芯片技术筛选夹脊穴预处理心肌缺血再灌注大鼠Wnt通路相关差异表达基因

Differentially Expressed Genes Related to Wnt Pathway in Rats with Myocardial Ischemia-Reperfusion Injury by Expression Microarray

目的:通过基因芯片技术筛选心肌缺血再灌注损伤(MIRI)大鼠差异表达的基因,富集分析关键通路,寻找电针夹脊穴预处理可能的保护途径。方法:72只大鼠,随机分为对照组、模型组、夹脊组、内关组、阳陵泉组、曲池组,每组12只。各针刺组电针预处理7 d后,制备MIRI模型。提取缺血区心肌组织总RNA并评估质量后,进行样品标记和芯片杂交,使用Agilent Feature Extraction软件获得芯片图,Gene Spring GX v12.1软件进行Quantile标准化及数据处理,使用标准的富集计算方法进行GO分析和Pathway分析。并采用荧光定量PCR对筛选出的差异基因进行部分验证。结果:电针预处理能够调控缺血再灌注大鼠的基因表达,其中夹脊穴组对Wnt通路相关基因影响显著,共涉及10个基因:上调细胞外因子Wnt9b、Wnt10b,蛋白受体Fzd2,磷脂酶C家族Plch1、plch2,DKK家族中Dkkl1及APC家族基因Amer1和Amer3;同时下调磷脂酶C家族Pclb1及Sapcd家族基因Sapcd2。这些基因在内关、阳陵泉、曲池组无显著性差异。PCR结果与芯片结果相符。结论:多条信号通路参与电针夹脊穴预处理对心肌缺血再灌注的调控,涉及多个基因。从分子水平阐明了其对MIRI的保护作用机制。

Objective： To differentially screen expressed genes in myocardial ischemia reperfusion injury （MIRI） rats by expression microarray and enrich the critical pathways to find out the possible protective pathway of electroacupuncture at Jiaji points. Method： Totally 72 rats were randomly divided into control group, model group, Jiaji group, Neiguan （PC6） group, Yanglingquan（GB34） group, Quchi（ LI11 ） group, and 12 rats in each group. Rats in acupuncture group were treated with pretreatment of electroacupuncture for 7 days, and then MIRI models were prepared. After extracting to- tal RNA from ischemic myocardium and evaluating quality, the test samples were tagged with the fluorescent dyes and hy- bridized wtih the structured biochip sufficiently. Then Agilent Feature Extraction was used to get the chip figures. And quantile standardization and data processing was carried out by GX v12.1 GeneSpring software. GO analysis and Pathway analysis were done according to standard enrichment calculation methods. The differentially expressed genes were screened by fluorescence quantitative PCR. Result： Electroacupuncture preconditioning could regulate gene expression in rats with ischemia reperfusion injury. The Wnt pathway related genes were significantly affected by Jiaji points. There were 10 genes involved： the expressions of Wwnt9b, Wntl0b, fzd2, plchl, plch2, Dkkll, Amerl and Amer3 were in- creased while the expressions of Pclbl and Sapcd2 decreased. These genes had no significant differences in Yanglingquan （GB34）, Neiguan（PC6） and Quchi（LI11 ） groups. The results of PCR were consistent with the results of the microar- ray. Conclusion： Multiple signal pathways are involved in the regulation of myocardial ischemia and reperfusion by the pretreatment of electroacupuncture at the acupoints of Jiaji, which involves multiple genes. The protective effect mecha- nism of MIRI was elucidated from the molecular level.