| Summary: | 碩士 === 國立成功大學 === 生物化學暨分子生物學研究所 === 103 === Autophagy is a survival mechanism against stress. It involves the degradation of cellular components to generate energy through the lysosome. However, autophagy overactivation under continuous stress leads to cell apoptosis. Recombinant thrombomodulin protects vascular endothelial cells and reduces the risk of atherosclerosis. Treatment with recombinant thrombomodulin increases endothelial cells Akt activation which also regulates autophagy initiation. We hypothesized that recombinant thrombomodulin might influence vascular endothelial cell behaviors through regulating autophagy. First, we observed the expression of autophagy related proteins in endothelial cells under starvation with and without recombinant thrombomodulin treatment. In starvation, we found that autophagy initiation signal was induced, and LC3-II and Atg5 expression contributing to autophagosomal formation increased. After treating recombinant thrombomodulin, Akt and mTOR was activated and autophagy-related proteins expression were inhibited. When treating Akt inhibitor, recombinant thrombomodulin effect on mTOR activation was abolished. Next, we observed the autophagosome by MDC and LC3 immunofluorescence staining. After treating recombinant thrombomodulin, the number of autophagosome puncta was significantly decreased than serum starvation group without recombinant thrombomodulin treatment. Promoting autophagy with autophagy activator could inhibit recombinant thrombomodulin-induced cell proliferation. Recombinant thrombomodulin could not reduce cell apoptosis when treating autophagy activator at the same time. Recombinant thrombomodulin could not regulate endothelial cells proliferation and apoptosis in endothelial cells with Atg5-knockdown. In carotid ligation model, we found neointima was induced and endothelial autophagy expression increased after carotid ligation. Recombinant thrombomodulin treatment suppressed endothelial autophagy expression after carotid ligation. For future study, recombinant thrombomodulin effect will be studied to see its influence on endothelial autophagy and atherosclerosis in apoE deficiency mice.
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