully make up rat spinal cord collision injury model;

6. after inducing the Olig2+-GFP+-OPC that obtains to be transplanted to spinal cord injury model, success is bred in host and is broken up, and is divided into oligodendrocyte and neurone, promotes the recovery of Remyelination and neural function.

Description of drawings

Fig. 1 is Olig2 among the present invention ⁺-GFP ⁺The aspect graph of-mES and MEF,

Wherein, A～C:Olig2+-GFP+-mES grows at MEF, clone's edge clear, and close structure, the protuberance growth, the iuntercellular boundary line is unclear; A:bar=100 μ m; B:bar=50 μ m; C:bar=10 μ m; ★ shows MEF, ▲ show Olig2+-GFP+-mES.

Fig. 2 is the identified by immunofluorescence figure of Olig2+-GFP+-mES among the present invention,

Wherein, the common mark of the Oct-3/4 of A:mESC and Sox-1; The common mark of the Oct-3/4 of B:mESC and Sox-2, bar=50 μ m; Immunofluorescence dyeing shows, the Oct-3/4 of Olig2+-GFP+-mES cell clone, Sox-1 and Sox-2 dyeing strong positive, and the MEF around the Olig2+-GFP+-mES is fully not painted.

Fig. 3 is the positive cell analysis of accounts figure of Olig2+-GFP+-mES immunofluorescence dyeing among the present invention,

Wherein, positive percentage=positive cell number/DAPI redyes total cellular score.

Fig. 4 shows the Olig2+-GFP+-positive cell in the expression of EB different steps,

Wherein, bar=50 μ m, the EB of different steps all is comprised of up to a hundred cells, and its form is spheroidal substantially, and is rough; EB2D expresses without Olig2, and EB6D begins to express Olig2, and along with induction time prolongs, the expression of Olig2 increases gradually, and is the highest to EB12D Olig2 expression amount.

Fig. 5 shows that Purmorphamine induces the expression of Olig2 and Nestin,

Wherein, the neural ball Olig2 of figure A:EB6D and Nestin coexpression, bar=100 μ m; Figure B: be the high power figure in the A figure square frame, arrow represents the neurocele sample shape of " rosettes ", bar=25 μ m.

Fig. 6 shows Immuncytochemical detection Olig2 and Hb9 in the expression of EB8D,

Wherein, figure A: Immuncytochemical detection Olig2 and the Hb9 expression in the different treatment group, bar=100 μ m; Figure B: statistic histogram shows EB8D, Olig2 and the Hb9 positive cell expression rate in the different treatment group; The Purmorphamine group is compared * * P＜0.01 with SHH group and Control group.

Fig. 7 shows Immuncytochemical detection Olig2 in the expression of neurone different steps,

Wherein, scheme A:Olig2 and Tuj1 at the immunofluorescence dyeing of neurone different steps, a: the expression neuronal precursor; B: expression mature neuron, bar=100 μ m; Figure B: statistic histogram shows that Olig2 is at the positive cell expression rate of neurone different steps; The neuronal precursor group is compared with the mature neuron group, * * P＜0.01.

Fig. 8 shows the expression of Immuncytochemical detection dynamoneure associated protein,

Wherein, figure A-a: observe bar=50 μ m under the inverted phase contrast microscope; B: neuronic Hb9 positive expression, bar=50 μ m; C: neuronic MNR2 positive expression, bar=100 μ m; D: neuronic Isll positive expression, bar=10 μ m; E: neuronic Olig2 is negative to express bar=10 μ m; F: neuronic Hoxb4 positive expression, bar=10 μ m; Figure B: statistic histogram shows, the positive expression rate of dynamoneure associated protein.

Fig. 9 shows that RT-PCR detects the expression of dynamoneure genes involved,

Wherein, 2% agarose gel electrophoresis of figure A:PCR product; Figure B:RT-PCR sxemiquantitative statistical graph.

Figure 10 is the form of Olig2+-GFP+-OPC under the phase microscope,

Wherein, a and b: observe the OPC of visible bipolar or three utmost points under the phase microscope; C and d are respectively under the fluorescence phase microscope of a and b and observe; A, c bar=100 μ m; B, d bar=50 μ m.

Figure 11 is the form of Olig2+-GFP+-oligodendroglia pedigree under the phase microscope,

Wherein, a: visible a large amount of OPC move out bar=50 μ m from neural ball; B: visible bipolar OPC, bar=50 μ m; C: arrow represents immature oligodendrocyte, bar=25 μ m; D: arrow represents ripe oligodendrocyte, bar=10 μ m.

Figure 12 is the form of Olig2+-GFP+-oligodendroglia pedigree under the fluorescence phase microscope,

Wherein, a: visible a large amount of OPC move out bar=50 μ m from ball; B: visible immature oligodendrocyte, arrow represents the prematurity oligodendrocyte, bar=50 μ m; C: be shown as ripe oligodendrocyte, bar=25 μ m; D is the high power figure in the c square frame, and arrow represents ripe oligodendrocyte, bar=10 μ m.

Figure 13 is the immunofluorescence dyeing of the ripe oligodendrocyte of Olig2+-GFP+-,

Wherein, a: the Olig2 of oligodendrocyte and CNPase coexpression, bar=25 μ m; B: the Olig2 of oligodendrocyte and GC coexpression, bar=25 μ m.

Figure 14 shows that astroglia cell do not express Olig2 (bar=25 μ m).

Figure 15 is that genes involved is expressed in Olig2+-GFP+-mES nerve-inducing atomization in the SHH signal path,

Wherein, 2% agarose gel electrophoresis of figure A:PCR product; Figure B: the statistics broken line graph shows that the expression of genes involved in the nerve-inducing atomization changes in the SHH signal path.

