Research Of In Vitro Cultivation Technology Of Rat Bone Marrow Mesenchymal Stem Cells

Chapter1:Cell Culture and Purification of SD Rat BMSCsObjective:The first part aims to optimize the culture condition and to establish a stable and rapid standard operation procedure of gaining large numbers of SD rat BMSCs with high proliferative quality.Method:The density gradient centrifugation and adherence method was used to isolate and purify BMSCs and the isolated cells was inoculated with gradient density of1.0×106,5.0×105,1.0×105,5.0×104,1.0×104/ml.The colony formation time and time of subculture was recorded.MTT method was used to observe cell proliferation in different serum concentrations:5%,10%,15%,20%,25%,30%and different time points:24,48,72hours.Results:Using density gradient centrifugation method to isolate BMSCs and inoculating isolated cells by the density of1.0×105/ml and culturing cells with25%fetal bovin serum can shorten the time of primary culture and can get BMSCs with highest proliferative activity.Conclusion:We optimized a protocol for isolation and culture of BMSCs that can be applied in the following experiments with minimal requirement.Chapter II:Identification of SD rat BMSCsObjective:The second part aims to identify and observe the multilineage differentiation potential of the isolated and purified cells in the first part.Methods:The expression of surface antigen:CD45, CD34, CD90, CD44by isolated cells were analysed by cell immunofluorescence. The osteogenic and adipogenic culture medium were used to induce obtained cells into osteoblast and adipocyte, the intracellular lipid droplets of adipocytes was stained by oil red O and the calcium nodules was stained by alizarin red.Results:Immunofluorescence assay showed positive expression of CD44, CD90and negative expression of CD34, CD45by obtained cells. After14days of adipogenic induction and25days of osteogenic induction, obtained cells performed adipocyte and osteobast morphology and can be satined by oil red O and Alizarin Red.Conclusion:The isolated and purified cells in the first part are BMSCs which have multilineage differentiation potential.Chapter III:The expression of all germ layers specific markers of undifferentiated SD rat BMSCsObjective:The third part aims to detect the neuron and hair cell and germline、 endodermal and mesodermal specific genes expressed by undifferentiated SD rat BMSCs.Methods:RT-PCR was used to detect the expression of Ceruloplasmin SM22, Protamine2, Aldolase, MAP2, Amyloid precursor protein, NMDA Glutamate binding subunit, Syntaxin, Neurofilament-M, Tau, NeuroD、 Calretinin, Brn3.a, Myosin7A, Mathl, Espin genes in transcription level by undifferentiated BMSCs.Results:The neuronal specific marker genes:Amyloid precursor protein, NMDA Glutamate binding subunit and hair cell specific marker gene Myosin7A and endodermal marker gene Ceruloplasmin, mesodermal marker gene SM22are expressed in some of the primary cells.Conclusion:The primary cells have differention potential across the germ layers and intrinsic predisposition to differentiate into neurons and hair cells of these cells, also the data can be used by the following experiments for identification of specific markers after specific induction.19figures,3tables,43references...