基于NGS和AAV基因技术的艾灸对APOE-/-小鼠血小板活化的特异性作用及P2Y嘌呤能受体机制研究

This project is based on the traditional Chinese traditional theory of moxibustion, "activating blood and removing blood stasis to prevent disease", and taking APOE-/- mouse as the model to define the effection of platelet activation infection or activating platelet In the method of gene therapy vector adeno-associated virus (AAV) specific infection or activating platelet. Application of a new Generation of high-throughput Sequencing technologies (Next Generation Sequencing (NGS), from P2Y purine and P2Y12 platelet activation P2Y1 receptor mediated - PLC - PI3K - Akt signaling pathways, and interpret molecular mechanism that moxibustion prevention and cure atherosclerosis, screening differential genes, and rich scientific connotation of "preventing disease", and to provide a scientific basis for clinical moxibustion prevention and cure atherosclerosis in clinic, meet the major needs of prevention cure of traditional Chinese medicine in our country.

本项目以艾灸“活血化瘀治未病”这一中医传统理论为出发点，以APOE-/-小鼠为模型，运用基因治疗载体腺相关病毒（AAV）感染血小板，特异性抑制或者激活血小板细胞，明确不同时间尺度下艾灸对血小板活化的特异性作用；应用新一代高通量测序技术（Next-Generation Sequencing (NGS)），观察艾灸后P2Y嘌呤能受体介导的血小板活化P2Y1-PLC和P2Y12-PI3K-Akt信号通路在mRNA和蛋白层次的表达，阐释艾灸防治动脉硬化的分子作用机制，筛选差异基因，丰富“中医治未病” 的科学内涵，满足我国中医药慢病防控的重大需求。

本项目以艾灸“活血化瘀治未病”这一中医传统理论为出发点，在不同时间点对干预4周和12周的ApoE-/-小鼠进行尾静脉注射100μl滴度为1.27×109 V.G./ml的GP6过表达慢病毒，对干预4周和12周的C57BL/6小鼠尾静脉注射100μl EGFP荧光表达质粒。取材后，用荧光显微镜观察镜下骨髓细胞的荧光转染情况以间接评估慢病毒在体内的转染成功与否，用流式细胞仪检测血浆中的血小板活化分子标志物CD63、CD62p、CD154的表达水平，结果提示艾灸治疗具有时间尺度，在动脉硬化早期对血小板活化的抑制作用可能较好；应用新一代高通量测序技术（Next-Generation Sequencing (NGS)），观察艾灸后P2Y嘌呤能受体介导的血小板活化P2Y1-PLC和P2Y12-PI3K-Akt信号通路在mRNA和蛋白层次的表达，Akt(Ser473)蛋白可能是艾灸调控血小板活化信号通路中的有效作用靶点。此研究丰富“中医治未病” 的科学内涵，满足我国中医药慢病防控的重大需求。