CN108060131A - A kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells - Google Patents
A kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells Download PDFInfo
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Abstract
The present invention relates to a kind of methods of inducing bone marrow mesenchymal stem cells into neurons differentiation.Belong to the method that stem cell is divided into neural stem cell.The present invention is derived from the stem cell and Olfactory essheathing cell of body in implementation process, can exclude foreign preteins rejection, avoids morals and ethical pressure, mitigates patient's financial burden；Inducing bone marrow stem cell differentiation stage by stage is taken in incubation, inducing nerve cell precursor is further divided into mature cell, stablizes so as to obtain biological character, survival time in vitro is long, the nerve cell of differentiation and maturation.A certain amount of trehalose and sucrose are added in into culture medium in incubation, being found surprisingly that the addition of trehalose and sucrose increases the ratio that cell differentiation is neural precursor, break up cycle time, so as to obtain large batch of ripe nerve cell in the short time.
Description
Technical field
It is specifically a kind of inducing bone marrow stroma stem cell to nerve the present invention relates to the technical field of induction stem-cell therapy
The method of cell differentiation.
Background technology
Spinal cord injury (spinal cord injury, SCI) often results in limbs of patient paralysis, gatism and sexual function
Obstacle is a kind of common disease for seriously affecting patient's physical and mental health, frequently-occurring disease.With modern transportation and the quick hair of industry
Exhibition, the incidence of spinal cord injury are just increasing year by year, and U.S.'s annual morbidity is 20-40 people/a population of one million, China's spinal cord injury
Annual morbidity is about 15-20 people/million, and latest data shows that Beijing area annual morbidity has reached 60 surprising people/million.
The reparation of spinal cord injury is the scientific research problem of long-standing problem medical field, and surgical intervention means (reset, vertebra including fracture dislocation
Pipe decompression, hematoma clearance etc.) with the effect of drug (such as heavy dose of methylprednisolone shock therapy) treatment spinal cord injury very
It is limited.With the progress of INVENTIONModern cell and Protocols in Molecular Biology, scientists are attempted will be external by the method for cell transplantation
The cell implantation spinal cord injury of culture sentences the nerve cell for substituting missing, and secretion promotes the neurotrophic factor of nerve regneration,
Neuron to be protected to reduce secondary injury, and is integrated with host's nervous system, Reconstruction of The Function cynapse promotes spinal function to recover,
Huge progress is had been achieved at present, becomes the spinal cord regeneration means of great prospect.
Seed cell currently used for spinal cord injury reparation includes embryonic stem cell, neural stem cell, Schwann cell, smells
Sheath cell, mesenchymal stem cell etc..Wherein embryonic stem cell, neural stem cell, Schwann cell exist in various degree
Materials are difficult, directed differentiation does not know, is related to the problems such as ethics, it is difficult to applied to clinic.Bone marrow stroma stem cell (bone
Marrow stromal cells, BMSCs), mesenchymal stem cell (mesenchymal stemcells) is also known as, is come
The undifferentiated mesenchymal cell of mesoderm is come from, there is the ability to a variety of mesoderm tissues cell differentiations, be such as divided into skeletonization
Cell, chondroblast, adipocyte etc..Discovered in recent years BMSCs has the potentiality across differentiation of germinal layers, is such as divided into entoderm
The cardiac muscle cell in source, the nerve cell of ectodermal origin etc..It is created across the application for being found to be BMSCs of differentiation of germinal layers ability
Broader prospect, BMSCs skin grafing and mendings central nervous system injuries such as cerebral ischemia, Parkinson's disease, traumatic cerebral spinal cord injury
Research is unfolded in succession.BMSCs has abundance, materials simplicity, is easy to the spies such as amplification in vitro and energy Induction of committed differentiation
Point, and autotransplantation can be carried out and immunological rejection is avoided to be argued with ethics, therefore treat SCI by transplanting BMSCs
Have become the hot spot of current spinal cord regeneration research field.
Although the undifferentiated BMSCs with differentiation can be used to repair spinal cord injury, through inducing the BMSCs broken up in advance
May be particularly suited for cell transplantation because the neural-like cells that are formed after pre- differentiation of BMSCs can preferably with spinal nerve
Damage location is integrated, and repairs lesion, and the BMSCs after pre- differentiation is in mitosis anaphase, can reduce into the several of tumour
Rate.
