| Objective:Maxillofacial muscle plays an important role in the deformit) of the occurrence, development and correction and curative effect of maintaining aspects. Skeletal muscle can follow the plasticity of the strong outside stimulation change happens by adaptive reconstruction. It is the basis that orthodonlic doctors through the orthopedic force to regulate reconstruction.In this process, myoblasi are the recepiors of stress-stimulating and the effectors of stress stimulation of skeletal muscle reconstruction.lt is very necessary that we deeply research into the mechanics of muscle cells in the process of signal transduction mechanism for clarifying functional orthopedic skeletal muscle remodeling mechanisms.Therefore, in thi.-research, successfully constructs myoblasts cells in vitro model of mechanical stimulation, to study on the effects of different magnitudes of cyclic stretch on apoptosis levels in myoblasts and to investigate the possible mechanisms involved. And the role of caspase-3in the cyclic stretch-induced apoptosis of myoblasts. These results suggest the existence of a novel mechanism for the regulation of myoblast apoptosis by cyclic stretch.This will helps clinical doctor used different of method and means induced or regulation muscle conversion, providing many theoretical guidance.Methods:In this study, cyclic stretch was applied on the fibrobtasts lor1,6,12and24hours,.myoblasts were subjected to15%surface elongation; with frequency of10cycles per minute, each cycleincluding the3-s stretch and3-s relaxation.. Immediately after the cyclic stretch, We used transmission electron microscopy (TPM) and Hoechst33258staining to observe the morphology of the apoptotic cells and we used flow cytometry to count the number of the apoptotic cells in each group,and the caspase-3activity was detected by Caspase-3Activity Assay Kit. We investigated the role of caspase-3in cyclic stretch-induced apoptosis in myoblasts.We directly measured the activity of caspase-3by observing changes of the35-kDa procaspase-3and the17-kDa cleaved caspase-3, and we indirectly measured the activity of caspase-3by observing changes in PARP levels. The expression levels of PARP mRNA and PARP protein were detected using RT-PCR and Western blot. And then,we used20uM of the caspase-3-specific inhibitor, z-DEVD-fmk to determine the of caspase-3in cyclic stretch-induced apoptosis in myoblasts.To assess the difference between each groups, we used statistical analysis software (SPSS18.0).Results:1. TEM, Hoechst33-58staining and Flow cytometry results showed that the test imposed by15%.10cycles/min cyclical tensile stress can induce apoptosis of myoblasts, and with cyclic stretch, the apoptosis of fibroblasl cells was increased, to24hours reached the peak. 2. Caspase-3Activity Assay results showed that the activity of the Caspase-3was rising with the extension of time,RT-PCR results showed, with cyclic stretch, the expression levels of PARP mRNA is rising, there is significant statistical difference (P<0.05).3. With cyclic stretch, quantitative analysis of the expressions levels of procaspase-3and PARP are reducing, the expressions levels of cleaved caspase-3and PARP is rising, there is significant statistical difference (P<0.05).4. When cells were incubated with z-DEVD-fmk, the numbei of apoptotic cells, the activity of the Caspase-3, the expressions levels of cleaved caspase-3and PARP was significantly lower than untreated stretched cells, the expressions levels of procaspase-3and PARP was higher than untreated stretched cells, there was significant statistical difference (P<0.05).Conclusions:Taken together, these results showed that cyclic stretch can induce apoptosis of myoblasts in amagnitude-dependent manner and this process was mediated by caspase-3. Caspase-3/PARP apoptotic signalling pathways played an important role in cyclic stretch-induced apoptosis in myoblasts, and the z-DEVD-fmk could completely inhibit caspase-3activity and apoptosis caused by activation of caspase-3/PARP signalling pathways. |
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