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Objective: To investigate the velvet antler polypeptide anabolic function of rabbit articular chondrocytes cultured in vitro in the normal state ; explore normal in vitro culture of rabbit articular chondrocytes apoptosis and antler polypeptides impact ; explore nitric oxide - induced in vitro cultured rabbit articular cartilage apoptosis and antler polypeptides its impact . Methods : 1 , rabbit articular chondrocytes in vitro culture system , and toluidine blue staining cartilage cells were identified by the establishment of chondrocytes in vitro culture system : under sterile conditions, cut rabbit bilateral hip , knee cartilage and cut it into pieces , using enzymatic digestion The cell suspension was collected in test tubes , 1000 rpm , 10min after centrifugation supernatant was added containing 10% fetal bovine serum of DMEM to 2 × 106/ml inoculated in culture flasks , placed in 37oC, 5% CO2 incubator for primary culture, and toluidine blue staining for identification of cartilage cells . The primary cells covered subculture . The chondrocytes used in the experiments for the first 1 to 2 substituting passaged cells . 2, sodium nitroprusside adding cartilage cell culture system , the NO-induced chondrocyte apoptosis model , using flow cytometry (Annexin V / PI dual parameter method ) and fluorescent staining ( acridine orange staining ) chondrocytes withered death for quantitative, qualitative detection . First , the establishment of rabbit articular chondrocytes apoptosis model : in chondrocyte culture system 3mmol / L SNP (NO donor) induced rabbit articular chondrocytes apoptosis rate reached a peak of about 20h chondrocyte apoptosis . Were divided into four groups: the velvet antler polypeptide of the normal group , the normal group , the apoptosis group , apoptosis group antler polypeptides , each of six wells . Flow cytometry and acridine orange staining : digested with 0.25% trypsin , collected from each group chondrocytes , washed 2 times with PBS , 1ml of cells , the cells was adjusted to 5 × 105 / ml of each , and the cells were resuspended 200μl Binding Buffer, adding 10μl Annexin-V-FITC 5μl PI, mix gently , and the dark at room temperature for 15min or of 4oC reaction 30min . Added the 300μl BindingBuffer, on the machine detected within 1h . In addition , the cells in each group were suspended in 50μl PBS , adding 100μl acridine orange staining solution , added to the cells staining after 30min fixative home ice was observed under an inverted fluorescence microscope . 3 , using the MTT assay chondrocyte proliferation .
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