Method currently used for external evoked BMSCs differentiating into nerve cells mainly includes chemicals method and growth factor
Two kinds of combined method, major defect is not high including cells transdifferentiate rate, often (common with cell degeneration, necrosis in atomization
In chemicals method), noble cells stability is poor, hold time under vitro it is short (be common in chemicals method with growth
Factor set is legal), neuron and Deiter's cells ratio are difficult to control in noble cells, thus clinical value is low.
For example, disclosing a kind of scutelloside in Chinese patent application 200810059560.9 medulla mesenchyma is being promoted to do carefully
Purposes and method in terms of born of the same parents' vitro directed differentiation, this application provide a kind of traditional Chinese medicine monomer compound scutelloside inducing bone marrow
The method of stem cell differentiation, although the compound can succeed differentiation of stem cells, chemical-induced process receives always
Dispute, because chemical substance also has toxin in addition to playing the role of Cell differentiation inducing activity.And chemicals method inducing cell point
Often with cell degeneration, necrosis during changing, cell differentiation stability is poor, and vitro is held time the shortcomings of short.
A kind of cerebrospinal fluid inducing self-body stem cell is for another example disclosed in Chinese patent application 200810020094.3
Be divided into the method for nerve cell, this application provide it is a kind of replace hyclone with autoserum, self celiolymph substitutes
Additive and stimulating factor originally obtains the method for nerve cell, but the nerve cell that differentiates of this method it is most for not into
Ripe neural precursor, mature cell ratio is relatively low, noble cells phenotype hold time it is short, then may influence transplanting after body
Interior time-to-live and action effective.
Present inventor is directed to the research in stem-cell therapy field all the year round, once explores self olfactory mucosa source for the first time
Olfactory essheathing cell (OECs) and BMSCs co-culture, induction BMSCs breaks up to neural-like cells, and result of study shows that OECs can be lured
Lead BMSCs differentiating into nerve cells (Neuroscience Letters, 2010,475 (2)：99-103).It is lured in vitro with previous
Guiding method compares, and OECs, which co-cultures mode, has the characteristics that easy to operate, cells transdifferentiate rate is high, and maximum advantage is then
OECs and BMSCs can be by obtaining self, can be to avoid immunological rejection and law, ethics dispute if being applied to clinic.
However, OECs co-cultures the defects of mode is there is also with chemicals method and similar combinations of growth factors method, mainly differentiation is thin
Most born of the same parents are immature neural precursor, and ripe nerve cell ratio is low；Noble cells phenotype is held time short, then may be used
It can influence internal time-to-live and action effective after transplanting；And above two aspects defect is the most main of influence BMSCs transplantation effects
Want reason.
Basic fibroblast growth factor (basic fibroblast growth factor, bFGF also known as FGF-2)
It is fibroblast growth family a member, is distributed widely in the Various Tissues and device of mesoderm and neuroectodermal origin
Official by being combined with tissue-specific receptors (FGFRs), activates tyrosine protein kinase in target cell and plays a role, have
Promote the various biologicals effects such as cell Proliferation, differentiation, migration, wide participation angiogenesis, tissue repair, tumour generation etc. raw
Life process.BFGF is as a kind of important growth factor with high efficiency and broad spectrum activity, in recent years in central nervous system
Function be gradually taken seriously, bFGF/FGFRs systems are embodied in the main function of nerve fiber：Nerve to occur, aixs cylinder life
Long, neuroprotection and regeneration etc..
BFGF plays an important role in nervous system development and injury repair, can promote nervous tissue source progenitor cells or
Precursor is proliferated and is divided into ripe nerve cell, therefore bFGF can provide application idea for the differentiation of nerve cell.
However, on the one hand, in the prior art, bFGF is commonly used in the induction to the neural precursor of nervous tissue source
Differentiation.On the other hand, cell culture experiments in vitro is found, in addition to necessary culture medium condition, addition bFGF is obviously promoted
BMSCs division growths, and it is influenced to multilineage differentiated, such as to nerve cell and osteoblast differentiation.In another aspect, bFGF is such as
How induction system is added in, when high ripe nerve cell ratio and cell phenotype maintenance can be obtained by which kind of condition carrying out induction using
Between long effect at present still in unknown state.
