【作者】 王芳；

【导师】 马向华；

【作者基本信息】 南京医科大学 ， 内分泌学， 2008， 硕士

【摘要】 目的探讨过氧化物酶体增殖物激活受体γ(PPAR-γ)激动剂吡格列酮对瘦素刺激的大鼠血管平滑肌细胞(VSMCs)增殖效应的影响及其作用机制。方法(1)以组织贴块法培养大鼠主动脉VSMCs,采用倒置相差显微镜和透射电镜观察的方法进行细胞鉴定;(2)分别使用0、12.5、25.0、50.0、100.0ng/ml瘦素作用于VSMCs24、48、72小时,以四甲基偶氮唑盐(MTT)法观察细胞的增殖情况;(3)100.0ng/ml瘦素作用于VSMCs72小时后,再分别加入1.0、10.0、100.0μmol/L的吡格列酮作用24小时,以MTT法观察细胞的生长情况;(4)提取细胞的总RNA和蛋白,分别用逆转录聚合酶链反应(RT-PCR)和免疫印迹法(Western Blot)检测增殖细胞核抗原(PCNA)和瘦素受体(OB-R)mRNA及蛋白的表达水平。结果(1)与空白对照组相比,各浓度瘦素(12.5、25.0、50.0、100.0ng/ml)均可促进VSMCs增殖,其中50.0、100.0ng/ml瘦素作用于VSMCs 72小时后差异具有统计学意义(P＜0.05),且该增殖作用呈现一定的时间和剂量依赖效应,以100.0ng/ml瘦素作用72小时后的细胞增殖率最大(22.4%)。(2)吡格列酮能抑制瘦素诱导的VSMCs的增殖效应,以浓度为100.0μmol/L时达最大抑制效应(P＜0.01)。(3)与空白对照组相比,100.0ng/ml瘦素作用于VSMCs72小时后,VSMCs PCNA及OB-R mRNA和蛋白的表达均明显升高,差异具有显著性(P均＜0.05)。(4)加入不同浓度的吡格列酮(1.0,10.0,100.0μmol/l)作用24小时后,与仅加瘦素组比较,可见VSMCs PCNA及OB-R mRNA和蛋白的表达量逐渐降低,其中以100.0μmol/l吡格列酮组作用最明显,差异具有统计学意义(P＜0.01)。结论高瘦素血症与动脉粥样硬化(AS)的发生、发展有相关性,吡格列酮可能通过降低OB-R和PCNA的表达抑制瘦素刺激的VSMCs增殖及表型转换,发挥一定的心血管保护作用。更多还原

【Abstract】 Objective:To investigate the effects and mechanism of peroxisome proliferators-activated receptorγagonist pioglitazone on leptin-induced proliferation of rat aortic vascular smooth muscle cells (VSMCs).Methods:(1)Primary rat aortic vascular smooth muscle cells were cultured with the adherent method of tissue explants.VSMCs were observed and verified by inverted phase contrast microscope and transmission electron microscope.(2)VSMCs were stimulated with leptin at different concentrations(12.5,25.0,50.0,100.0ng/ml)for 24,48 and 72 hours,the proliferation of which was determined by Methyl thiazolyl tetrazolium(MTT)assay.(3)After induction with 100.0ng/ml leptin for 72 hours,VSMCs were treated by pioglitazone at different concentrations (1.0,10.0,100.0μmol/L),the proliferation of which was observed by MTT.(4)Total RNA and protein were extracted and the expression of PCNA and OB-R at mRNA level as well as protein level were measured by Reverse transcription-polymerase chain reaction(RT-PCR)and Western blot,respectively.Results:(1)Compared with controls,leptin (12.5,25.0,50.0,100.0ng/ml)stimulated VSMCs proliferation,of which 50.0 ng/ml and 100.0ng/ml were statistically significant(P＜0.05).The effect was also time and dose-dependent,reaching optimal proliferation rate(22.4%)at 100.0ng/ml for 72h.(2)Pioglitazone antagonized leptin-induced VSMCs proliferation,with the strongest effect at 100μmol/L(P＜0.01).(3)Leptin treatment(100.0ng/ml)for 72h significantly promoted PCNA and OB-R expression of VSMCs at both mRNA and protein levels(P＜0.05).(4)Compared with leptin only group, pioglitazone at different concentrations(1.0,10.0,100.0μmol/l)for 24 hours attenuated the increase of PCNA and OB-R expression in VSMCs induced by leptin,especially at 100μmol/L(P＜0.01).Conclusion: Hyperleptinemia was related to the pathogenesis of atherosclerosis. Pioglitazone may suppress leptin-stimulated VSMCs proliferation and influence the phenotypic change of VSMCs by reducing OB-R and PCNA expression,which may be beneficial for cardiovascular system.更多还原