細胞自噬是一種用來對抗逆境的生存機制，主要是透過溶酶體分解掉細胞一些物質來獲得能量。然而，長期的逆境引發過度細胞自噬會導致細胞走向死亡階段。重組凝血酶調節素能夠保護血管內皮細胞並且降低罹患動脈粥狀硬化的風險。給予重組凝血酶調節素處理能夠活化內皮細胞Akt的表現，而Akt也能夠去調控細胞自噬的起始因子，因而我們推測重組凝血酶調節素是否是透過調控細胞自噬來影響內皮細胞的上述行為。首先我們將內皮細胞經過飢餓處理，並且觀察再有或者沒有重組凝血酶調節素處理狀態下的細胞自噬相關路徑蛋白的表現差異。當細胞經過飢餓處理，細胞自噬起始因子被誘導，並且相關蛋白如LC3或Atg5也被誘導活化，當給予重組凝血酶調節素處理，Akt和mTOR會活化並且抑制細胞自噬相關蛋白，我們也利用給予Akt的抑制物來確認重組凝血酶調節素是透過活化Akt來活化mTOR表現。接著我們利用MDC和LC3免疫螢光染色來觀察autophagosome數量差異，將細胞經過飢餓處理，有處理重組凝血酶調節素的組別所染到的autophagosome數量明顯比沒有經過重組凝血酶調節素的組別來的少。此外我們也觀察到當給予內皮細胞自噬的活化物能夠抑制重組凝血酶調節素所誘導的細胞增生，並且也抑制重組凝血酶調節素保護內皮細胞免於細胞凋亡的現象。當內皮細胞經過Atg5 knockdown處理讓細胞自噬表現恆定很少，此時給予重組凝血酶調節素無法去調控內皮細胞的增生與凋亡。在動物實驗當中，我們觀察到將小鼠給予頸動脈結紮手術能夠誘導小鼠血管內皮細胞的細胞自噬相關蛋白表現，而給予重組凝血酶調節素治療則能夠降低小鼠血管內皮細胞的細胞自噬相關蛋白表現。在未來我們會再利用apoE缺陷小鼠來觀察重組凝血酶調節素對於小鼠內皮細胞的細胞自噬與動脈粥狀硬化的影響。

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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