Therefore, how efficiently to be divided using the bone marrow stroma stem cell in bFGF induction non-nervous tissues source to nerve cell
Change, the Neural Cell Phenotypic for forming more ripe nerve cells and being formed length of holding time is technology that this field needs solve
Problem.
The content of the invention
To solve the above-mentioned problems, the self Olfactory essheathing cell inducing bone marrow stroma stem cell differentiating into nerve cells of the present invention
For neural precursor, and promote neural precursor using exogenous basic fibroblast growth factor (bFGF also known as FGF-2)
Cell proliferation and differentiation, the neural-like cells that BMSCs is made to be formed after OECs and the induction of bFGF stageds, so as to obtain biological character
Stablize, survival time in vitro is long, the nerve cell of differentiation and maturation.
It can make the new method that stem cell directed differentiation is neural stem cell it is an object of the invention to provide a kind of, be
Human neural stem cell transplant techniques open up new approach.
The present invention provides a kind of methods of inducing bone marrow stroma stem cell differentiating into nerve cells, comprise the following steps：
(1) culture of bone marrow stroma stem cell；
(2) culture of Olfactory essheathing cell；
(3) bone marrow stroma stem cell and Olfactory essheathing cell co-culture
Obtained bone marrow stroma stem cell will be cultivated and Olfactory essheathing cell is co-cultured in Tranwells systems (non-direct contact)
In, simulation human body environment is co-cultured in DMEM/F12In culture medium, training that bone marrow stroma stem cell culture is handled in poly-D-lysine
On piece is supported, Olfactory essheathing cell is incubated in the tube core for the perforated membrane that aperture is 0.4m；A culture solution was replaced per 2-3 days, is co-cultured
Two weeks, the variation of microexamination cytomorphology, and neuron is detected using immunohistochemistry and real-time fluorescence quantitative PCR (RT-PCR)
Specific marker nestin, NSE, β-III-tubulin and MAP2 and Deiter's cells Specific marker GFAP with
p75NTRVariation, it was demonstrated that through the external evoked BMSCs of OECs Godwards through precursor cell differentiation, obtain the neural precursor in BMSCs sources
Cell；
(4) bFGF induces the further differentiation of the neural precursor in BMSCs sources
The neural precursor in the BMSCs sources that step (3) is obtained is inoculated in the DMEM trainings containing 2-20ng/mLbFGF
It supports in base, inoculum concentration is 4-6 × 105A cell/mL culture mediums, incubation time are 5-7 days, obtain ripe nerve cell.
Any method commonly used in the art may be employed in the culture of the step of above method (1) bone marrow stroma stem cell
Culture obtains passage bone marrow stroma stem cell.
The culture of above method step (2) Olfactory essheathing cell may be employed any method culture commonly used in the art and obtain
Pass on Olfactory essheathing cell.
Applicant has been surprisingly found that the training in bone marrow stroma stem cell and Olfactory essheathing cell co-cultivation during cell culture
It supports in base and adds in a certain amount of trehalose and sucrose, and it is 3 to control trehalose and sucrose mass ratio：1, bone marrow matrix can be increased
The ratio of ripe nerve cell in the neural precursor that cell differentiation obtains, and Apoptosis can be reduced.
The addition of the trehalose is every liter of DMEM/F120.05-3mg in culture medium；Preferably 0.05-2mg；Into one
Step is preferably 0.05-1mg.
The addition of the sucrose is every liter of DMEM/F120.02-1mg in culture medium；Preferably 0.02-0.6mg；Into one
Step is preferably 0.02-0.3mg.
Equally, in step (4) bFGF induce BMSCs sources neural precursor further break up contain 2-
It is 3 that mass ratio is added in the DMEM culture mediums of 20ng/mL bFGF：1 trehalose and sucrose, can accelerate neural precursor
To mature cell break up speed, make neural precursor to the cycle time of ripe neural cellular differentiation, break up into
Ripe nerve cell stability is good, can obtain large batch of ripe nerve cell in a short time, and obtained maturation nerve
Cell vitro is held time length.
The addition of the trehalose is in every liter of DMEM culture medium plus 0.05-3mg；Preferably 0.05-2mg；Into one
Step is preferably 0.05-1mg.
