The Role Of P53 In Cyclical Stretch Induced Apoptosis Of Myoblasts And Its Regulating Mechanism

Objective: In this research,using an in vitro mechanical stimulation model,we apply appropriate cyclic stretch to the cells to investigate the effect of stress on the growth of myoblasts and to explore the role of p53 in in myoblast apoptosis and its mechanism.In order to elucidate the micro regulation mechanism of maxillofacial skeletal muscles reconstruction and provide theoretical guidance for functional orthopedic treatment.Method: L6 rat myoblasts,as the research objects,were subjected to mechanical stimulation using an established in vitro model.The experiment included control group and tension-force group,cyclic strain stress of 2h,6h,12 h and 24 h were applied to the tension-force group cells at a 10% loading frequency and 15% cell elongation deformation,control groups were cultured in the same incubator with non-stretch.The in vitro growth status of myoblasts and its changes under stress were observed by inverted phase contrast microscope.Apoptosis morphology changes were observed by DAPI staining.Western Blot technique was used to detect the expression of p53 and Cyt-c from the level of protein.The activity of Caspase-9 was detected by Caspase-9 activity assay.Use p53 inhibitors to further observe the effect of p53 in apoptosis.In addition,through the separation of mitochondria,the p53 protein expression inside and out side mitochondria were detected to in-depth study the regulation mechanism of p53 involved in the apoptosis.SPSS17.0 statistical software was used to analyze the data of the results.Result:1.Myoblasts showed good proliferative ability in suitable environment in vitro observed by inverted phase contrast microscope.2.After applying tensile stress of 10% loading frequency and 15% cell elongation deformation to cells,we observed that the arrangement of myoblasts could be adjusted with the stress stimulus.3.DAPI staining results showed that cyclical tensile stress can successfully induce myoblasts apoptosis,and the apoptosis rate was increased with the extension of loading time.4.The results of Western Blot showed that the protein expression of p53 and Cyt-c increased with time extension,and after the addition of p53 inhibitors,the expression of p53 and and Cyt-c decreased but it was still higher than control group.The difference was statistically significant(p <0.05).5.The Caspase-9 activity assay kit detected that the activity of Caspase-9 was enhancedwith time extension,p53 inhibitors decreased the activity of Caspase-9 to some extent,but it is still higher than the control group,the difference was statistically significant(p<0.05).6.After extracting the cell mitochondria,the Western Blot results showed that the expression of mitochondrial p53 was increased with time increasing,the difference was statistically significant(p<0.05),while the p53 expression outside of mitochondria was with no statistically significant.Conclusion:1.The cyclic-stretch of 10 cycles/min and 15% cell elongation deformation could induce the myoblast apoptosis.2.p53 was involved in the apoptosis induced by cyclic-stretch.3.The role of p53 may be associated with mitochondria.