Figure 16 is the expression of genes involved in Olig2+-GFP+-mES nerve-inducing atomization in the back of the body-abdomen axle,

Wherein, 2% agarose gel electrophoresis of figure A:PCR product; Figure B: the statistics broken line graph shows that the expression of the genes involved in the back of the body-abdomen axle in the nerve-inducing atomization changes.

Figure 17 shows the scoring of SCI study of behaviour,

Wherein, A:BBB scoring; The B:Tarlov scoring; N=6 compares * P＜0.05 with sham operated rats.

Figure 18 shows funiculus posterior medullae spinalis myelin relative optical density value (n=12, * * P＜0.01).

Figure 19 shows that transplanted cells breaks up in the myeloid tissue behind SCI,

Wherein, A:Olig2 and GFAP coexpression; B:Olig2 and NG2 coexpression, arrow represent that transplanted cells is oligodendrocyte precursor cells; C:Olig2 and MAP2 coexpression, arrow represent that transplanted cells and neurone mark altogether; D:Olig2 and MBP coexpression, arrow represent transplanted cells expression MBP, bar=10 μ m.

Figure 20 is the ultrastructure of funiculus posterior medullae spinalis myelin before and after transplanting,

Wherein, A and B: sham operated rats, B is the high power figure of A,

Show compact; C and D: before the Transplanted cells (being 7d behind the SCI), D is the high power figure in the square frame among the C, and ☆ shows the sex change of cavity sample, ▲ showing that microglia, ★ show the myelin of sex change, arrow represents naked axon; E and F: the Transplanted cells group, * shows oligodendrocyte, arrow represents that the myelin of regenerating is loosely organized.

Figure 21 is Tarlov and the BBB scoring figure of SCI rat after the Transplanted cells,

Wherein, A:Tarlov scoring; The B:BBB scoring, n=6, ↓ show transplant time, * P＜0.05vs sham operated rats, ▲ P＜0.05vs operative control group.

Figure 22 is SCI rat MEP detection figure before and after the Transplanted cells,

Wherein, A: sham operated rats; B: before the Transplanted cells (being 7d behind the SCI); C: 1w after the Transplanted cells (being 14d behind the SCI); D: 2w after the Transplanted cells (being 21d behind the SCI); E: 4w after the Transplanted cells (35d behind the SCI); The latent period of F:MEP (ms); The wave amplitude of G:MEP (mv); N=6.

Embodiment

Embodiment 1Olig2 ⁺-GFP ⁺The culture ﹠ identification of-mES

1, test used cell: mouse (CF-1) embryo fibroblast (MEF), former culture;

, 2, Olig2 ⁺-GFP ⁺-mES clone is provided by the Changjiang river scholar professor invited specially professor Zhang Suchun of Fudan University.

, 3, main agents:

Gelatin Gelatin, DAPI, DMSO: available from Sigma (U.S.);

DME/F12 glutamine, beta-mercaptoethanol, non-essential amino acid, Sodium.alpha.-ketopropionate, pancreas enzyme-EDTA, foetal calf serum (FBS): available from Gibco BRL company (U.S.);

6 holes and 24 porocyte culture plates, centrifuge tube, pasteur pipet: available from Greiner company (Germany);

Fluorescence mountant: available from Vector company (U.S.);

The anti-Oct-3/4 antibody of mouse (Santa Cruz, SC-5279), the anti-Sox2 antibody of goat (R﹠amp; D, AF2018), the anti-Sox1 antibody of goat (R﹠amp; D, AF3369);

The anti-goat fluorescence of donkey Alexa 594 mark IgG two are anti-, the anti-mouse fluorescence of donkey Alexa

488 mark IgG two are anti-: available from Invitrogen company (U.S.);

Other reagent is import or domestic analytical reagent.

4, key instrument equipment:

Phase microscope (Nikon-TS 100), fluorescent microscope (Nikon-Eclipse TE 2000-S), humidity CO ₂Cell culture incubator (Heraus), Bechtop (An Sen company of Su Jing group), Milli-Q ultrapure water production system (Millipore, D-5623), whizzer (Eppendorf), liquid nitrogen container (Mve), freezing storing box (Nalgene).

5, the main agents preparation

Table 1 mouse embryo fibroblast substratum

Table 2MEF frozen storing liquid

Table 3 embryonic stem cell substratum

Table 4 embryonic stem cell frozen storing liquid

6, experimental technique

1) cultivation of mouse embryo stem cell

CF-1 is that mouse is purchased from cell institute of the Chinese Academy of Sciences, male and female adult mice was mated in about 5:00PM by 1: 2, check cloudy bolt before the second day 7:30AM, the inspection bolt is designated as pregnant 0.5d noon on the same day, gets pregnant 13.5d mice embryonic and separates mouse embryonic fibroblast (MEF).

2) preparation MEF feeder layer

MEF separates:

(1) the pregnant mouse of conventional processing 13.5d obtains the embryo, removes the embryo and organizes outward, washes the embryo three times with PBS;

(2) conventional processing embryonic tissue;

(3) tissue with (2) moves in the 15ml centrifuge tube, add about 5ml 0.25% pancreatin solution digestion 30min after, stop with the MEF substratum; 1000rpm, centrifugal 5min collecting cell;

(4) inoculating cell: according to the ratio inoculation of 3 bottles of T25 Tissue Culture Flasks of every embryo's inoculation;

(5) change fresh MEF substratum behind the inoculation 24h.When cell converged to 90%, part was frozen in liquid nitrogen, gives over to seed cell; Part continues amplification cultivation (in 3 generations), and is frozen in liquid nitrogen after the irradiation, directly uses as feeder layer cells after the recovery.