The addition of the sucrose is in every liter of DMEM culture medium plus 0.02-1mg；Preferably 0.02-0.6mg；Into one
Step is preferably 0.02-0.3mg.
The present invention has the following advantages compared with prior art：
(1) stem cell and Olfactory essheathing cell of body are derived from, foreign preteins rejection can be excluded, avoids morals and ethics
Pressure mitigates patient's financial burden；
(2) inducing bone marrow stem cell differentiation stage by stage is taken, inducing nerve cell precursor is further divided into mature cell,
Stablize so as to obtain biological character, survival time in vitro is long, the nerve cell of differentiation and maturation.
(3) a certain amount of trehalose and sucrose are added in incubation, being found surprisingly that the addition of trehalose and sucrose makes
Cell differentiation for neural precursor ratio increase, break up cycle time, so as to obtained in the short time it is large batch of into
Ripe nerve cell.
Specific embodiment
Basic embodiment
Preparing experiment：
The bone marrow stroma stem cell and Olfactory essheathing cell passed on using method culture commonly used in the art.
(1) culture of bone marrow stroma stem cell
(2) culture of Olfactory essheathing cell
(3) bone marrow stroma stem cell and Olfactory essheathing cell co-culture
By above-mentioned steps (1) and step (2) the obtained bone marrow stroma stem cell of culture and Olfactory essheathing cell co-culture in
In Tranwells systems (non-direct contact), simulation human body environment is co-cultured in DMEM/F12In culture medium, in every liter of culture medium
The sucrose of the middle glucose and 0.4mg for adding in 1.2mg, the culture piece that bone marrow stroma stem cell culture is handled in poly-D-lysine
On, Olfactory essheathing cell is incubated in the tube core for the perforated membrane that aperture is 0.4m；A culture solution was replaced per 2-3 days, is co-cultured two weeks,
Microexamination cytomorphology changes, and detects neuron-specific using immunohistochemistry and real-time fluorescence quantitative PCR (RT-PCR)
Property marker nestin, NSE, β-III-tubulin and MAP2 and Deiter's cells Specific marker GFAP with
p75NTRVariation, it was demonstrated that through the external evoked BMSCs of OECs Godwards through precursor cell differentiation, obtain the neural precursor in BMSCs sources
Cell；Found by detecting, the ratio of neural precursor that culture obtains after 2 weeks account for the 95% of primitive marrow stroma stem cell with
On.
(4) bFGF induces the further differentiation of the neural precursor in BMSCs sources
The neural precursor in the BMSCs sources that step (4) is obtained is inoculated in the DMEM cultures containing 15ng/mLbFGF
In base, the glucose of 0.9mg and the sucrose of 0.3mg are added in every liter of culture medium, inoculum concentration is 4 × 105A cell/mL cultures
Base, incubation time are 5 days, obtain more than 90% ripe nerve cell.
Zoopery
1st, animal model
Female sd inbred rats are selected, weight 250-300g, ketamine (90mg/kg) intraperitoneal injection anesthesia, prone position is fixed
Exposure 10 vertebral plate of chest is cut on operating table, under microscope and cuts off it, appears dura mater, using Allens WD methods, with 10g ×
2.5cm cause injury power damage spinal cord, cause contusion and laceration of spinal cord model.
2nd, cell transplantation
Six groups of animal point is respectively simple BMSCs injections group, BMSCs+ single bFGF injections group, BMSCs+ bFGF twice
Injection group, simple bFGF injections group.Postoperative 4 weeks in modeling, worn with reference to the method for the foundation such as Bakshi through waist 3-5 intervertenral spaces
Thorn, subarachnoid injection BMSCs or/and bFGF, wherein BMSCs injection volumes (contain 10 for 20 μ L6A cell), bFGF injection volumes
For 150 μ g/kg, the wherein BMSCs+ groups of bFGF injections twice carry out bFGF injections again 1 week after injecting for the first time.
