Effect And Mechanism Study Of Hypoxia On The Differentiation Of Precartilaginous Stem Cells

Part I Isolation, culture and identification of precartilaginous stem cellsObjective: To isolate and purify precartilaginous stem cells from growth plate of SD rats.Methods: 1. Growth plate chondrocytes were isolated from the femural and tibial growth plate of rats. Precartilaginous stem cells were purified with immunomagnetic separation system. FGFR-3 phenotype was identified by immunofluorescence. 2. Precartilaginous stem cells were stained with alcian blue.Results: 1. Precartilaginous stem cells were successfully isolated and purified, most of which were spindle or triangular like and were FGFR-3 positive. 2. The micropellet of precartilaginous stem cells were positive to alcian blue.Conclusion: precartilaginous stem cells can be successfully isolated and purified with immunomagnetic separation system.Part II The effect of physical and chemical hypoxia on the differentiation of precartilaginous stem cellsObjective: To study hypoxia on the differentiation of precartilaginous stem cells. Methods:1. Precartilaginous stem cells were cultured with 1% oxygen in different time, and then m RNA expression of HIF-1α, SOX9, COL-II, AGG, and protein expression of HIF-1α, SOX9, COL-II, AGG, ALP were detected using RT-PCR and western blot methods. 2. Precartilaginous stem cells were cultured with 1% oxygen for 4 hours and then cultured with 21% oxygen for 1 hours,after that the protein expression of HIF-1α, SOX9, COL-II, AGG, ALP were detected. 3. Precartilaginous stem cells were cultured with different concentrations of cobalt chloride, and then m RNA expression of HIF-1α, SOX9, COL-II, AGG, and protein expression of HIF-1α, SOX9, COL-II, AGG, ALP were detected.Results: 1. After Precartilaginous stem cells have been cultured with 1% oxygen a period of time, the m RNA expression of HIF-1α, SOX9, COL-II, AGG increase, and the protein expression of HIF-1α, SOX9, COL-II, AGG also increase. But the protein expression of ALP has not significant change. 2. High concentration of oxygen downregulated the protein expression of HIF-1α, SOX9, COL-II, AGG which were upregulated by hypoxia effect.But the protein expression of ALP has not significantly changed. 3. After precartilaginous stem cells have been cultured with certain concentration of cobalt chloride, m RNA expression of HIF-1α, SOX9, COL-II, AGG increased, and protein expression of HIF-1α, SOX9, COL-II, AGG also increase. But the protein expression of ALP has not significantly changed.Conclusion: Hypoxia can promote chondrogenic differentiation of precartilaginous stem cells.Part III Physical and chemical hypoxia on the expression of YAP in precartilaginous stem cellsObjective: To study the effect of hypoxia in regulating the YAP expression in precartilaginous stem cells.Methods: 1. Precartilaginous stem cells were cultured with 1% oxygen with different time, and then m RNA and protein expression of YAP were detected using RT-PCR and western blot methods. 2. Precartilaginous stem cells were cultured with 1% oxygen for 4 hours and then cultured with 21% oxygen for 1 hour, after that m RNA and protein expression of YAP were detected. 3. Precartilaginous stem cells were cultured with different concentrations of cobalt chloride, and then m RNA and protein expression of YAP were detected.Results: 1. After Precartilaginous stem cells have been cultured with 1% oxygen a period of time, the m RNA and the protein expression of YAP increase. 2. High concentration of oxygen downregulated the expression level of YAP m RNA and protein which were upregulated by hypoxia. 3. After precartilaginous stem cells have been cultured with certain concentration of cobalt chloride, the m RNA and protein expression of YAP increase.Conclusion: hypoxia promoted the expression of YAP in precartilaginous stem cells.