MEF is frozen: MEF grows to 90% cell that merges, and sucks nutrient solution, and PBS cleans, and adds the digestion of 0.25% pancreas enzyme-EDTA, ends digestion with the MEF substratum that contains serum, and cell suspension is transferred in the 10ml centrifuge tube, 1000rpm, centrifugal 5min; Cell precipitation is resuspended with the MEF frozen storing liquid, moves into cryopreservation tube, and-80 ℃ are spent the night, and moves into the medium-term and long-term preservation of liquid nitrogen container next day, and frozen cell density is 1 * 10 ⁶Cells/cm ²

The MEF recovery: take out cell cryopreservation tube from liquid nitrogen, immerse 37 ℃ of water-baths, ice crystal is melted, the cell suspension of thawing is transferred in the centrifuge tube that nutrient solution is housed, the piping and druming mixing, normal temperature, 1000rpm, centrifugal 5min, cell precipitation is resuspended with the MEF substratum, and cell density is according to 0.7 * 10 ⁵Cells/cm ²Be inoculated in 6 well culture plates; Second day is changed nutrient solution, removes dead cell, and 2-～3d can go down to posterity, or the preparation feeder layer.

MEF irradiation: MEF uses after going down to posterity and cultivating 2～4 times ⁶⁰Co γ processes irradiation, and total dose reaches 8000rads, and its mitogen activation of deactivation renews bright nutrient solution after the irradiation, put at least 4h of 37 ℃ of balances, digestion, with 1 * 10 ⁶Cells/cm ²Branch is frozen in liquid nitrogen; MEF needs in advance recovery during use, is seeded in the 6 coated porocyte culture plates of 0.1% gelatin to get final product, and cell density is according to 0.7 * 10 ⁵Cells/cm ²Inoculation.

3) Olig2 ⁺-GFP ⁺The cultivation of-mES:

With Olig2 ⁺-GFP ⁺-mES cell is seeded on the MEF feeder layer, adds the mESC substratum, and 37 ℃, 5%CO ₂Cultivate under the condition, the next day change nutrient solution, 3～4d goes down to posterity.

(1) Olig2 ⁺-GFP ⁺The digestion of-mES, go down to posterity

1d prepares the MEF feeder layer in advance, sucks nutrient solution in 2d, after PBS cleans 3 times, add the mESC cell culture fluid, balance 1h at least sucks the mESCs cell culture fluid in 37 ℃ of incubators, and PBS cleans 3 times, add 0.05% pancreas enzyme-EDTA 1ml, after placing 5min in 37 ℃ of water-baths, add substratum and stop digestion, 1000rpm, centrifugal 5min is with 1 * 10 ⁴Cells/cm ²Density be passaged on six well culture plates that contain the mESC cell culture fluid, remaining cell is used for the nerve-inducing differentiation of EB.

(2) Olig2 ⁺-GFP ⁺The freezing and thawing of-mES

Trypsin process digestion Olig2 ⁺-GFP ⁺-mES, 1000rpm removes supernatant behind the centrifugal 5min, and add the mESC frozen storing liquid and make cell suspension, the cryopreservation tube of packing into ,-80 ℃ are spent the night in the freezing storing box, transfer to the medium-term and long-term preservation of liquid nitrogen next day;

Cryopreservation tube by taking out in the liquid nitrogen, is immersed 37 ℃ of water-baths, when only remaining a fritter ice crystal, cryopreservation tube is taken out, the sucking-off cell suspension joins in the 15ml centrifuge tube that has in advance 9ml mESC cell culture medium, and featheriness is beaten and made the cell mixing; Normal temperature, 1000rpm, centrifugal 5min removes supernatant liquor, repeats to wash once with substratum again; Add 2ml cell culture medium re-suspended cell, dropwise be seeded in 6 orifice plates that are covered with in advance after MEF and the balance; Behind 24h, change liquid to cell.

4) immunocytochemical stain

The Olig2 of vitro culture 5～7d ⁺-GFP ⁺-mES cell is with the expression of Immunofluorescence method detection cell-surface antigens Oct-3/4, Sox-1, Sox-2, to determine its totipotency and not divide voltinism.

(1) sucking-off substratum is washed 3 times with PBS;

(2) sucking-off PBS adds fixedly 20min of 4% Paraformaldehyde 96 room temperature;

(3) sucking-off stationary liquid is washed 3 times with PBS, each 10min;

(4) add 0.3%Triton X-100, room temperature leaves standstill 15min; PBS washes 3 times, each 10min;

(5) add the sealing of donkey serum, room temperature leaves standstill 15min;

(6) sucking-off serum adds corresponding primary antibodie (1: 2000 anti-Oct-3/4+1: 2000 anti-Sox-1 and anti-Sox-2), 4 ℃ of refrigerator overnight; PBS washes 3 times, each 10min;

(7) add the anti-goat fluorescence of donkey Alexa

594 mark IgG, the two anti-and anti-mouse fluorescence of donkey Alexa

488 mark IgG two are anti-, and room temperature leaves standstill 60min;

(8) add DAPI (dilution in 1: 1000) transfect cell nuclear 10min; PBS washes 3 times, each 10min;

(9) mountant mounting, fluorescence microscopy Microscopic observation, photograph.

5) positive cell counting

Use ImageJ software the fluorescence photo that takes is carried out cell counting.After counting respectively the positive cell number of DAPI, Oct-3/4, Sox-1, Sox-2, carry out data statistics.

Experimental data mean ± standard deviation Expression is carried out analyzing and processing with the SPSS statistical software, relatively checks with t between group.

