Research On Transplantation Of Neural Stem Cells And Dopaminergic Neurons In Treatment Of Parkinson's Disease Mouse

Background and Objective: The discovery of neural stem cells(NSC) is the most important breakthrough in neurobiology. It not only challenge the opinion of traditional regeneration of capacity of central nerve system,but also bring the hope for the treatment of many neural nervous disease. Isolation,cultivation,purification and identification of NSC is the basical work of its research and clinical therapy. Objective of this paper is to obtain NSC,observe its biological characteristics and establish the foundation for further study on its proliferation and differentiation. Methods : 1. Isolation, cultivation and purification of the primary NSC from embryo brain,the cells are passaged repeatedly in vitro in order to let the NSC survive for long time. 2. Identification of NSC: ①By using the technique of single-cell clone culture,the capacity of NSC self-renewing is confirmed; ②Immunocytochemistry(ICC) analysis is emploied to detect the expression of nestin which is the specific mark of NSC;③Through ICC assay of NSE and GFAP in the NSC, detect the differentiation of NSC. 3. Cytometry is used to evaluate the capability of proliferation after different number of planted NSC,and the growth curve is ploted. 4. Evaluation of the capability of proliferation and stability of biologic characteristics of NSC after prolonged passage or being cryopreserved and thawed repeatedly. Results: 1. We have isolated NSC from the 7～12w embryo cerebral cortex which could survive in vitro for long time. 2. By using the suspensory cell culture,we have purifyied and cultured NSC for long time.NSC have greatly expanded.The total number of NSC expanded 200 folds after 15 times passage. 3. We have succeeded in cryopreserving and thawing NSC.The average survival rate of NSC is 80%,after thawing, the growth behavior of NSC still keep good. Conclusion: We have isolated NSC from embryo cerebral cortex.These cells could be culture in vitro, and have the ability of self-renewing and differentiation into neurons and astrocyte.