microRNA-29b对早期AS动脉内皮功能的影响及其调控机制

The vascular endothelium is a semipermeable barrier between blood and vascular tissue. A major function of the endothelial cell is to serve as a barrier to fluid and solute flux across the blood vessel wall. Arterial endothelial dysfunction is the first step for Atherosclerosis (AS), which showed that endothelial dependent diastolic function (EDD) decreased. our previous researchs demonstrated that myosin light chain kinase (MLCK) expression and activity increased significantly in AS. The expression and activity of MLCK can cause arterial muscle contraction, and increase along with the cholesterol diet feeding time increase. MLCK inhibitor ML7 can significantly increase the vascular EDD in AS. It is suggested that the decrease of EDD maybe related with increase of MLCK expression, activity and enhance of systolic function of arterial wall in AS. Recent studies demonstrate that miRNA-29b expression is up-regulated in the development and formation of atherosclerosis. Further target gene prediction also indicates that miRNA-1 maybe targeted negative regulator of Mitogen-activated protein kinase (MAPK) signal pathway, which is an endothelial cell specific mitogen and chemotactic agent. Thus, these promising data strongly suggest that the effects of miRNA-29b on EDD of atherosclerosis maybe mediate through MAPK. this project aims to This project aims to reproduce high cholesterol and AS rabbit model, to analyze the changes of EDD using high frequency ultrasound; to detect the endothelial function using artery ring analysis and to observe the expression of diastolic function related protein in arterial endothelial cells cultured with miRNA-29b, finally to elucidate the effect of miRNA-29b on EDD wehether through the expression and activity of rabbit arterial wall MLCK and its regulation mechanism. This project will provide solid evidence and basis for development new drug target for atherosclerosis therapy and prevention.

动脉内皮功能受损是动脉粥样硬化(AS)的启动环节，表现为动脉内皮依赖性舒张功能（EDD）下降。前期研究发现早期AS形成过程中肌球蛋白轻链激酶（MLCK）表达和活性出现明显升高，并伴随时间的增加而增强，MLCK 抑制剂ML7 明显增加AS 血管EDD。提示早期AS 时，EDD 降低可能与动脉壁MLCK 表达和活性增加有关。近期发现miRNA-29b在AS过程中异常高表达，靶标预测MAPK通路负性调节因子是其潜在靶标，提示其可能通过激活MAPK通路影响血管EDD，进而调控AS进程。本项目拟复制高胆固醇血症和AS模型，高频超声检测EDD变化；动脉环离体实验检测内皮舒张功能；并观察miRNA-29b对动脉内皮细胞舒张功能相关蛋白表达等影响，探讨AS 发生发展过程中miRNA-29b 是否通过MLCK 的表达和活性变化对EDD 产生影响及相关调控机制，为寻找新的抗AS药物靶点及AS预防提供实验依据。

动脉内皮功能受损是动脉粥样硬化(AS)的启动环节，表现为动脉内皮依赖性舒张功能 （EDD）下降。前期研究发现早期AS形成过程中肌球蛋白轻链激酶（MLCK）表达和活性出 现明显升高，并伴随时间的增加而增强，MLCK 抑制剂ML7 明显增加AS 血管EDD。提示早期AS 时，EDD 降低可能与动脉壁MLCK 表达和活性增加有关。近期发现miRNA-29b在AS过程中异常高表达，靶标预测MAPK通路负性调节因子是其潜在靶标，提示其可能通过激活MAPK通路影响血管EDD，进而调控AS进程。本项目复制高胆固醇血症和AS模型，高频超声检测EDD变化；动脉环离体实验检测内皮舒张功能，用Real-time PCR、Western blot和免疫组织化学从mRNA和蛋白质两个角度分析了实验性MAPK信号转导途径相关蛋白（p38、ERK、JNK）及其核内转录因子等相关蛋白的变化；分析影响NO合酶、MLCK转录、表达的因素，在整体水平分析实验性高胆固醇血症和早期AS动脉壁NO合酶、MLCK转录、表达增加与哪些信号转导通路相关。测定实验性早期AS apoE-/-小鼠动脉壁NO合酶活性、NO含量、Ca2＋浓度、MLCK活性、MLC磷酸酶活性、MLC磷酸化水平，综合分析MAPK信号转导途径相关蛋白磷酸化水平（pp38、pERK、pJNK）与MLCK活性、MLC磷酸化之间的关系；并观察miRNA-29b对动脉内皮细胞舒张功能相关蛋白表达等影响，探讨AS 发生发展过程中miRNA-29b 是否通过MLCK 的表达和活性变化对EDD 产生影响及相关调控机制，为寻找新的抗AS药物靶点及AS预防提供实验依据.