The result shows that as shown in Figure 1, former generation MEF cell is impure, except fibroblast-like cells is arranged, also has neural like cell, myocardial cell and the indefinite cell of some types; Reduce rapidly through heteroproteose cell behind the passage, passed for 2～3 generations after cellular constituent be tending towards single, be inoblast.Warp ⁶⁰Behind the Co gamma-radiation, cell loses multiplication capacity, and adherent culture can be kept 10～14d, and postradiation cell ordered arrangement is spindle shape or ribbon, is in contact with one another between the cell to be monolayer alignment.Tenuigenin central authorities are oval nuclear, periphery protruding fibrous pseudopodium (seeing ★ among Fig. 1).Suitable cell density as the mESC feeder layer is 0.75 * 10 ⁵Cells/cm ²

Olig2 ⁺-GFP ⁺-mES cell is that feeder layer is the growth of reunion shape at l cell, and cell clone is typical colony shape growth, and is rounded or oval, clone's edge clear, smooth, cell volume is little and arrange closely, and is rounded, nuclear is large, kernel is clear, and nucleus/tenuigenin is than high.Clone periphery and MEF feeder layer boundary clear are without differentiation sign (see among Fig. 1 ▲).Colony need go down to posterity near converging state behind general inoculation 2～3d.The ratio of going down to posterity is generally 1: 6～and 1: 8.

By the Olig2 that long-term cultivation is gone down to posterity ⁺-GFP ⁺Totipotency, minute voltinism (as shown in Figure 2) of this cell identified in the expression that-mES regularly detects its specific antigens, and by its Specific marker of cell counting Oct-3/4, Sox-1, the positive rate of Sox-2 (as shown in Figure 3).Statistics shows: Olig2 ⁺-GFP ⁺The Oct-3/4 of-mES, Sox-1, the positive cell rate of Sox-2 is respectively 84.24 ± 0.04%, and 83.28 ± 0.04%, 85.18 ± 0.05%, all above 80%, show the Olig2 under this culture system ⁺-GFP ⁺-mES can keep good totipotency and not divide voltinism.

The result of present embodiment shows that adopting has the vitro culture mode of feeder layer to cultivate Olig2 ⁺-GFP ⁺-mESC, by the detection to embryonic stem cell genes involved Oct-3/4, Sox-1, Sox-2, the cell positive rate of Oct-3/4, Sox-1, Sox-2 of finding shows the Olig2 under this culture system all above more than 80% ⁺-GFP ⁺-mESC is keeping undifferentiated state, for subsequent experimental provides believable seed cell.

Embodiment 2Purmorphamine induces Olig2 ⁺-GFP ⁺-mESC directed differentiation is dynamoneure

1) cell

Identical with embodiment 1.

2) main agents and articles for use

RA: available from Sigma company (U.S.);

N2, B27, Neurobasal substratum: available from Gibco BRL company (U.S.);

Purmorphamine: available from Calbiochem company (U.S.);

BFGF, NT3, GDNF, BDNF, PDGF-AA: available from R﹠amp; D company (U.S.);

RNA enzyme inhibitors, Oligo (dT), dNTP, archaeal dna polymerase: available from Takara company (DaLian, China);

Primer: it is synthetic that worker company is given birth in Shanghai;

M-MLV reverse transcription test kit, Total RNA extraction agent Trizol: available from Invitrogen company (U.S.);

The anti-Tuj1 antibody of rabbit (Convance, PRB-435P), the anti-Tuj1 antibody of mouse (Sigma, T8660), anti-Hb9 antibody (the Santa cruz of goat, SC-22542), the anti-MNR2 antibody of mouse (DHSB, 81.5C10), the anti-Isl1 antibody of rabbit (Chemicon, AB5754), the anti-Hoxb4 antibody of mouse (R﹠amp; D, AF3369), the anti-Nkx2.2 antibody of mouse (DHSB, 74.5A5), the anti-Olig2 antibody of goat (Santa cruz, SC-19969) the anti-Nestin antibody of mouse (Chemicon, MAB353);

The anti-rabbit fluorescence of donkey Alexa

594 mark IgG two are anti-, the anti-mouse fluorescence of donkey Alexa

594 mark IgG two are anti-: available from Invitrogen company (U.S.);

Without RNA enzyme rifle head, centrifuge tube: available from Axyge company (U.S.);

All the other articles for use are identical with embodiment 1.

3) main laboratory apparatus:

EPICS ALTRA flow cytometer (Beckman); PCR instrument (Bio-Rad); ultraviolet gel image analyser (Syngene); GS-800 scanning densitometer (BIO-RAD); constant temperature freezing microtome CM1900 (LEICA); the desk-top refrigerated centrifuge of high speed (Eppendorf), electrothermostat DHP-9052 (medicine equipment company limited is entered by Shanghai China), Nanodrop2000 ultramicrospectrophotometer (Thermo).

4) main agents preparation

The neural substratum (ModifiedNS Medium, MNS) of table 5 improvement

The neurone substratum (Modified Differentiation Medium of Neuron, Mdiff) of table 6 improvement

5) experimental technique

The removal of MEF:

Utilize Olig2 ⁺-GFP ⁺The difference of-mES and MEF adherent speed on gelatin to remove adherent faster MEF cell, obtains highly purified Olig2 ⁺-GFP ⁺-mES.Step is: the Olig2 that trysinization is cultivated at MEF ⁺-GFP ⁺-mES, with the mES nutrient solution stop digestion and blow and beat into unicellular after, spread to the pretreated 100mm culture dish of gelatin, after leaving standstill 30min, 37 ℃ of cell culture incubators draw supernatant liquor, be transferred to the pretreated 100mm culture dish of another gelatin, leave standstill 30min in 37 ℃ of cell culture incubators again, draw supernatant liquor, use the resuspended and counting of nutrient solution behind the centrifugal collecting cell.

Inducing of neural precursor:

Embryoid (EB) forms: get 1 * 10 ⁵Cells/cm ²Olig2 behind the removal MEF ⁺-GFP ⁺-mES is suspension culture in 100mm low-adhesion culture dish, and used medium is for containing the mES substratum of LIF (1: 5000).Can form two days later the less EB ball of a large amount of individualities, change the Neural Differentiation substratum, in Neural Differentiation training liquid, add the Purmorphamine (0,0.25,0.5,0.75 and 1 μ M) of all-trans-retinoic acid (RA, 0.5 μ M) and different concns, induce 4d.Observe the expression of GFP every day, observe the EB ball (EB12D) to the 12 day.

