The Research On Neural Stem Cells Survival And Differentiation After Transplantation In Parkinson Rat Striatum

Background and Purpose: Parkinson's disease (PD) is a common neurodegenera- tive disease in the elderly. The pathologic feature is selective degeneration of dopaminergic neurons in the substantia nigra leading to loss of dopamine in the striatum. At present cell transplantation has been an ideal therapy to PD, and NSCs is an ideal seed for cell replacement for neurodegenerative disease and show great potential in treatment of Parkinson disease. NSCs transplantation has obtained some therapeutic effect. However, because of lack of the reliable tracer technology, it was difficult to trace and recognize these transplanted cells in vivo after transplantation. Accordingly, it was also difficult to provide an effective evidence for the clinical application of NSCs. In order to investigate the cellular mechanism of NSCs transplantation for the treatment of PD, in this experiment, NSCs were isolated from the E14 rat ventral mesencephalons and GFP transgenic mouse hippocampus, the cells were cultured and amplified in vitro, then transplanted into the rat striatum stereotaxicly, the behavioral changes were observed in order to investigate the survival and differentiation of the graft cells and provide theoretical basis for clinic use in future.Materials and Methods: (1)NSCs were isolated from the E14 rat ventral mesencephalons mechanically, cultured in the serum-free medium containing bFGF, EGF, B27 and DMEM/F12, and identified by the nestin staining which was a mark of neural stem cells; NSCs were frozen through D-MEM/F12 supplemented with 10%DMSO, 10%FBS and 15%B27, and cell viability and biological characters of NSCs were detected after thawed; In order to evaluate the potential differentiation ability of neural stem cells, the cells were induced by the 5% fetal bovine serum, then the differentiated cells were identified by immunohistochemical staining with the antibodies to GFAP(special marker of astrocytes) and NSE(special marker of neurons). (2)Cells amplified and marked by BrdU were transplanted into the rat striatum through the stereotaxis instrument, the behavioral changes at different time after the surgery were observed, and the survival and differentiation of the transplanted cells were measured by Immunohistochemistry. (3)NSCs were isolated from the GFP transgenic mouse hippocampus and cultured in the free-serum medium. The ability of multi-directional differentiation and specific antigen, nestin, were detected by immunocytochemistry. The amplified cells expressing GFP were transplanted into the rat striatum. The changes of rotation induced by APO were observed at different time after surgery, cell survival and migration were detected by fluorescence microscope directly, and TH expression in striatum after transplantation was detected by immunohistochemistry.Results: (1)The average survival rate of the NSCs by mechanical separation was 90%. Cells were passaged after culturing 6 days, the passaged cells grew rapidly, in the experiment they reached the sixth generation. NSCs obtained from the hippocampus of the GFP transgenic mouse grew well in the same way. The expression of GFP protein did not change in single cell and neurosphere after passaging 5 times serially, cells expressed nestin. (2)The isolated cells have potential differentiation ability, they could differentiate into neurons and astrocytes in the culture medium with 5% fetal bovine serum. (3)The property of NSCs was stable after cryopreserved, and the average survival rate of the NSCs was between 54% and 66% after thawed, there were no significant differences among them. (4)The rotation of rats that have received NSCs revealed decreasing apparently 4-6 weeks after tranplantation comparerd with the control graft group. (5)The survival, differentiation, and migration of transplanted cells in host brain.â‘ Survival: the GFP cells and marked by BrdU cells could be observed in the striatum after transplantation, cells could survive 8 weeks at last.â‘¡Differentiation: most of the cells kept undifferentiated while a few TH cells appeared in the striatum at the 4th week after transplantation, it proved the transplanted cells could differentiate into dopaminergic neurons in host brain.â‘¢Migration: the two sources of cells is not limited to injection site after transplantation, they could migrate widely around a certain direction, but the GFP cells could be better observed in migration than NSCs. (6)NSCs were isolated from the GFP transgenic mouse after transplantation, it could be observed in survival, differentiation, and migration of transplanted cells in host brain dynamicly. So it may be valuable for the tracer of the seed cells in cell transplantation for treatment of nervous system diseases.Conclusions: In the experiment, we obtained NSCs mechanically and cultured in the free-serum medium, they have reproductive activity and retain the property of NSCs. The rotation of rats that have received NSCs revealed decreasing apparently after transplantation, and the transplanted cells could survive, migrate and differentiate into dopaminergic neurons, and replace injured neurons of the kind to treatment the PD. The results provide the experimental basis for the treatment of PD and other neurodegenerative diseases.