| Part I. Research of culture and biological characteristics of bone marrow mesenchymal stern cells from SD rat in vitroObject:To explore the approaches to isolate, culture and identify the bone marrow mesenchymal stem cells (BMSCs) of SD rat in vitro.Methods:The BMSCs were enriched and expanded by whole bone marrow culture. The morphology of the BMSCs was examined and analyzed by light microscopy and the purity and phemotype were analyzed by flow cytometry. The ability of osteogenic differentiation of the BMSCs under different induction conditions was assessed.Results:The BMSCs expanded by whole bone marrow culture were adherent cells with a similar spindle-shaped morphologh during different passages. The phemotypes are CD44(96.49%), CD90(95.63%), CD45(0.64%), CD34(0.39%) respectively. The BMSCs could be induced to differentiate into osteoblasts.Conclusion:Rat BMSCs can be isolated and expanded by whole bone marrow culture, which is an efficient and simple method with high purity and good stability. Part Ⅱ. GDNF, NT-3dual genes transfection promote rat bone marrow mesenchymal stem cells differentiation into neural-Iike cellObjective:Use glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3(NT-3) gene transfected rat bone marrow mesenchymal stem cells (BMSCs), to provide the experiment basis of treating neurological disease;Methods:BMSCs were isolated by the whole bone marrow culture, Characterized by flowcytometry and osteogenic induction. BMSCs with GDNF and NT-3gene were transfected, and morphological changes were measured by microscope. The expression of neural specific markers were detected by RT-PCR and immunofluorescence assay;Results:BMSCs were cultured and purified in vitro. After the induction of differentiation, BMSCs became round or cone-shaped with distinctive outgrowth of protrusions. These cells revealed neuron-like morphological changes, and most of these cells were intertwined into a network structure. RT-PCR showed positive expressions of GDNF, NT-3, NSE, nestin and MAP-2, and immunofluorescence assay showed MAP-2and GFAP expression;Conclusions:Genetically modified BMSCs of co-expressing GDNF and NT-3were able to differentiate into neuronal-like cells and express nerve markers. There provides all experimental basis for gene therapy to treat nervous system-related disorders, such as Hirschsprung disease. Part Ⅲ. Changes in electrophysiology characteristic of BMSCs before and after differentiationObject:The study which compare ion channel current between BMSCs and induced neuron-like cells by path clamp technique, and affirm the induced-neuron electrophysiological function, provide experimental data for enteric nervous system diseases therapy;Methods:Whole-cell patch and perforated patch technique were performed to elucidate the electrical behavior of MSC and induced neuron-like cells by inductor GDNF and NT-3;Results:Before differentiation, The IKDR current peak is583.6536±74.75945pA at+60mV, current density is10.25393±1.313413pA/pF, the EKCa current peak is370.775±49.57507pA, current density is6.513967±0.87096pA/pF, the Ito current peak is467.03±68.44461pA, current density is8.205025±1.20247pA/pF; after differentiation, The DCDR current peak is583.6536±74.75945pA at+60mV, current density is10.25393±1.313413pA/pF, the IKCa current peak is370.775±49.57507pA, current density is6.513967±0.87096pA/pF, the Ito current peak is467.03±68.44461pA, current density is8.205025±1.20247pA/pF; after differentiation, the IKDR current peak is850.32±53.5708pA, current density is18.72207±1.578505pA/pF, the Kca current peak is452.6455±13.4805pA, current density is8.058586±0.943178pA/pF,the Ito current peak is621.194±66.039pA, current density is15.00152±1.918339pA/pF. The changes of current peak and current density between before and after differentiation are significant different (P<0.05);Conclusion:the neuron-like cells differentiated from BMSCs called preliminary electrophysiology characteristic of neuron cells, it is in the process from immature neuron to mature neuron gradually. |
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