Olig2 ⁺-GFP ⁺-mES is induced to differentiate into dynamoneure:

The EB6D recession is added bFGF (10ng/ml), behind the inducing culture 2d except RA and Purmorphamine, with 0.05% pancreas enzyme-EDTA digestion 5min, stop digestion with pancreatin inhibitor, repeatedly blow and beat into the minicell ball, centrifugal 1000rpm, 5min removes the garbage collection cell.The cell seeding of collecting is coated with glutinous 24 well culture plate that connects albumen (laminin) of layer, carried out monolayer culture with the neurone substratum.Be that the observable neurone is moved out from ball next day, fixing behind cultivation 7～14d, carries out the dyeing of cell fluorescence immunochemistry and cell counting.

Do following detection at different time points: 1. at first detect the expression that has or not Nestin at EB6D; 2. detect the expression of motor neuron Specific marker Hb9 at EB8D; 3. after adherent 7 and 14d detect respectively the coexpression situation of Olig2 and the Tuj1 of neuronal precursor and mature neuron; 4. adherent rear 14d detection is Hb9 with the gene that participates in toward motor neurons, MNR2, and Isl1, Olig2, the coexpression situation of Hoxb4 and neurone mark Tuj 1 is (because that the present embodiment employing is Olig2 ⁺-GFP ⁺-mES is so the expression of Olig2 need not to use the Olig2 antibody test).

Flow cytometry (cell sorting of fluorescence-activation):

Because Olig2 ⁺-GFP ⁺-mES only just begins to express Olig2 when being induced to differentiate into neural precursor, for clear and definite this cell strain in when begin to express (i.e. visible green fluorescence under fluorescent microscope) and expression amount how much, adopt fluorescence microscope in the present embodiment, and carry out viable cell with flow cytometer and detect the Purmorphamine of different concns to the impact of Olig2 expression.

Respectively at EB0D, EB2D, EB4D, EB6D, EB8D, EB10D, seven time point collecting cells of EB12D, carry out flow cytometer and detect.Step is as follows: after the EB ball is cleaned one time with 0.01M PBS, add 0.05% pancreas enzyme-EDTA digestion 10min, stop digestion with pancreatin inhibitor, screen filtration with 30 μ m, be not digested to single celled cell ball to remove, the centrifugal 5min of 1000rpm abandons supernatant liquor; Add Neural Differentiation substratum (substituting DMEM/F12 and Neurobasal with PBS) and as FACs buffer, dispel cell; Cell transfer to fluidic cell is detected in the dedicated pipe, detect at EPICS ALTRA flow cytometer.

The extraction of cell total rna:

Respectively at EB0D, EB2D, EB4D, EB6D, EB8D, EB10D, seven time point collecting cells of EB12D, extract cell total rna with Trizol reagent, observe Purmorphamine to participating in the relevant gene of motor neuron in the variation of nerve-inducing process: step is as follows:

(1) with cell (5～10 * 10 ⁶) clean 2 times with PBS, adding 1ml Trizol, piping and druming makes its mixing; Cell after the cracking and Trizol liquid are moved in the 1.5ml EP pipe together, and room temperature (15～30 ℃) leaves standstill hatches 10min;

(2) add the 0.2ml chloroform, thermal agitation 15s, room temperature (15～30 ℃) leaves standstill 15min;

(3) precooling whizzer to 4 ℃, 12000rpm, centrifugal 15min;

(4) draw the colourless water in upper strata, add 500 μ l Virahols, mixing, room temperature leaves standstill 10min;

(5) 4 ℃, 12000rpm, centrifugal 10min abandons supernatant, and sheet RNA is sunken to the pipe end;

(6) 1ml 75% ethanol (preparation of DEPC water) washing is 2 times, the vortex vibration, and the precipitation that suspends, 8000rpm, centrifugal 5min abandons supernatant, and RNA is sunken to the pipe end, natural drying at room temperature 5min, natural drying at room temperature 5min;

(7) DEPC with 30～50 μ l processes water (or 0.01mM TE; 0.5%SDS) dissolving RNA, or 60 ℃ of water-bath 10min are short molten;

(8) with the integrity of 1% agarose gel electrophoresis checking R NA, and survey the OD value with the Nanodrop2000 ultramicrospectrophotometer, RNAA260/A280 ratio 1.6～1.8 is that quality is good, RNA concentration (μ g/ml)=OD260 * K (40) * extension rate (50).-80 ℃ save backup, or directly carry out next step experiment.

Reverse transcription reaction:

(1) gets the total RNA of 5 μ g, 1 μ l Olig (dT) ₂₀, 1 μ l 10mM dNTP mixture and proper volume DEPC water be mixed to 13 μ l, 65 ℃, be put on ice at least 1min behind the 5min;

(2) add 4 μ l, 5 * First-Strand Buffer, 1 μ l 0.1MDTT, 1 μ l RNA enzyme inhibitors (RNase OUT) and 1 μ l M-MLV reversed transcriptive enzyme (SuperScript ^TMIII RT) mix, 25 ℃, 5min;

(3) change 50 ℃ over to, 30～60min;

(4) 70 ℃, 15min, termination reaction, it is for subsequent use that cDNA puts-20 ℃ of storages.

Table 7 is reaction system; Table 8 is the RT-PCR reaction system; Table 9 is primer sequence and reaction conditions.

Table 7

Table 8

The PCR response procedures is:

Table 9

Amplified fragments is identified with 2% agarose gel electrophoresis, 120V, 20-30min.

The cellular immunofluorescence chemistry:

In order to detect Purmorphamine to the impact of neurone different steps, the collecting cell ball detects respectively the expression of Nestin and Hb9 when EB6D and EB8D; Neuronal precursor behind the adherent 7d detects the coexpression of Olig2 and Tuj1; Adherent 14d after ripening motor neuron detects respectively the coexpression situation of Hb9, MNR2, Isl1, Olig2, Hoxb4 and Tuj1.

