Directly Induction Of Bone Mesenchymal Stem Cells To Neuron-like Cells And Preparation Of The Cell Models Of Parkinson’s Disease

Bone marrow mesenchymal stem cells (BMSCs), one kind of the adult stem cells in bone marrow, have the potential to differentiate into cells of multiple directions, such as muscles, bones, nerves and so on. Studies have shown that BMSCs can be transplanted into brain to treat neurodegenerative diseases such as PD. It is supposed that BMSCs can differentiate to neuron-like cells so as to replace damaged neurons and play an important role in brain. There is no research on directly differentiation of BMSCs. After amplification in vitro, BMSCs are induced to neuron-like cells. The pattern of BMSCs is explored by testing specific markers of neuron-like cells and studying the mechanism of treating neurodegenerative diseases such as PD through differentiating into neuron-like cells, which is a thought-provoking topic. BMSCs can be used for cell transplantation, which lays a solid experimental foundation of cell and gene therapy for the diseases.PD is a common central neurodegenerative disease characterized by neuron degeneration, apoptosis and the formation of inclusion body. At present, animal models of PD are used to observe behavioral changes at the overall level and the pathological characteristics of histologically. It is difficult to grasp the knowledge of cytological and molecular mechanism of the disease and the research of medication is restricted to some extent. The cell models of PD can make up for the disadvantages. The cell models of PD require cell apoptosis and inclusions of Lewy body. PD cell models can be studied in the aspects of cellular level. The cell models of PD, which own two pathological characteristics of PD, are prepared to study all changes in molecular-level deeply in this experiment and provide platform for transplantation in treatment and drugs screening.PD cell models can be used to explore the pathogenesis of PD and study the biological changes of nerve cells in animal models, which suggests the function of enlightenment at cellular level. BMSCs used to treat PD are similarly associated with PD cell models in the mechanism of cellular level in aim of making a certain foundation for cell transplantation and drug treatment from now on.Part I Induction of Bone Mesenchymal Stem Cells to Neuron-like CellsRat BMSCs isolated and purified in vitro had the potential to be induced to neuron-like cells and provide technology procedure and experimental basis of cell transplantation in the treatment of neurodegenerative diseases. BMSCs grew well after inoculated into the completed medium; BMSCs were purified through whole bone marrow cultivation; The cell growth curves of the first, third and fifth generation of BMSCs were drawn; Specific factors were detected on BMSCs surface; Preinducer was added to induce BMSCs to the neuron-like cells; the morphological changes of BMSCs could be observed under microscope; Cells’specific markers could be detected through cell immunchemical dyeing to exam that BMSCs had the potential to be induced to neuron-like cells. BMSCs were collected by the method of whole bone marrow cultivation. BMSCs sticked quickly and grew well after inoculated with completed medium; The growth curve of BMSCs was in the shape of S and BMSCs in logarithmic phase grew faster than other phases; The vitality of BMSCs was high; The expression rate of CD29and CD90positive through flow cytometry were high, which fit the characteristics of BMSCs surface antigen; The cell immunochemical staining showed that inductions of nestin and NSE were positive and GFAP was negative, which showed that cells expressed specific enzyme and the specific marker of the neural stem cells and did not express glial fibrillary acidic protein in vitro. BMSCs could be successfully isolated and cultured with high purity through the method of whole marrow cultivation. The phenotype corresponded with characteristics of BMSCs. BMSCs had the potential to differentiate into neuron-like cells.Part II The Preparation of cell models of Parkinson’s Disease with PC12cellsCell models of PD after appropriate treatment with PC12cells were successfully made. System of PC12cells was created; PC12cells were inoculated into completed medium; Cell growth curve of PC12cells was drawn; PC12cells were treated with different concentrations of MPP+; The most appropriate MPP+concentration was chosen to prepare cell models of PD; The cell vitality, apoptosis and specificity protein alpha synuclein were used as indexes to detect PD cell models. MTT method was used to detect the cell vitality; Apoptosis could be observed through Hoechst33342staining as well as AO and EB staining; Immunofluorescence staining was used to detect the presence of specific protein alpha synuclein. The system was stable and the operation was simple; PC12cells adhered quickly and grew well; the vitality of PC12cells was high, the growth curve of PC12cells was in the shape of S; The different concentrations of MPP+had different influences on PC12cells; When the concentrations of MPP+were more than500micromoles, the vitality of PC12cells was no longer significantly reduced; Apoptosis could be observed and blue fluorescent fragments were found under microscope; Cell apoptosis was observed through AO and EB staining, the cell apoptosis rate of experimental group was higher than the control group; PC12cells treated with MPP+expressed specifically protein alpha synuclein. PC12cells treated with MPP+expressed two pathological features of PD:cell apoptosis and the emergence of Lewy body, and expressed specific protein alpha synuclein as well, suggesting that PD cell models was successfully prepared.