抵抗素样分子β在2型糖尿病大血管病变发病机制中的作用

Atherosclerosis(AS),which was the macrovascular complication of diabetes(DM), was the leading cause of death in type 2 DM patients. Resistin like molecule β(RELMβ)had been observed to be expressed in the atherosclerotic plaques of type 2 diabetic patients. TRAILR2, which was encoded by TNFRSF10B, had been screened out from G protein coupled receptors by Homogeneous Time Resolved Fluorescence(HTRF) for its interaction with RELMβ. Plasma RELMβ had been proved to be affected by glucose and lipid metabolism. TRAILR2 was expressed in vascular smooth muscle cells(VSMCs), which had been reported in some articles. Therefore, we hypothesize that RELMβ might be produced in the artery walls by the stimulations of hyperglycemia and hyperlipidemia. It would result in VSMCs dysfunction and vascular homeostasis damage.We are going to reveal the RELMβ mediated pathophysiological effects in the local lesions by using RELMβ overexpressed and silenced AS animal models, to explore the mechanism of RELMβ-induced vascular homeostasis damage by using recombinant RELMβ stimulation and lentivirus mediated RELMβ overexpressed or knockdowned VSMCs, to identify the specific spot or domain where RELMβ and TRAILR2 interact and to screen the downstream molecules. We wish to improve the prevention and treatment of diabetic macrovascular disorders by providing some new targets.

动脉粥样硬化(AS)是DM的主要致死原因。项目组前期观察到抵抗素样分子β(RELMβ)在DM患者斑块中表达，并应用均相时间分辨荧光技术在GPCR全基因表达库中筛选到TNFRSF10B编码的TRAILR2与RELMβ存在相互作用，且发现社区人群血浆RELMβ受糖脂影响。而文献报道，TRAILR2在粥样斑块的血管平滑肌细胞(VSMCs)表达。故设想高糖高脂刺激动脉壁产生RELMβ，经TRAILR2致VSMCs功能紊乱及血管稳态受损。本研究拟通过RELMβ沉默和过表达AS动物模型及人体标本研究RELMβ在病灶的表达和作用；通过重组RELMβ刺激、慢病毒介导RNAi或过表达RELMβ研究其对VSMCs的影响；通过免疫共沉淀、特异突变、蛋白芯片等技术鉴定RELMβ与TRAILR2的相互作用位点及下游信号分子；最后检测2型DM患者血浆RELMβ水平与AS的关系；期望能为糖尿病大血管病变防治提供新靶点。

为研究高糖环境中RELMβ对人主动脉平滑肌细胞表型转换、迁移功能等的影响及其参与动脉粥样硬化的机制，本研究选取原代人主动脉平滑肌细胞分为对照组、高糖组、RELMβ组及高糖＋RELMβ组；Transwell及划痕实验检测RELMβ对平滑肌细胞迁移功能的影响，cck8检测细胞增殖变化，油红O检测细胞摄脂功能改变；检测细胞表型标志物、细胞周期蛋白、迁移分子、清道夫受体、炎症因子及信号通路关键分子的表达变化。结果提示高糖和RELMβ均可以促进HASMC的表型转换，与对照组相比，OPN及CD31在高糖+RELMβ组中表达最多，高糖组及RELMβ组次之，而SM-α表达变化趋势则与OPN及CD31相反；高糖或RELMβ单独干预HASMC或者是两者协同干预，均可以促进细胞迁移，且基质金属蛋白酶MMP-2、MMP-9表达增高，其中高糖+RELMβ的促进作用较其他两组显著增高；炎症因子IL-1β、TNF-α及TGF-β的表达在仅RELMβ干预时无明显变化，而加入高糖或高糖+RELMβ后表达均上调，高糖+RELMβ更明显；高糖环境下加入RELMβ后细胞摄脂的能力显著提高。清道夫受体CD36、LOX-1的表达也呈增高趋势；高糖和RELMβ单独干预或协同作用于HASMC时均促进细胞增殖。高糖环境下细胞周期蛋白CDK2、cyclin D1、CDK4、cyclin E在HASMC中的表达较对照组显著上调，加入RELMβ后表达增高更明显；高糖干预下MAPK 通路的关键分子p38、ERK磷酸化表达较正常组增高。高糖基础上再加RELMβ时增高更明显。在高糖+RELMβ组中，使用p38抑制剂RWJ64809和ERK抑制剂PD98059干预HASMC后，细胞数量明显减少，增殖作用也被抑制。提示在高糖环境中RELMβ可促进HASMC的增殖、迁移、表型转换及摄脂功能及炎症反应的发生，且可能通过MAPK调控。