| Bone marrow stromal cells(BMSCs) are cells got rid of hemopoietic stem cells in marrow cell of mature individual, which is approved to be multi-differentiation potential including transforming into neural stem cell, and differentiation into neurs and neuroglial cell. Inducing BMSCs into nerve cell in vitro aims at applying that to clinical and therapeutics. Under this condition, the cardinal way is that drawing the materials from BMSCs, and then culture and proliferate and induce it into neural cell, planting differentiated cell into body and setting forth its biological role and clinical therapy. The cell stability of differentiated phenotype after induction is largely unknown.Objective: Studying methods of detachment and culture in BMSCs in vitro, determining the optimal schem that BMSCs differentiate into neural cells and observing the phenotype stability in induced-BMSCs aim at application of BMSC in future.Methods: (1) BMSCs were proliferated in vitro after seperated and cultured them in GFP mice.(2) then induce them by basic fibroblast growth factorb(bFGF), retinoic acid (RA), β-mercaptoethanol(BME), brain-derived neurotrophic factor(BDNF), nerve growth factor (NGF)and dimethylsulfoxid (DMSO). Compare the role of RA and/or BME at different concentration in induction process. The ratio which BMSCs differentiated into neural cell was examined through neurofilament protein 200(NF-200), gliad fibrillary acidic protein(GFAP), neuronspecific enolase(NSE) and fibronectin by immunofluorescence and immunohistochemistry assays. Inducer and induced concentration was choosed. (3) the proportion that BMSC in GFP mice differentiated into neural cell was determined by immunofluorescence and immunohistochemistry assays at different induced-time to determine optimal induced time.(4) the proportion of cell possesing neurocyte phenotype was determined by immunofluorescence and immunohistochemistry assays after added brain homogenate extracts.Results: (1) Cultured BMSC in GFP mice can proliferate and go down to the generation in vitro after induction. Doubled time is 41h. homogeneous cell in conformation can form after 3 generations. Keeping splitting-capability after 10 generations, BMSCs posseses capability of cultivation and proliferation in vitro. (2) compared with control, positive cell in NF-200 ,GFAP and NSE increased but fibronectin positive cell decline when RA concentration from 0.25μM to 5.0μM. RA(1.0μM) which is at the lowest concentration can induced NF-200 and NSE positive cell proportion is 20.5% and 22.1%(P<0.05);compared with control, the proportion of positive cell in NF-200 and NSE increased to 32% and 40.3%at the lowest concentration of BME is 0.5mM;compared with simplex RA, the proportion of positive cell is higher in combination of RA(1.0μM) and BME(0.5Mm)(P<0.05).therefore, induced effects can be got through the combination of RA and BME at their lower concentration. (3) Raising in time, The proportion of positive cell in NF-200, GFAP and NSE indreased gradually within 3h～3d. compared with control, the proportion which BMSCs differentiated into positive cell in NF-200, GFAP and NSE increased to 18.5%,19.7% and 20.5% within 8h (P<0.01) by immunofluorescence assays. 12h is the result of immunohistochemistry assays . (4) the proportion of positive cell descended more markedly at 7d than at 1d. after change liquid(P<0.05). the proportion of positive cell in NF-200, GFAP and NSE descended more markedly at 7d than at 1d after change liquid in group of brain homogenate extracts(P>0.05).Conclusion: (1) The BMSCs can be induced to differentiate into neural cells with RA,BME,bFGF,DMSO,NGF and BDNF in vitro. Conbination of RA(1.0μM) and BME(0.5mM) by lower concentration can significantly differentiate BMSC into nerve cell. (2) compared with other groups, inducing for 8～12h can significantly differentiate BMSC into nerve cell within shorter time.(4) changing fluid after induction, getting rid of inducer, brain homogenate extracts can maintain the stability in differentiated phenotype.
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