The result shows, uses the Neural Differentiation that Purmorphamine induces, and can simulate the internal milieu in the embryo development procedure, induces Olig2 ⁺-GFP ⁺-mES directed differentiation is dynamoneure, and the more ways of regeneration of high score rate is provided.

It is oligodendrocyte that embodiment 3Purmorphamine induces the Olig2+-GFP+-mES directed differentiation

Experimental cell is identical with embodiment 1.Main laboratory apparatus is identical with embodiment 2.

Main agents and articles for use:

Insulin, Transferrin, BSA, Progeserone, Na Solenite, Putrescine, T3, Biotin, NAC: available from Sigma company (U.S.);

The anti-NG2 antibody of rabbit (Chemicon, MAB5320), the anti-O4 antibody of mouse (Chemicon, MAB345), the anti-MBP antibody of mouse (Chemicon, MAB389), the anti-PDGFR-a antibody of rabbit (Santacruz, SC-338), the anti-Nkx2.2 antibody of mouse (DHSB, 74.5A5), the anti-GFAP antibody of rabbit (Invitrogen, 709135A);

The anti-rabbit fluorescence of donkey Alexa

594 mark IgG two are anti-, the anti-mouse fluorescence of donkey Alexa

594 mark IgG two are anti-: available from Invitrogen company (U.S.);

The main agents preparation:

Table 10100 * SATO substratum

SATO substratum after table 11 improvement

Table 12 astroglia cell substratum (ASM)

Experimental technique:

1) Olig2 ⁺-GFP ⁺-mES breaks up to oligodendrocyte precursor cells

It is oligodendrocyte precursor cells that present embodiment adopts 4 step adjective law to induce the Olig2+-GFP+-mES directed differentiation: postdigestive ES is being contained the embryonic stem cell substratum suspension culture 2d of LIF (20U/ml), Neural Differentiation substratum suspension culture 4d (this stage begins to add RA and 0.5 μ M Purorphamine), (this stage is removed RA and Purmorphamine in EB6D to Neural Differentiation substratum suspension culture 6d, add 10ng/ml bFGF and PDGF-AA), adherent culture 7d in the SATO substratum of improvement, continue to cultivate 7 days after removing PDGF-AA and NT-3, impel oligodendrocyte precursor cells ripe, differentiation position oligodendrocyte;

Wherein, the neural precursor ball with EB12D is divided into three groups: control group (without Purmorphamine, containing DMSO), Purmorphamine group (0.5 μ M) and SHH group (100ng/ml).

Table 13 primer sequence and reaction conditions

All the other experimental techniques are identical with enforcement 2.

The result shows that Purmorphamine starts the expression of spongiocyte differentiation associated gene by participating in SHH signal path and the back of the body-veutro axle path among the present invention, induces Olig2 ⁺-GFP ⁺-mES is divided into oligodendrocyte,

Embodiment 4 sets up rat spinal cord collision injury model

1) laboratory animal

Cleaning level healthy adult Wistar is female, and (200～250g) rats, available from this Leco Corp. of the Chinese Academy of Sciences (Shanghai), are raised the Experimental Animal Center in Medical Center of Fudan University by totally 60.Keep the illumination (7:00AM-7:00PM) of 12h every day with fluorescent lamp, room temperature remains on about 25 ℃, and all laboratory animal are fed and the equal adhere rigidly to of experimental arrangement Fudan University laboratory animal safeguard clause.

2) main agents

The anti-NF-200 antibody of rabbit (Chemicon, AB1989), large mouse-anti MBP antibody (Abcam, ab7349), the anti-NG2 antibody of rabbit (Chemicon, MAB5320), the anti-rabbit fluorescence of donkey Alexa

594 mark IgG two are anti-, the anti-mouse fluorescence of donkey Alexa

594 mark IgG two are anti-, the anti-rabbit fluorescence of donkey Alexa

488 mark IgG two anti-(Invitrogen company, the U.S.).

3) key instrument equipment

NYU spinal cord instrument (the NYU-Impactor modle-II Spinal cord contusion system that causes injury, the U.S.), spinal fixation system (Clamping systems, USA), paraffin slicing machine (FJ-200, Shanghai), freezing microtome CM1900 (LEICA), constant water bath box.

4) experimental technique

Make according to a conventional method experimental rat spinal cord collision injury model, the animal of experiment is all study of behaviour training before the art of 7～10d before formally testing, comprise BBB (Basso, the Beattie and Bresnahan) scoring of being familiar with spacious, swash plate Tarlov experiment and footprint Blot experiment.

The grouping of table 14 laboratory animal,

(A group) damage group (44): open vertebral plate, carry out spinal cord T10 impact damage;

(B group) sham operated rats (10): only open vertebral plate, do not clash into spinal cord;

(C group) Normal group (6): be left intact.

The study of behaviour of Spinal Cord Injury in Rats is identified: sham operated rats and operation group get at random 6 rats respectively at art before, postoperative 1,3,7,14,21 and 28d improve the motorius function score of Tarlov and BBB associating.

Table 15Tarlov standards of grading

Score value	Feature
				0 and 1 minute	Do not have activity, only limit to the nonreflective motion of hip joint
2 minutes	The motion in limbs hip, knee, three main joints of ankle
		3 minutes	Can initiatively support body weight and inharmonious gait

4 minutes	The gait that forelimb and hind leg are coordinated has the motion of interphalangeal joint during walking
		5 minutes	Normal gait

Rat is exposed to first in the open experimental situation before modeling, makes rat be familiar with environment.Postoperative is marked to the hind leg motor capacity of every rat with the BBB point system, observes 5min at every turn.

