| Objective To explore the study of rat bone marrow mesenchymal stem cells (BMSCs) in the order of chemical induction medium induced to dopaminergic (DA) neurons in the capacity of cell differentiation and transplantation in the treatment of Parkinson's disease model rats, evaluation and discussion transplantation in the treatment effect. Methods: 1, BMSCs were isolated from bone marrow of rats with density gradient centrifugation and adherent culture. Expression of the BMSCs surface marker was detected by flow cytometer. 2, BMSCs of P4 were induced and differentiated into dopaminergic neuron by three steps, the first seps: the P4 BMSCs were made in Single cell suspension, adjusting the cell concentration was 106/L, was inoculated in the poly-lysine-coated 6-well plate;the second steps: Application of basic fibroblast growth factor (bFGF),epidermal growth factor (EGF) and nerve conditioned medium (NCM) induced by the combined total of 7d ; Continue induction 7d to join the fibroblast growth factor (FGF8),sonic hedgehog (SHH),Dopamine associated protein and genes in the treated cells were examined by Western blot, immumofluore-scence and RT-PCR identification of cells induced. 3, Obtained the model of Parkinson's disease from adult SD rats 26, the rat model were randomly divided into 3 groups: experimental group A after transplantation induced differentiation of dopaminergic neurons; experiment group B bone marrow mesenchymal stem cells in (BMSCs); Transplantation experiment C PBS as control group. After transplantation to a single cage of rats fed for 8 weeks, weekly rotation line of animal testing. Every 3 weeks, 3 rats from each group were sacrificed by anesthesia, paraformaldehyde fixed brain tissue removed after sacrificing parts of the midbrain and striatum slices for TH immunohistochemical staining and dopamine content. Comparison of each group were positive for TH protein expression and to explore the distribution of dopamine nigrostriatal function repair. Result: 1, Isolation and culture of adherent cells (P4) with a typical fibroblast-like morphology and cell surface markers BMSCs. Joint bFGF, EGF and NCM 1 week after induction of the expression in cell morphology and neuronal cells similar to it. And the expression of neuron-specific markerβ-tubulin isotype III (TUJ1), Continue to induced by FGF8 and SHH 1 week, induction cells of gene expression levels of dopamine neuron-specific genes: Nurr1, AADC, VAMT, DAT and TH; Expression in protein levels of dopaminergic neuron-specific marker tyrosine hydroxylase (TH), And TUJ1 more expression. Inverted phase contrast microscope showed that: After 2 weeks of induction and differentiation, cells became round or were polygonal, and extend a number of long axons and dendrites, with the induction time, the gradual extension of axons and between cells, even into the net in. That early induction of in vitro bone marrow mesenchymal stem cells can induced into dopaminergic neuron-like cells. 2, Inverted phase contrast microscope revealed that: induction of differentiation, the cells became round or were polygonal, and out of a long axon and several dendrites, with the induction time, the gradual extension of axons and cells connected into a network between. Immunohistochemistry identified as markers of dopamine neuron-specific TH and TUJ1 antibody positive, indicating that the early induction of in vitro bone marrow mesenchymal stem cells can be induced into dopaminergic neuronal cells. 3, Test results show that the rotational behavior in rats: 2 to 12 weeks after injury, experimental group A and B rotational behavior in rats than in the control group C was significantly lower (P < 0.05), and the experimental group A was lower than experiment B groups (P < 0.05), the difference was statistically significant. 4, Determination of dopamine in the striatum displayed: the experimental group A than group B both in brain tissue contains more dopamine, the control C group only a small amount of dopamine. 5, immunohistochemical staining: the experimental test group A and group B tissue TH content was better than the control C group, the content of group A more than the B group. Conclusion: 1, Joint bFGF, EGF, FGF8, SHH order to be effective BMSCs induced dopaminergic neurons to the induction of directional differentiation method, the cells have been induced by dopaminergic neurons in the shape and functional characteristics. 2, We are neural precursor cells in the induction of intermediates arising in the process, As the following characteristics:①able to pass on behalf of 8 to 10;②can at any time and cryopreservation;③from neural progenitor cells to induce cells into DA neurons need only 6 to 8 days, a greater degree of shortening the induction time;④nerve by passage selection precursor cells, can effectively remove those who can not induce dopamine neurons, thereby increasing the efficiency of induction. 3, In vitro induction of dopaminergic neurons after transplantation in vivo, can promote Parkinson's disease striatum of rat brain neuronal regeneration, morphology of the repair and functional recovery. BMSCs significantly better than simple cell transplant.
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