【作者】 王磊；

【导师】 马礼坤；

【作者基本信息】 安徽医科大学 ， 内科学（心血管病）， 2016， 硕士

【摘要】 研究背景和目的：心肌纤维化是许多心血管疾病的终末期共同表现,是以心肌成纤维细胞的过度增殖及Ⅰ型、Ⅲ型胶原蛋白等细胞外基质的过量沉积为特征。目前抑制心肌纤维化的主要方法是使用RAAS系统抑制剂如ACEI和ARB类药物等。MiRNA是真核细胞内一类具有负性调控作用的小RNA,通过与其靶基因的特异性结合,抑制基因的表达。MiRNA参与了真核生物基因表达的几乎全部过程,越来越多的研究表明miRNA在心血管疾病的发生发展过程中发挥了重要作用。本研究主要探讨微小RNA-9 (miRNA-9, miR-9)在心肌纤维化中的作用及其相关机制。方法：PDGF-BB (20ng/ml、50ng/ml)或血清(10%)处理大鼠新生心肌成纤维细胞48小时,采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测各组大鼠心肌成纤维细胞中miRNA-9的表达量。MiRNA-9、miRNA-9+miRNA-9抑制剂(anti-miR9)分别转染大鼠心肌成纤维细胞,采用噻唑蓝细胞增殖活性检测法(MTT)、qRT-PCR、蛋白免疫印迹法(Western blotting)分别检测各组大鼠心肌成纤维细胞增殖率、血小板衍生生长因子受体-β (PDGFR-β)及Ⅰ型、Ⅲ型胶原蛋白(collagen Ⅰ、 collagen Ⅲ)表达量。PDGFR-β过表达质粒、PDGFR-β过表达质粒+miRNA-9分别转染大鼠心肌成纤维细胞,检测各组PDGFR-β及collagen Ⅰ、collagen Ⅲ表达量。SiPDGFR-β转染大鼠心脏心肌成纤维细胞,检测PDGFR-β及collagen Ⅰ、collagen Ⅲ表达量。PDGFR-β双荧光素酶报告基因质粒+miRNA-9转染大鼠心肌成纤维细胞,检测荧光素酶活性。结果：PDGF-BB或血清处理大鼠心肌成纤维细胞48小时后,miRNA-9表达量较对照组明显下调(P<0.05)。MiRNA-9组细胞增殖率,PDGFR-β、collagen Ⅰ、 collagen Ⅲ mRNA及蛋白表达量较对照组均明显下调(P<0.05), miR-9+ miRNA-9抑制剂组上述指标较对照组变化无统计学意义(P>0.05)。PDGFR-β过表达质粒组PDGFR-β及collagen Ⅰ、collagen Ⅲ mRNA较对照组明显上调(P<0.05),较PDGFR-β过表达质粒+miRNA-9组变化无统计学意义(P>0.05)。SiPDGFR-β组PDGFR-β、collagen Ⅰ、collagen Ⅲ mRNA及蛋白表达量较对照组明显下调(P<0.05), siPDGFR-β与miRNA-9对大鼠心肌成纤维细胞具有相似的抑制作用。PDGFR-β双荧光素酶报告基因质粒+miRNA-9组荧光素酶活性较对照组明显下调(P<0.05)结论：MiRNA-9通过抑制靶基因PDGFR-β表达进而抑制心肌成纤维细胞的增殖和胶原蛋白的表达,发挥抑制心肌成纤维细胞的纤维化效应。更多还原

【Abstract】 Objective:Cardiac fibrosis is common at the end stage of many cardiovascular diseases, which is characterized by the proliferation of CFs and excessive deposition of ECM proteins such as collagen Ⅰ and Ⅲ. At present the main method of inhibiting cardical fibrosis is the use of RAAS inhibitors such as ACEI and ARB class of drugs. MicroRNAs are a diverse group of endogenous RNA-based molecules, which can regulate important functions in many cardiovascular cells types. This study focused on miRNA-9 and its associated mechanisms of cardical fibrosis.Methods:Treatment of cultured rat neonatal CFs with PDGF-BB or serum,the expression of miRNA-9 was detected by qRT-PCR. Rat neonatal CFs was transfected with miRNA-9 mimic、miRNA-9 mimic+ miRNA-9 inhibitor, MTT assay, qRT-PCR and Western blotting was used to verified the proliferation rate and PGDFR-β、 collagen expression of CFs. The expression of PGDFR-βand collagen Ⅰ、collagen Ⅲ in rat neonatal CFs was revealed after transfection with PGDFR-βoverexpression plasmid、PGDFR-βoverexpression plasmid+ miRNA-9. The rat neonatal CFs was transfectd with siPDGFR-βand after that, the expression of PGDFR-β、collagen Ⅰ and collagen Ⅲ was detected. Dual-luciferase assay was used to determined the target gene of miRNA-9.Results:The expression of miRNA-9 was significantly down-regulated after treated with PDGF-BB or serum. Overexpression of miR-9 suppressed the proliferation、 PDGFR-βand collagen expression in rat neonatal CFs. The effect of miR-9 in CFs was abrogated by co-transfection with miRNA-9 inhibitors. Transfection with PDGFR-βoverexpression plasmid generate a markedly increase in the expression of PDGFR-βand collagen Ⅰ、collagen Ⅲ, while co-transfetion with miRNA-9 failed to inhibit this phenomenon. After siPDGFR-βtransfection, the expression of PDGFR-βwas markedly supressed, collagen Ⅰ and Ⅲ were inhibited at the same time. Both miRNA-9 and siPDGFR-βhas similarly role in CFs. Dual-luciferase assay results showed that PDGFR-Pwas a direct target of miRNA-9.Conclusions:MiRNA-9 can inhibited cardiac fibrosis by targeting PDGFR-β.更多还原