Table 16BBB standards of grading

The morphology research of model mouse Spinal injury is observed:

According to a conventional method, get Spinal injury position frozen section, be used for follow-up immunohistochemical staining, phenodin, Yihong dyeing, fast blue Luxol Fast blue dyeing, the myelin staining relative optical density is measured; Experimental data mean ± standard deviation

Expression is carried out analyzing and processing with the SPSS16.0 statistical software, relatively checks (two groups relatively above) with t check (comparing between two groups) or one-way analysis of variance F between group; P＞0.05 is for difference is meaningless statistically; There is significant P＜0.05 statistically for difference; P＜0.01 is very significant statistically for difference.

Experimental result shows, study of behaviour scoring and the histological observation of the present invention behind the rat SCI used the NYU spinal cord instrument of causing injury and successfully set up the rat acute spinal cord injury model; Experimental animal model of the present invention is easy to make, and the operation in the Modelling process is little to the wound of rat, and the damage of formation is stable, good reproducibility, can reflect preferably the pathophysiological process of human Spinal injury.

Embodiment 5Olig2 ⁺-GFP ⁺-OPC transplantation treatment experimental rat Spinal injury

Setting up of laboratory animal, main agents and plant and instrument and spinal cord collision injury model is same as the previously described embodiments.

Cell is processed: collect the oligodendrocyte precursor cells ball of EB12D, through Trypsin-accutase be digested to unicellular after, FACs filters out Olig2 ⁺-GFP ⁺-OPC expects that with 0.4% tongue blue solution detects cell viability, adjusts cell concn to 1 * 10 simultaneously ⁶/ μ l is for subsequent use.

Living cell rate detects: get 9 cell suspensions and drip 1 0.4% tongue and expect blue solution mixing, with cell counting count board difference living cell counting (non-staining cell) and dead cell (painted cell), calculate cell viability according to following formula in the 3min: living cell rate (%)=viable cell sum/(viable cell sum+dead cell sum) * 100%.

Transplanted cells: press table 17 experiment grouping,

Table 17

(A group) Olig2 ⁺-GFP ⁺-OPC transplantation group: the Olig2 that transplants screening ⁺-GFP ⁺-OPC;

(B group) operative control group: 1w transplants substratum (DMEM) behind the SCI;

(C group) sham operated rats (sham group): only open vertebral plate, do not implement SCI;

(1) before the transplanting cell concn is adjusted into 1 * 10 ⁶Cells/ μ l places for subsequent use on ice; The flat mouth end of transplanting used glass electrode is socketed the microsyringe of 5 μ l by plastic casing, make first glass electrode be full of aseptic physiological saline, the tip of glass electrode is by air seal, then the cell of preparing is inhaled 3 μ l cells by microsyringe and enter in the glass electrode, be fixed on the stereotaxic instrument glass electrode for subsequent use;

(2) experimental rat art injecting immune the day before yesterday inhibitor C sA (10mg/kg), anaesthetize with 3% vetanarcol abdominal injection (30mg/kg body weight), in the art, the spinal cord upper and lower ends of selected distance Spinal injury position 5mm, two sites about i.e. damage is respectively got with normal intersection, Transplanted cells is carried out in totally 4 sites;

(3) regulate stereotaxic instrument slow decreasing glass electrode, insert spinal cord with 15 ° of oblique angles, slow manual rotation microsyringe, the speed that the adjusting Transplanted cells enters spinal cord is 0.5 μ l/min, treat that cell injects complete fully, then original position let the acupuncture needle remain at a certain point 5min slowly extracts pin the layer-by-layer suture wound with stereotaxic instrument.Every animal co-transplantation 3 * 10 ⁶Cell (4 sites).Operative control group is injected the substratum of equivalent as stated above.The result shows, sees in the transplantation that the tissue of Damage of Rats is inflammation reparation change, and the more contiguous normal spinal cord sections of the quality of Spinal Cord tissue is obviously partially soft.Cell suspension slowly is injected into Spinal injury and normal intersection, in migration process, has no intravenous extravasation and local swollen tissue, guarantee that the cell of transplanting can successfully enter in the myeloid tissue.

Postoperative is processed the local penicillin that drips of wound rat and is protected from infection, and injecting immune inhibitor C sA (10mg/kg), to alleviate the rejection after the Transplanted cells, observes until rat self urinary reflex is recovered.

The present invention adopts the study of behaviour methods such as Tarlov scoring, BBB scoring and footprint analysis, and observation of cell is transplanted the impact on functional rehabilitation after the Spinal Cord Injury in Rats:

Tarlov and BBB scoring methods of marking are the same;

Footprint analysis: the experiment gained is printed on the blank sheet of paper of rat footprint, respectively removes 15cm with before and after it, and (70cm) analyzes to remaining interlude.In experimentation, if rat has stop in the middle of passage in advancing, then this time experiment calcellation is processed;

With rat footprint analysis data before the art as its basal level numerical value, then behind the SCI behind 7d (before being Transplanted cells), the SCI behind 14d (being 7d after the Transplanted cells), the SCI behind 21d (being 14d after the Transplanted cells) and the SCI 35d (being 28d after the Transplanted cells) carry out footprint analysis.The index that detects in the footprint analysis comprises step size (Stride length, the distance at adjacent two the hind paw centers of homonymy), outward turning angle (Angle of rotation, the straight line of hind paw central point and the formation of the 3rd toe and an angle through forming between the sole central point straight line parallel with direct of travel).In the experimentation, every rat is repeated 3 footprint analysis, then gets its mean value.At last, the basic data of footprint analysis gained before the data of postoperative footprint analysis gained and the art is compared the percent value that obtains, as the data of statistical analysis.

