【作者】 于心亚；

【导师】 赵连友；

【作者基本信息】 第四军医大学 ， 内科学， 2004， 硕士

【摘要】 目前认为，左心室肥厚是影响原发性高血压患者病残率和病死率的一个重要的独立危险因素。因此，逆转左心室肥厚已成为当前治疗原发性高血压的重要目标之一。左心室肥厚的细胞病理学基础不仅表现在心肌实质细胞的肥大，还有心脏成纤维细胞(CFs)的过度增殖和细胞外基质大量沉积，导致心肌纤维化。原发性高血压心肌纤维化可引起心室功能不全、冠脉储备下降和严重的室性心律失常，使原发性高血压患者发生心力衰竭和猝死的危险显著增加。因此，深入研究间质改建与CFs增殖、胶原合成分泌的关系，采取有效手段抑制或促进间质改建，是改善心脏重构和提高心血管功能的可能途径之一。 目前，国内外学术界公认原发性高血压与内分泌功能紊乱和糖代谢障碍有密切关系，特别是原发性高血压人群有明显的胰岛素抵抗。有学者认为，胰岛素抵抗是原发性高血压的发病原因之一。盐酸吡格列酮(PIO)属于格列酮类新一代胰岛素增敏剂，能够增强胰岛素的敏感性，改善胰岛素抵抗。晚近，国外学术界研究发现格列酮类药物具有强大的心血管保护作用，如抑制血管平滑肌细胞增殖、改善血脂代谢、改善内皮功能、降低纤维蛋白溶解酶原激活抑制物-1和减少动脉粥样硬化发生以及抑制心肌细胞肥大等。但是，关于PIO对于心脏间质细胞有何作用，能否抑制第四军医大学硕士学位论文CFs的增殖，逆转心肌纤维化，目前尚不十分清楚。 本研究以培养的自发性高血压大鼠(S HR)和正常血压对照大鼠(WIStar大鼠)的CFs为试验模型，采用MTT法测定细胞增殖数目，流式细胞仪(FCM)分析技术检测细胞周期，轻脯氨酸比色法测定CFs培养上清胶原含量，硝酸还原酶法测定NO浓度，分光光度法测定NOS活性，免疫组化染色法检测PIO干预下的SHR CFs中NF一KB表达量。 本课题观察PIO对SHR和Wistar大鼠的CFs增殖和胶原合成的影响;研究PIO与SHR CFs一氧化氮合酶/一氧化氮伽05一o)系统活性的关系;探讨PIO调控SHR CFs的NF一KB表达的作用，旨在阐明PIO逆转心脏重构的作用机制，为防治原发性高血压提供理论依据和新的方法，同时也为PIO的临床应用提供一个新的途径。 研究结果表明:(l)不同浓度Plo对sHR、wistar的cFs增殖均具有抑制作用，且随着浓度的增加，抑制作用逐渐增强，即具有浓度依赖性。1 x 10一7mol/L、1 x 10一6 mol/L、5 x 10一6 mol/L、lx1o一sm。比的PIO作用24小时后，SHR的A490值分别为0.19士0.01、0.18士0.01、0.16士0.01、0.16士0.02，与对照组(0.22士0.01)相比，具有非常差异性显著(只0.01)。Wistar的A4卯值分别为0.21士0.01、0.19士0.01、0.18士0.01、0.17士0.01，与对照组(0.21士0.02)相比，亦具有非常差异性显著(只0.01)。(2)不同作用时间PIO对SHR、Wistar的CFs亦具有抑制作用，且随着作用时间的延长，抑制作用逐渐增强，具有时间依赖性。5xlo一6mo比的PIO作用12h、24h、36h、48h、6oh、72h后，SHR的A，卯值分别为0.19士0.01、0.16士0.01、0.16士0.02、0.15士0.01、0.15士0.01、0.15士0.02，同一时间点的对照组A、，。值分别为0.21士0 .02、0.21士0.01、0.22士0.01、0.23士0.02、0.23士0.01、0.23士0.02，两者相比，有非常显著性差异(P<0.001)。5 x 10一6 mo比第四军医大学硕士学位论文的PIO作用1 Zh、24h、36h、48h、60h、72h后，Wistar的A，，o值分别为0.19士0.01、0.18士0.01、0.17士0.01、0.16士0.02、0.16士0.01、0.15士0.01，同一时间点的对照组A妇。值分别为0，20士0.01、0.21士0.02、0.22士0.02、0.22士0.01、0.23士0、02、0.23士0.01，两者相比，差异非常显著(P<0.OOI)，其A妇。值亦随着作用时间的延长而逐渐减低。(3)1 x 10一，mol几、2 x 10一6 mol几、SXlo一6mol几、lxlo一5 mo比的PIO作用48小时后，SHR、Wistar CFs的G。/G，期细胞百分率较对照组显著增高(尸均<0.01)，S期细胞百分率、q/M期细胞百分率和增殖指数则显著低于对照组(尸均<0 .01)，且随着作用浓度的增加，PIO对细胞周期的影响逐渐增强。(4)不同浓度PIO对SHR、Wista大鼠CFs培养上清胶原合成具有明显的抑制作用，随着作用浓度的增加，上清轻脯氨酸含量逐渐降低。lxlo一7moljL、lxlo一6mol几、5火10一6mol/L、lxzo一smol几的PIO作用48小时后，SHR CFs上清轻脯氨酸含量为:2.25士0.08、2.82士0.05、1.35士0.05，2.26士0.07，除lx一。一7mol几的PIO组和对照组(2 36士0.11)相比，无明显差异(P二0.19)外，余药物干预组与对照组相比，具有非常显著性差异(尸<0 .01)。Wistar CFs的上清轻脯氨酸含量为:1.87士0.14、1.76士0.06、1.44士0.05、1.37士0.09，除1 x 10一7mo比的PIO组和对照组(1 .58士0.16)相比较，无明显差异(P二0.19)外，余药物干预组与对照组相比，亦有非常显著性差异(P<0.01)。(5)不同时间PIO对SHR、Wista大鼠CFs培养上清胶原合成亦具有明显的抑制作用，随着作用时间的延长，上清轻脯氨酸含量逐渐减低。5 x 10一“mol几的PIO作用24h、48h、72h后，SHR CFs培养上清经脯氨酸含量分别为:1 .47士0.11、1.35士0.08、1.43士0.21，与同一时I’ed点的对照组(2.15更多还原

