Research Of In Ischemia And Hypoxia Model Rabbit Bone Marrow Mesenchymal Stem Cells Apoptosis Protein Change

Mesenchymal stem cells (MSCs) are adult stem cells with multipotentdifferentiation potential, which has many characteristics,suchas immunemodulation, easy expansion in vitro culture and the evading of surveillance ofthe host immune system. It has extensive application prospect on cellular tissueengineering science. But the tissue reconstruction is limitd by the apoptosis andinactivation of transplant cellular. And one keydeath mechanism of stem cellstransplanted into the organ is due to oxygen deficit and ischemia.CASPASE is one kind of protease with similar structure existing in cytoplasm,which can selectively incise some protein and cause apoptosis consequently. Itis proved that partial anoxia and ischemia are the important adjusting elementsof apoptosis, which can induce active change of cell Caspase-3、8、9to adjustapoptosis.The apoptosis effect of oxygen deficit and ischemia has been researched athome and abroad. This project bases on the other researches on experiments invitro and makes use of the technologies of cellar and Modern MolecularBiology to detect the biological impact on rabbit mesenchymal stem cellsinin-vitro environment through the model of oxygen deficit and ischemia constructed by in-vitro serum-free and anaerobic culture. And then through thetreatment of naringin, the change of apoptin of rabbit mesenchymal st-em cellsaffected by naringin is detected. Through the respective testing on theexpressionof apoptosis-related proteinwith manymethods,includingWestern-blot,the project makes apreliminary study on the protectionmechanismof naringin on rabbit mesenchymal stem cells under the oxygen deficit model.Part I Culture, identification and biological character analysis of rabbitBMSCsOBJECTIVE: To establish a method of isolation, cultivation and purification ofrabbit BMSCs, to observe cell morphology and assess surface markers andmulti-directional differentiation capacity.METHODS: rabbit BMSCs were isolated, cultured and purified bythe wholebone marrow adherence method for morphology observations, drawing thegrowth curve, detecting cell surface marker with FCM and the assessing ofosteogenic induced differentiation.RESULTS: BMSCs from rabbits were spindle cell-based, showing radial colonyarrangement. Cells kept strong growth and could passage incontinuous and stable manner. The growth curve demonstrated that BMSCswere consistent with the growth characteristics and good activity of normal cells.At the third passage, BMSCs were negative for CD45；but positive for CD44（97.6士1.81%）, CD29（98.42士1.01%）. Following induction, alkalinephosphatase staining and alizarin red staining produced a positve reaction incells. Whole bone marrow adherence method is simple and can isolate, purify and amplify BMSCs in vitro.The obtained cells have general biologicalcharacteristics of mesenchymal stem cells, and also have potentiality ofmulti-directional differentiation after induction culture.CONCLUSION：Whole bone marrow adherence method in experiment hasimportant practicalsignificance to provide adequate source of seed cells fortissueengineering. Part II The induction of apoptosis of BMSCs under the model of ischemiaand anoxia.OBJECTIVE: To build in-vitro model of ischemia and anoxia and to induce theapoptosis of BMSCs.METHODS: Cell anoxia model(serum starvation method+anoxia cultivationmethod) is built. Cell apoptosis condition and expression of related protein ofcell Casapse3.RESULTS: The in-vitro ischemia and anoxia model can cause up-regulation ofthe expression of related protein of cell Casapse3.Conclusion: The in-vitro ischemia and anoxia model can cause the rising ofapoptosis rate of BMSCs,which has relevance with time.