3rd, functional evaluation
Rat model is modeling is preoperative, modeling is postoperative and the postoperative row functional evaluation in 1,2,4,8 week of cell transplantation.Functional evaluation
Using Basso Beatlie Bresnahan motor function scores.Experimental rat is positioned on the circular platform of diameter 2m, seen
Examine the walking for recording its hind leg and limb activity.Divide three parts：It first portion 0-7 points, judges each joint of animal hind leg and lives
It is dynamic；8-13 points of second portion judges the gait and coordination function of hind leg；14-21 points of Part III judges the fine of movement median claw
Action, three full marks are 21 points, and test result see the table below 1
Table 1
4th, electro physiology is tested
Rat model is modeling is preoperative, modeling is postoperative and postoperative 1,2,4, the 8 week row SEP of cell transplantation is checked.It, will after anesthesia
Stimulating electrode is bound on the skin outside the lower limb gastrocnemius of one side, is around moistened with warm saline.Through Nihon Kohden electro photoluminescence
Instrument gives the stimulation of direct current square wave electric pulse, intensity 50mA, ripple wide 0.1ms, frequency 3Hz.Recording electrode is placed in animal offside sense
Feel cortex surface, it is subcutaneous that reference electrode is inserted into left volume.Signal is amplified with FA292 amplifiers, filters 100~1000Hz of band logical, is put down
Equal 200 times, computer samples and stores signal.Every animal surveys double lower limb.
Testing result is analyzed, the neural precursor in bFGF inductions BMSCs sources is further broken up into obtained maturation
Nerve cells transplantation is to carrying out SEP and check to show that result has substantially returned to the preoperative level of modeling in rat body, after 4 weeks, 8 weeks
SEP is carried out afterwards checks that display result has been completely recovered to the preoperative level of modeling.
Shorten the cell culture time to further prove that culture medium provided by the invention has, it is thin to improve neural precursor
The ratio of born of the same parents and ripe nerve cell a little, and has carried out comparative testing below.
Comparative example 1-9
Comparative example 1
It is DMEM/F in step (3) with the difference of basic embodiment12Without dextrose and saccharose, other party in culture medium
Method is identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 70% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 5 days, obtains 65% or so ripe nerve cell.
Comparative example 2
It is with the difference of basic embodiment in step (4) in DMEM culture mediums without dextrose and saccharose, other methods
It is identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 93% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 8 days, obtains 70% or so ripe nerve cell.
Comparative example 3
It is DMEM/F in step (3) and step (4) with the difference of basic embodiment12In culture medium and DMEM culture mediums all
Without dextrose and saccharose, other methods are identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 68% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 7 days, obtains 41% or so ripe nerve cell.
Comparative example 4
It is DMEM/F in step (3) with the difference of basic embodiment12The ratio of dextrose and saccharose is 1 in culture medium：
1, other methods are identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 85% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 5 days, obtains 78% or so ripe nerve cell.
Comparative example 5
It is that the ratio of dextrose and saccharose in DMEM culture mediums in step (4) is 1 with the difference of basic embodiment：1,
His method is identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 92% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 7 days, obtains 73% or so ripe nerve cell.
Comparative example 6
It is DMEM/F in step (3) and step (4) with the difference of basic embodiment12Portugal in culture medium and DMEM culture mediums
The ratio of grape sugar and sucrose is 1: 1, and other methods are identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 83% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 6 days, obtains 70% or so ripe nerve cell.
Comparative example 7
It is DMEM/F in step (3) with the difference of basic embodiment12The ratio of dextrose and saccharose is 1 in culture medium:
5, other methods are identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 76% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 5 days, obtains 67% or so ripe nerve cell.
Comparative example 8
It is that the ratio of dextrose and saccharose in DMEM culture mediums in step (4) is 1: 5 with the difference of basic embodiment,
His method is identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 92% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 5 days, obtains 74% or so ripe nerve cell.
Comparative example 9
It is DMEM/F in step (3) and step (4) with the difference of basic embodiment12Portugal in culture medium and DMEM culture mediums
The ratio of grape sugar and sucrose is 1: 5, and other methods are identical with basic embodiment with step.
Experimental result：Bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured into neural precursor after two weeks in step (3)
Ratio account for the 76% of archaeocyte, the further differentiation culture of the neural precursor in bFGF induction BMSCs sources in step (4)
Time is 8 days, obtains 69% or so ripe nerve cell.
