| Objective:The reconstruction of maxillofacial muscle plays an very important role in the developing, treating and retention of dentofacial deformity. Myoblast is one of the most important part of the maxillofacial muscle, and the main embodiment of adaptive reconstruction. Previous study is focused on the proliferation and differentiation of cell, but the study of apoptosis is neglected. As apoptosis plays a role as important as the proliferation and differentiation, we study on the apoptosis levels in myoblasts which is imposed cyclic stretch in vitro. So we can investigate the role of CaN-NFAT pathway in the apoptosis of myoblasts induced by cyclic stretch and the possible mechanism involved. Then we can clarify the possible molecular regulation mechanism of the adaptive reconstruction and provide the theoretical support for the functional orthopedic therapy.Methods:Rats L6myoblasts were cultured in vitro and constructed mechanical stimulation model successfully. The myoblasts were imposed tension for0,1,2,6,12and24hours respectively by multi-channel cells stress loading system. The force value was15%cell deformation and the frequency was10cycles/min. Each cycle was consisted of stretch for3seconds and relaxation for3seconds, and the0h group was the control group. The morphological changes and growth of the muscle cells were observed under the inverted phase contrast microscope; the apoptotic myoblasts were dyed by Hoechst33258and observed through the fluorescence microscopy; the apoptosis rate was detected by flow cytometry analysis; the mRNA and protein levels of calcineurin (CaN) and NFAT in different groups were detected by real-time PCR and Western blot; the CaN inhibitor, cyclosporine (CsA), was used to clear the role of CaN in apoptosis induced by cyclic stress and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic stress in further. SPSS17.0statistical software was used to analyse the data statistically.Results:1. The Rat L6myoblasts could maintain a good condition, no degeneration, and extremely low loss rate after imposed by cyclic stress of15%cell deformation and10cycles/min, which can demonstrate the vitro-mechanical stimulation model was constructed successfully.2. The arrangement of the myoblasts converted from the original haphazard arrangement to comply with the direction of stress after continuous loading, and this tendency becomes more pronounced after continuous loading, which can imply the alignment direction of myoblasts was affected by duration of stress.3. The results of Hoechst33258staining and flow cytometry analysis showed that cyclic stress could induce apoptosis of the Rat L6myoblasts. The apoptosis rate increased with the extension of loading time, and reached the peak when loaded24hours.4. The results of Real time-PCR showed the mRNA of calcineurin (CaN) and NFAT were increased with the extension loading time. The results of Western blot showed the protein level of NFAT was also increased with the extension of loading time, which is consistent with the results of PCR. So the CaN-NFAT signaling pathway may be involved in signal transduction of the L6rat myoblasts apoptosis imposed by mechanical cyclic tensile stress.5. When the myoblasts were incubated with cyclosporine (CsA, the inhibitor of CaN) and loaded24hours, the apoptosis rate decreased, and the expression of CaN and NFAT decreased too. And the difference had significant statistical meaning (P<0.01).Conclusions:1. The cyclic stress of15%cell deformation and10cycles/min could induce the apoptosis of the Rat L6myoblasts.2. CaN-NFAT signaling pathway may be involved in the myoblasts apoptosis induced by cyclic stretch.
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