金属蛋白酶ADAMTS13表达调控的机制研究

Coronary artery disease (CAD) is the major threat to the healthy life. The thrombi formed inside the vessels block the coronary arteries, which leads to myocardial infarction. Previous studies have shown that the metalloprotease ADAMTS13 specifically cleaves VWF to regulate the thrombus formation. The reduction of ADAMTS13 has been associated with the initiation and progress of CAD. Hepatic stellate cells and endothelial cells are the major source for ADAMTS13 in plasma, but the regulation of ADAMTS13 expression is still unknown. According to database, miR882 and miR185, which are predicted to bind the 3'UTR of ADAMTS13, might be two major miRNAs regulating ADAMTS13 transcription. In line with the prediction, we observed increased miR882 and miR185 levels in hepatic stellate cells of mice with liver cirrhosis, while ADAMTS13 expression in the livers was decreased. Exosomes, small vesicles containing nucleic acid and protein, are important in intercellular communication. Our preliminary data has also shown that the changes of ADAMTS13 mRNA, miR882 and miR885 in exosomes isolated from circulation were consistent with that in cirrhotic livers, indicating that the information carried by exosomes reveals the liver conditions. In the present study, we will further determine the regulatory mechanism of miRNAs on ADAMTS13 transcription. Furthermore, we will elucidate the intercellular communication by exosomes between hepatic stellate cells, as well as hepatic stellate cells and vascular endothelial cells. Lastly, we will investigate whether the infusion of exosomes containing miRNAs and ADAMTS13 protein will alter ADAMTS13 expression and play a role in myocardial ischemia/reperfusion injury in mice.

冠状动脉心脏病（冠心病）是威胁人类生命健康的重要疾病。血栓形成造成冠状动脉的堵塞是导致心肌梗死的直接原因。研究表明，金属蛋白酶ADAMTS13可以通过剪切VWF蛋白调节血栓形成，其功能降低与冠心病密切相关。ADAMTS13主要合成于肝星状细胞和血管内皮细胞，但调节机制尚不清楚。本课题组利用小鼠肝纤维化模型，证明肝组织中miR882和miR185升高,而ADAMTS13 mRNA与蛋白含量相应降低。因可在细胞间传递生物学物质，外泌小体正成为研究热点。本课题组发现外周血的外泌小体中，ADAMTS13 mRNA及相关miRNA的变化与肝组织中一致，提示外泌小体中的生物学信息可反映肝脏的功能状态。本申请将阐明与miRNA对ADAMTS13转录的调控机制，揭示肝星状细胞间及其与血管内皮细胞间外泌小体介导的信号传递，并研究外泌小体进行机体内物质转运来调节ADAMTS13的表达以及心肌保护的可行性。

冠状动脉心脏病（冠心病）是威胁人类生命健康的重要疾病。血栓形成造成冠状动脉的堵塞是导致心肌梗死的直接原因。研究表明，金属蛋白酶ADAMTS13可以通过剪切VWF蛋白调节血栓形成，其功能降低与冠心病密切相关。ADAMTS13主要合成于肝星状细胞和血管内皮细胞，但调节机制尚不清楚。最近研究结果显示microRNAs (miRNAs)可调控ADAMTS家族蛋白转录。因可在细胞间传递核酸，蛋白等物质调控细胞间交流，外泌体已成为研究的热点。为了进一步解释ADAMTS13产生的内在机制，本课题组的研究聚焦于通过外泌体传递miRNAs调控ADAMTS13这一方向。本课题组利用小鼠肝纤维化模型，观察到ADAMTS13 mRNA与蛋白在肝纤维化发展过程中含量降低。随后，miR-185-5p被预测可结合到ADAMTS13的3′非翻译区(3'-UTR)调控其转录，并且进一步证实miR-185-5p与ADAMTS13的下调相关。本课题组结果显示进展期纤维化可导致ADAMTS13 mRNA下调，而miR-185-5p上调，这显示两者存在相互关联。使用mimic转染实验也证明miR-185-5p可以调控ADAMTS13转录。本课题组发现外周血的外泌小体中，ADAMTS13 mRNA及相关miRNA的变化与肝组织中一致，提示外泌小体中的生物学信息可反映肝脏的功能状态。将转染了miR-185-5p mimic的外泌体传递到肝星状细胞系HSC-T6中可导致ADAMTS13的下调。本申请证明了miRNA对ADAMTS13转录的调控机制，揭示了肝星状细胞间外泌体介导的信号传递。综上，本研究证实通过外泌体传递miR-185-5p也许是ADAMTS13转录调控的一种策略。ADAMTS13调控机制的研究对冠心病预防与治疗研究有一定提示作用。