血管内皮细胞自噬的剪切应力调控及其在As中的作用研究

Atherosclerosis is a serious risk to human health. The dysfunction of vascular endothelial cells induced by shear stress is a key event of early atherosclerosis. However, the mechanisms are not fully understood. Our previous studies have shown that vascualr endothelial cells autophagy was upregulated by laminar shear stress, while downregulated by low shear stress and oscillatory shear stress. Therefore, we hypothesized that endothelial cells autophagy dysfunction induced by shear stress is the key step of endothelial cells dysfunction and early atherosclerosis. To test this hypothesis, parallel plate flow chamber system, electron microscopy，western blotting, quantitative reverse transcriptase PCR（qRT-PCR）, laser scanning confocal microscope and fluorescent tracer were used to detect the effect of shear stress on endothelial cells autophagy and autophagy intervention on endothelial cells functon under shear stress. Furthermore, based on Tet methylcytosine dioxygenase 2 (TET2), molecular mechanisms of endothelial cells autophagy regulated by shear stress were explored with in invivo and in vitro modle compling with bioinformatics, siRNA，chromatin immunoprecipitation and gene transfection. The accomplish of this study will further explore the mechnasim of atherosclerosis with a new perspective, which will provide new interventional strategies and targets for the prevention and treatment for atherosclerosis.

动脉粥样硬化（As）严重危害人类健康。剪切应力诱导的血管内皮细胞（VEC）损伤是As的早期核心事件，但其确切机制尚未阐明。我们前期研究发现：层流剪切应力上调VEC自噬，低剪切应力以及振荡剪切应力抑制VEC自噬，而自噬具有VEC保护功能。据此，我们推测"剪切应力诱导的VEC自噬不足是VEC功能失调和As发生发展的关键环节"。为证实该假说，本课题拟采用平行平板流动腔系统，结合电镜、免疫印迹、qRT-PCR、激光共聚焦以及荧光示踪等实验技术，探讨剪切应力对VEC自噬的调节以及自噬干预对剪切应力环境中VEC功能的影响；再以DNA甲基化修饰酶TET2为切入点，采用体外培养系统以及整体动物模型，结合生物信息学、siRNA、染色体共沉淀、基因转染等方法，探讨剪切应力调节VEC自噬的分子机制。本研究的完成将从新的视角阐明As的发病机制，为As的有效防治提供新策略和新靶点。

血管内皮细胞（ECs）直接暴露于血流剪切应力环境，其生物学行为受到血流剪切应力调控。剪切应力诱导的ECs功能障碍是动脉粥样硬化（As）病变的重要环节。自噬是一个进化上高度保守的生物学过程，参与细胞组分的更新及组织的重塑。在本项目中我们研究了剪切应力对ECs自噬的影响及其在As中的作用，发现血管细胞自噬障碍贯穿于As病变过程，并且和病变程度正相关；As病变中DNA羟甲基转移酶TET2水平降低，TET2水平与As病变进程负相关。低剪切应力诱导ECs自噬障碍、抑制TET2表达, TET2过表达改善低剪切应力下ECs自噬及功能障碍并抑制高脂饮食apoE-/-小鼠As病变。对其作用机制的研究表明，TET2通过上调自噬相关基因Beclin1启动子的去甲基化修饰，上调ECs的自噬及自噬流，抑制炎症因子的表达、保护血管内皮细胞功能。α-酮戊二酸通过上调TET2的表达改善血管细胞自噬及自噬流、抑制高脂饮食apoE-/-小鼠As病变。以上结果表明低剪切应力诱导ECs自噬障碍是As病变的生物力学机制之一，TET2可能为防治As新的干预靶点。