Neural precursor proportion in each embodiment and ripe nerve cell proportion are listed in the table below 2
Table 2
| Example | Neural precursor proportion % | Ripe nerve cell proportion % |
| Basic embodiment | 95 | 90 |
| Comparative example 1 | 70 | 65 |
| Comparative example 2 | 93 | 70 |
| Comparative example 3 | 68 | 41 |
| Comparative example 4 | 85 | 78 |
| Comparative example 5 | 92 | 73 |
| Comparative example 6 | 83 | 70 |
| Comparative example 7 | 76 | 67 |
| Comparative example 8 | 92 | 74 |
| Comparative example 9 | 76 | 69 |
In order to further prove that method culture provided by the invention can obtain the ripe nerve cell of good properties, to base
The phenotype for the ripe nerve cell cultivated in plinth embodiment and comparative example 1-9, which is held time, to be classified,
6 ranks are distinguished according to time length, it is 1 grade that phenotype, which is held time shortest, and phenotype is held time up to 6 grades, tests number
According to as shown in the following table 3：
Table 3
Neural Cell Phenotypic prepared by basic embodiment is can be seen that by above-mentioned data hold time be considerably longer than comparison
Embodiment.And noble cells phenotype is held time short, may then influence after transplanting time-to-live and action effective in vivo, therefore
Nerve cell prepared by method provided by the invention has more practicability.
Only as described above, is only the preferred embodiments of the invention, when the model that implementation of the invention cannot be limited with this
It encloses, i.e., generally according to simple equivalent changes and modifications made by the content recorded in the claims in the present invention and description of the invention,
All it is still covered by the claims of the invention.It is searched in addition, summary part and title are intended merely to auxiliary patent document
It seeks and being used, be not used for limiting the interest field of the present invention.
Claims (10)
1. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells, it is characterized in that, it concretely comprises the following steps：
(1) culture of bone marrow stroma stem cell；
(2) culture of Olfactory essheathing cell；
(3) bone marrow stroma stem cell and Olfactory essheathing cell are co-cultured
The bone marrow stroma stem cell and Olfactory essheathing cell that step (1) and step (2) culture are obtained are co-cultured in Tranwells systems
In (non-direct contact), simulation human body environment is co-cultured in DMEM/F12In culture medium, bone marrow stroma stem cell culture relies in poly
The culture on piece of propylhomoserin processing, Olfactory essheathing cell are incubated in the tube core for the perforated membrane that aperture is 0.4m；It replaced per 2-3 days and once trains
Nutrient solution co-cultures two weeks, the variation of microexamination cytomorphology, and using immunohistochemistry and real-time fluorescence quantitative PCR (RT-
PCR) detect neuronal specificity marker nestin, NSE, β-III-tubulin and MAP2 and Deiter's cells is special
Property marker GFAP and p75NTRVariation, it was demonstrated that through the external evoked BMSCs of OECs Godwards through precursor cell differentiation, obtain BMSCs
The neural precursor in source；
(4) bFGF induces the further differentiation of the neural precursor in BMSCs sources
The neural precursor in the BMSCs sources that step (3) culture is obtained is inoculated in the DMEM containing 2-20ng/mL bFGF
In culture medium, inoculum concentration is 4-6 × 105A cell/mL culture mediums, incubation time are 5-7 days, obtain ripe nerve cell.
2. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
DMEM/F in above-mentioned steps (3)12Also containing trehalose and sucrose in culture medium, trehalose and sucrose mass ratio are 3: 1.
3. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of trehalose is every liter of DMEM/F in above-mentioned steps (3)120.05-3mg in culture medium.
4. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of trehalose is every liter of DMEM/F in above-mentioned steps (3)12It is 0.05-2mg in culture medium.
5. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of trehalose is every liter of DMEM/F in above-mentioned steps (3)12It is 0.05-1mg in culture medium.
6. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of sucrose is every liter of DMEM/F in above-mentioned steps (3)120.02-1mg in culture medium.
7. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of sucrose is every liter of DMEM/F in above-mentioned steps (3)12It is 0.02-0.3mg in culture medium.
8. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
In DMEM culture mediums also containing the trehalose and sucrose that mass ratio is 3: 1 in step (4).
9. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, it is characterized in that：
The addition of trehalose is to add 0.05-3mg in every liter of DMEM culture medium in above-mentioned steps (4).
10. a kind of method of inducing bone marrow stroma stem cell differentiating into nerve cells according to claim 1, feature
It is：The addition of sucrose is to add 0.02-1mg in every liter of DMEM culture medium in above-mentioned steps (4).
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