Electrophysiology Function detection: SCI causes spinal motor, sensory nerve conduction path impaired usually, cause Motion Evoked Potential (Motor Evoked Potentials, MEPs) and somatosensory evoked potential (Somato Sensary Evoked Potentials, SSEPs) change, MEPs is by stimulating the motor center of akrencephalon, while can directly be reflected the functional status of the descending conductive beam of spinal cord or peripheric movement nerve at the electrical signal that spinal cord, peripheral nerve or muscle are recorded to.Therefore, this experiment is further adopted the Electrophysiology Evaluation of detection methods, is analyzed Olig2 ⁺-GFP ⁺-OPC transplantation treatment is on the impact of spinal nerves conduction function; Experiment is respectively to the changing conditions of 7d (14d behind the SCI), 14d (21d behind the SCI) and 28d (35d behind the SCI) time detection rat MEPs after (7d behind the SCI), the Transplanted cells before sham operated rats, the Transplanted cells.

Immunohistochemistry: behind the transplanted cells 28d, get Olig2 ⁺-GFP ⁺6 rat perfusions of-OPC transplantation group are drawn materials, and the position of drawing materials is up and down each 1cm of SCI position, and row spinal cord transection face carries out immunohistochemical staining.Because transplanted cells is Olig2 ⁺-GFP ⁺-OPC, carry out two marks with the anti-GFP antibody of goat and neuronic mark (MAP2), astroglia cell mark (GFAP), oligodendrocyte precursor cells mark (NG2) and myelin basic protein (MBP) respectively, observe transplanted cells integration in the host, migration and differentiation situation.

Transmission electron microscope: get respectively after (7d behind the SCI) before sham operated rats (being pre-injury), the Transplanted cells, the Transplanted cells each two of three groups of rats of 28d (being 35d behind the SCI), cut the damage zone tissue and carry out transmission electron microscope observing, mainly observe the morphological structure of tractus corticospinalis.

Protein immunoblot (Western blot): get respectively after 2w after 1w after (7d behind the SCI) before sham operated rats (be sham group), the Transplanted cells, the Transplanted cells, the Transplanted cells, the Transplanted cells behind the 3w and Transplanted cells each two of six groups of rats of 4w, the myeloid tissue that cuts damage zone and transplanting zone carries out Western blot and detects, observe and transplant front and back, the protein expression of the NF-200 of myeloid tissue and MBP.

Table 18 determination of protein concentration application of sample amount

Component	1	2	3	4	5	6	7	Testing sample
									BSA(μl)	0	2.5	5	10	15	20	25	10
H 2O(μl)	25	22.5	20	15	10	5	0	15
									AB(μl)	200	200	200	200	200	200	200	200
Con(μg/ml)	0	50	100	200	300	400	500

The prescription of table 1912% separation gel 5ml and 5% concentrated glue 3ml

Before and after observation that present embodiment adopts is transplanted, the experimental technique that the test method of the protein expression of the NF-200 of myeloid tissue and MBP can be known with reference to those skilled in the art.

Experimental data is with mean ± standard deviation

Expression is carried out data analysis with the SPSS16.0 statistical software and is processed, and relatively with t check (comparing between two groups) or one-way analysis of variance F check (two groups relatively above), there is significant P＜0.05 statistically for difference between group.

The present invention is by morphology, Western blot, study of behaviour and neural electrophysiology research experiment, and the result shows, Olig2 ⁺-GFP ⁺After-OPC transplantation treatment rat SCI can significantly improve more; Described Olig2 ⁺-GFP ⁺-OPC will become another cell derived of cellular transplantation therapy after the Spinal injury.

Claims (10)

1. an external evoked ES cell differentiation becomes the neurocyte method, it is characterized in that, adopts the nerve-inducing method of embryoid body mediation, with Olig2-GFP ⁺-mES is as cell model, with purine derivative Purmorphamine in conjunction with the described Olig2-GFP of all-trans-retinoic acid directional induction ⁺-mES cytodifferentiation is dynamoneure and oligodendrocyte precursor cells; Comprise the steps:

1. with Olig2 ⁺-GFP ⁺-mES puts into the embryonic stem cell substratum suspension culture 2 days that contains leukaemia inhibitory factor LIF;

2. putting into the Neural Differentiation substratum that contains all-trans-retinoic acid RA and purine derivative Purmorphaine cultivated 4 days;

3. put into the Neural Differentiation substratum that contains bFGF and cultivated respectively 2 days and 6 days, adopt the cell screening Olig2 of fluorescence-activation ⁺-GFP ⁺-positive cell;

4. continue to cultivate at neurone substratum and oligodendrocyte substratum respectively, induce to generate dynamoneure and oligodendrocyte.

2. by method claimed in claim 1, it is characterized in that described step Olig2 1. ⁺-GFP ⁺-mES is the method for utilizing genetic engineering, by the tsiklomitsin inducible expression, inserts GFP at the Olig2 of mouse embryo stem cell locus and makes.

3. by method claimed in claim 1, it is characterized in that the concentration that contains leukaemia inhibitory factor LIF in the described step embryonic stem cell substratum 1. is 20u/ml.

4. by method claimed in claim 1, it is characterized in that the concentration that contains all-trans-retinoic acid RA in the described step Neural Differentiation substratum 2. is 0.5 μ M.

5. by method claimed in claim 1, it is characterized in that the content of bFGF is 10ng/ml in the described step Neural Differentiation substratum 3..

6. by method claimed in claim 1, it is characterized in that the cultivation in the neurone substratum 4. of described step is adherent culture 7～14d.

7. by method claimed in claim 1, it is characterized in that, described step 4. be adherent culture 14d in the oligodendrocyte culture medium culturing.

8. by method claimed in claim 1, it is characterized in that described Olig2-GFP ⁺-mES cytodifferentiation is dynamoneure, and its differentiation rate surpasses 50%.

9. by method claimed in claim 1, it is characterized in that described Olig2-GFP ⁺-mES cytodifferentiation is the oligodendrocyte differentiation, and its differentiation rate surpasses 80%.

10. method claimed in claim 1 is for the preparation of the purposes in the preparation of repairing Spinal injury.

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