| Objectives:The use of bone marrow mesenchymal stem cell differentiation,bone marrow mesenchymal stem cells to differentiate into like corneal endothelial cells;to investigate the purification and cultivation of tree shrews and Ganges RIver monkey bone marrow mesenchymal stem cells(BMSCs)biological characteristics:morphology,growth curve,activity,identification of differentiation the potential and phenotype,and compared the results of tree shrews and monkeys in rhesus monkey.The tree shrews BMSCs and the crystal,iris,corneal endothelial cells co cultured with non contact,become the feasibility of like corneal endothelial cells induced by in vitro analysis,and further explore the rhesus monkey bone marrow mesenchymal stem cells differentiate into possibility and experimental scheme of like corneal endothelial cells.Methods:The tree shrews and rhesus monkey tibia and femur,removal of epiphyseal bone marrow out collected by density gradient centrifugation and adherent culture of whole bone marrow culture method using BMSCs extraction,MTS detection of cell activity and draw two generation seven generation cell growth curve,using FITC Annexin V-PI apoptosis kit to detect cell apoptosis method,using the analysis method to detect the expression of cell surface markers CD29,CD34,CD45,CD90,CD 147,flow cytometry.Results:Tree shrews and rhesus monkey extract cells were long fusiform shape with uniform size,small cell size ranging from the number of tentacles,or a star;the second and seventh generation of the cell growth curve is in the shape of S,P7 cell proliferation ability was weaker than P2 generation;tree shrews apoptosis detection can estimate the proportion of living cells 90.12%,cell death was 8.77%and 0.54%apoptotic cells;cultured tree shrews BMSCs third generation,flow cytometry,CD45 negative expression of CD34 were 0%,0%,CD90,CD29 and CD 147 positive expression were 96.4%,95.8%,91.3%.After two weeks of induction of bone marrow mesenchymal stem cells induced by corneal endothelial cells,NSE staining was negative,and there were a lot of red lipid droplets in oil red O staining.BMSCs NSE staining was positive in the co cultured lens,and BMSCs NSE staining was negative in iris and corneal endothelial cells.Detection of rhesus monkey BMSCs cell apoptosis can be estimated by living cells ratio of 73.83%,cell death was 21.76%and 4.27%apoptotic cells;cultured rhesus monkey BMSCs third generation,flow cytometry and CD45 negative expression of CD34 were 0%,0%,CD90,CD29,CD 147 positive expression was 98.8%96.4%,89.7%.Conclusions:The biological characteristics and phenotypes were separated cells cultured with BMSCs coincide,osteoblasts and adipocytes induced by the results showed that the cells have multilineage differentiation potential of mesenchymal stem cells.The results demonstrated that the isolated cells were mesenchymal.stem cells;cell apoptosis detection showed good cell activity,that tree shrews and rhesus monkey BMSCs easily isolated and cultured in vitro and amplification.Tree shrews induced corneal endothelial cell culture medium,iris and corneal endothelial cells were co culture induced by BMSCs results to be corneal endothelial cells;co culture BMSCs crystal was confirmed as NSE positive staining,corneal endothelial cells. |
