Lin28a在糖尿病下肢血管病变球扩后再狭窄中的作用及分子机制研究

Peripheral vascular disease is a commom chronic complication of diabetes, which is characterized by distal, multiple obstructions with a higher percentage of occlusions with respect to stenoses. In our protocol for treatment of diabetic patients with TransAtlantic Inter-Society Consensus (TASC) criteria of lower extremity arterial disease (LEAD), percutaneous transluminal angioplasty (PTA) is the first choice revascularization procedure. Intimal hyperplasia leading to restenosis significantly limits the long-term success of PTA for LEAD. Our preliminary studies showed that compared with the hypocellular status in atherosclerotic plaque, restenotic lesions are hypercellular with foci of vascular smooth muscle cells (SMCs), and the increased activity of RNA binding protein Lin28a was closely related to the restenosis. Though Lin28 protein is emerging as important regulator in vascular systems, but the exactly mechanism in restenosis is remain unclear. It is reported that Lin28a，member of Lin28, acts as a suppressor of the let-7 microRNA biogenesis and enhance translation of certain mRNA, thereby promoting cell proliferation. Because Lin28a is itself let-7 target, this Lin28a/let-7 axis establishes a double-negative feedback loop in body development. Once Lin28a increased or let-7 decreased, the balance between them broke, following disregulation of downstream genes. Based on the analysis above, Lin28a/let-7 axis was speculated to play an important role in promoting the migration and proliferation of smooth muscle cell through regulating specific target genes in restenosis. So, we are going to clear Lin28a’s function and its molecular mechanism in inducing restenosis..The significance of the study is to put forward a new mechanism and potential target for the treatment of diabetic lower extremity arterial disease on the basis of previous discovery and reasonable evidences. This study will help us to get a better understanding of restenosis in LEAD.

球囊扩张术（PTA）是糖尿病下肢血管病变的重要治疗手段，而扩张部位再狭窄严重影响其疗效。与动脉粥样硬化显著不同，平滑肌细胞（SMCs）迁移增殖是再狭窄的主要发病机制。研究发现，Lin28a在促进细胞再生增殖中发挥重要作用。我们前期发现，与动脉粥样硬化比，Lin28a在再狭窄中呈差异性高表达，参与再狭窄发生，但机制不明。let-7是Lin28a的重要下游分子，可促进SMCs迁移增殖，且与Lin28a呈双重负反馈调节。由此提出假说：Lin28a在再狭窄斑块中高表达抑制let-7功能，同时let-7负反馈调节维持Lin28a的异常高表达，促进大量SMCs迁移增殖，致再狭窄迅速进展。本研究拟建立PTA术后再狭窄动物模型，运用生物信息学、双荧光素酶报告系统及基因转染等方法，聚焦“Lin28a-let-7双重负反馈通路” 促再狭窄，揭示Lin28a在再狭窄中作用路径，为再狭窄的治疗提供新思路、新靶点。

糖尿病下肢血管病变是糖尿病非外伤截肢的首要原因，球囊扩张术（PTA）是治疗该病的主要手段，然而扩张部位再狭窄严重影响其疗效。前期发现，与动脉粥样硬化显著不同，血管平滑肌细胞（VSMCs）过度迁移增殖是再狭窄发生的主要原因。通过构建再狭窄及动脉粥样硬化大鼠模型，取各自病变血管，联合蛋白芯片筛选及分子生物学验证发现，介导再狭窄与动脉粥样硬化VSMCs差异性的主要因子Lin28a，随后免疫组化及免疫荧光共定位实验显示，Lin28a在再狭窄斑块中VSMCs高表达，同时，体内外实验均证实：Lin28a显著促进VSMCs迁移增殖，介导再狭窄的发生。随后，对Lin28a的作用机制进行探究发现：再狭窄中高表达的Lin28a是通过抑制let-7特定亚型促VSMCs迁移增殖，同时明确了介导这一过程的主要let-7亚型：let-7c、let-7g和miR98。基于Lin28a-let-7双重负反馈环路，我们明确了let-7c、let-7g均可负反馈调节维持Lin28a的异常高表达，促进大量VSMCs迁移增殖，致再狭窄迅速进展。此外，并非所有的let-7亚型均受Lin28a的调控，let-7c和let-7g是参与该双重负反馈环路的主要亚型，而不受Lin28a调控的let-7b和let-7d却可拮抗Lin28a，在再狭窄中起一定的保护作用。同时，在再狭窄发生机制探究过程中，我们发现再狭窄中VSMCs虽大量迁移增殖，却非“恶性”失控状态，且没有表现出明显异型性，提示再狭窄中潜在自我保护机制，由此入手，结合高通量测序数据，找到拮抗Lin28a效应分子DEPP1，从另一角度探讨再狭窄病变的潜在“挽回效应”，从而为双管齐下干预再狭窄、恢复血管稳态提供思路。综上所述，本课题找到了介导再狭窄发生的特异性分子Lin28a，明确了Lin28a-let-7c/g双重负反馈环路介导VSMCs过度迁移增殖致再狭窄的重要机制。同时发现Lin28a上游阻遏因子let-7b/let-7d，在体内具有拮抗Lin28a的作用。此外，基于再狭窄中VSMCs增殖迁移可控性，我们转换思路发现DEPP1在再狭窄病变过程中潜在“挽回效应”。项目资助发表SCI论文6篇，中文期刊1篇，培养博士研究生1人，硕士研究生２人。项目投入经费51.0000万元，支出35.9757万元，剩余经费15.0243万元，剩余经费将用于本项目的后续研究支出