【Abstract】 Background Left ventricular hypertrophy is regarded as a strong independent risk factor for the morbidity and mortality of essential hypertension. Nowdays the reversal of left ventricular hypertrophy is one of main targets for the treatment of essential hypertension. Besides cardiomyocytes hypertrophy, the pathological basis of left ventricular hypertrophy shows cardiac fibroblasts proliferation and excessive extracellular matrix protein accumulation, which leads to myocardial fibrosis. Hypertensive myocardial fibrosis has been shown to facilitate ventricular dysfunction, diminished coronary reserve and ventricular arrhythmias that adversely affect the clinical outcome of hypertensive patients. Therefore, matrix remodeling is pathologic basis of myocardial remodeling. We should study matrix remodeling carefully and take effective measures to inhibit or promote matrix remodeling, they may give us a possible way to improve cardiovascular function.Nowadays, the acknowledged concept that the essential hypertension are strongly correlated with insulin resistance. The metabolic syndrome was defined as glucose intolerance, hypertension,low high-density lipoprotein cholesterol and hypertriglyceridemia. Some believed insulin resistance played an important role in the aetiology of essential hypertension.Pioglitazone hydrochloride, the thiazolidinediones which are novel insulin-sensitizing agents,as the high affinity ligands for peroxisome proliferator-activated receptor y (PPARy,also as pecific PPARr agonists), has been demonstrated to improve insulin sensitivity so as to attenuate insulin resistance. Recently, studies about thiazolidinediones found that they have the powerful cardiovascular protection, such as attenuate vascular smooth muscle cells prolifertion inhibit pathological cardiac hypertrophy regulate endothelial cell functions ameliorate intriacylglycerol and cholesterol metabolism and reduce plasminogen activator inhibitor-1, and also retard the process of atherosclerosis. Till now, the role of PIO in the pathogenesis of left ventricular hypertrophy associated with hypertension has not been clearly defined. PIO has been proved to attenuate vascular smooth muscle cells prolifertion and inhibit pathological cardiac hypertrophy; however, whether PIO is effective in cardiac interstitium is still obscure.The aim of this study was to evaluate the antiproliferative effects of PIO on cardiac fibroblasts isolated from SHR and Wistar rat, and its relationship with nitric oxide synthase-nitric oxide system activity and the expressing of NF- k B protein. Furthermore,this studies also want to investigate the effects of PIO on preventing myocardium remodeling so as to provide a valuable theory and. a novel pharmacological strategy for treatment of essential hypertension and find a new way for application of PIO.Methods Isolated and cultured CFs of SHR and Wistar rats wereused as experiment model. (1) CFs number was measured by MTT assay. FCM determine cell cycle. Collagen content of CFs culture supernatant was estimated by hydroxyproline chromatogragy. We want to investigate the effects of PIO with different concentration and time on proliferation and collagen synthesis of SHR CFs and their relations to cardiac remodeling. (2)Nitrate enzyme reverting method and spectrophotometer were also adopted to estimated the content of NO and activity of NOS in CFs of SHR with or without PIO. We also want to investigate the effects of PIO on the changing of NOS-NO system activity. (3) Using immunohistochemistry, we investigate the effects of PIO on the expressing of NF- k B protein.Results The results showed that:(1)PIO can inhabit proliferation of CFs from SHR in a concentration-dependent manner with different concentrations of PIO. A490 absorbant value of CFs treated by PIO for 24 hours (1X10-7 1X10-6 5 X 10-6 1X 10-5 mol/L were: SHR 0.19+0. 01 0.18+0. 01 0.16 + 0.01 0.16 + 0.02; Wistar0. 21 + 0. 01 0.19 + 0. 01 0.18 + 0. 01 0.17 + 0. 01) were significantly lower than those of corresponding control groups (0. 22 + 0. 0更多还原