【作者】 高海成；

【导师】 任立群；

【作者基本信息】 吉林大学 ， 病理与病理生理学， 2009， 博士

【摘要】 心肌纤维化(myocardial fibrosis,MF)是一种常见的心肌病理改变。无论何种原因引起心肌损害,只要病变持续和病程慢性化,终将导致心肌纤维化。重者可发展成为难治性心力衰竭、心律失常甚至猝死。由于心肌纤维化发生机制较为复杂,目前尚缺乏有效治疗方法。因此,深入研究心肌纤维化的发生机制及防治措施,一直是心血管病领域的研究热点。阿魏酸钠(sodium ferulate, SF)是阿魏酸钠盐,具有扩张血管、抑制血小板聚集、改善微循环、抑制炎症反应、抗氧化等药理学作用,目前研究最多的是其抗氧化和内皮素受体拮抗作用。然而,有关SF对心肌纤维化的保护作用及其对Rho/Rock信号通路的影响,迄今国内外尚未见报道。本实验应用一次性大剂量皮下注射盐酸异丙基肾上腺素(Iso),建立了心肌缺血缺氧性坏死诱发的心肌纤维化动物模型,应用免疫组化、RT-PCR、ELISA及Western blot等技术,研究了Rho/Rock通路在大鼠MF发生机制中的作用及SF在体内及体外对Rho/Rock通路的影响,以观察Rho/Rock通路在心肌纤维化发生机制中的作用及SF对MF大鼠心脏结构和功能的改善效果。研究结果表明,注射Iso后,心肌组织TGF-β1、CTGF、AT1R、COX2及tTG mRNA表达均在24 h达到高峰,其蛋白表达随Iso注射时间的延长而逐渐增加,至3 w蛋白表达量最高,SF能下调上述促纤维化物质的基因和蛋白表达,减轻心肌纤维化大鼠心肌ECM主要成分的沉积;Rho A及RockI的基因表达和蛋白表达与TGF-β1、CTGF等呈平行性表达,提示Rho/Rock信号通路可被多种促纤维化物质激活,该信号通路的激活促进了心肌纤维化的发生发展;SF能减少CFB培养上清中由Iso引起的ColⅠ分泌增加,下调培养上清中促纤维化物质的基因与蛋白表达; SF可改善MF大鼠心脏功能,减轻心肌纤维化大鼠心脏组织的病变程度,说明SF可能具有抑制Rho/Rock信号通路,下调促纤维化物质的表达和保护心脏的多靶点治疗作用。更多还原

【Abstract】 Mocardial fibrosis involves to various kinds of originality pathogeny, which regulative mechanism is influenced by many factors, and its severity has a close correlation with impairment of myocardial function. No matter what causes myocardial damage, the course of sustained and chronic diseases will eventually lead to myocardial fibrosis and develop into severe refractory heart failure, arrhythmia and even sudden death. The result of complex mechanisms of MF occurrence and development lacked effective treatment. Therefore, the occurrence of MF-depth study of the mechanism and prevention measures has been research in the field of cardiovascular disease hot spots.Myocardial fibrosis is the result of excessive deposition of myocardial collagen. Fibroblasts is the main cell by direct synthesis and secretion of collagen. In fibroblast proliferation, differentiation and the formation of collagen will involve a variety of cytokines, especially growth factors, such as transforming growth factor (TGF), fibroblast growth factor (FGF), connective tissue growth factor (CTGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and so on. These growth factors can be generated in the tissue, myocardial damage may also arise from a number of inflammatory cells. A large number of studies have shown that transforming growth factor (TGF-β) and connective tissue growth factor (CTGF) is involved in the formation of multi-organ fibrosis in two important cytokines. AT1R, COX2 and tTG in the liver, kidney and lung fibrosis development also plays an important role, but the relationship between the occurrence of myocardial fibrosis is still a lack of in-depth study. Therefore, to explore these substances in promoting fibrosis have the great significance in MF’s role of the developmentRho and its downstream effectors Rho kinase (Rock) composed of Rho/ Rock signaling pathway and play an important role in fibrosis of vitro and vivo. And Rho/Rock signaling pathway is considered the body of the organization are prevalent in a signal transduction pathway, which may be accompanied by a variety of inflammatory mediators and cytokines after receptor activation and coupling mechanisms activated kinase cascade through the directly involved in the response of bone cells microfilament-based control framework.Cell contraction, chemotaxis, wavering, adhesion, invasion, such as the biological behavior must be adopted by the polymerization of actin and extension to achieve. Therefore, some scholars think that Rho/Rock signaling pathway is to control these cells acts directly upstream signaling pathway. Rho stimulation signal in the upper reaches, which can never combined GDP form of activity into the activity of GTP binding form and the role of the target in the downstream pathway. Research has confirmed that the application of Rho/ Rock pathway selective inhibitor Y-27632 can improve the kidney, lung, liver fibrosis model which was the extent of fibrosis on Rho / Rock pathway and involved in the development of fibrosis. However, Rho / Rock pathway is unclear in the role of myocardial fibrosis.Sodium ferulate (SF) is the sodium salt of ferulic acid, with the expansion of blood vessels, improve microcirculation, inhibit inflammatory response, antioxidant effect, such as pharmacology. The present study is the largest of its anti-oxidation and the role of endothelin-receptor antagonist, and through the intervention of endothelin and oxygen free radicals and other mechanisms to protect the heart.However, the SF of the MF and its impact on the protective effect of Rho / Rock signaling pathway, has not been reported so far at home and abroad.The experimental application of a one-time high-dose subcutaneous injection of 15 mg / kg body weight of isoproterenol hydrochloride (Iso), the establishment of a hypoxic-ischemic myocardial necrosis induced animal models of myocardial fibrosis, combined with in vitro cultured rat cardiac fibroblasts immunohistochemical, high-performance liquid chromatography, RT-PCR, ELISA and Western blot techniques, through in vitro and in vivo studies confirm each other’s methods, a systematic study of the Rho / Rock pathway in the development of MF in rats the role of mechanisms and SF of in vitro and in vivo Rho / Rock pathway in order to observe the Rho / Rock pathway in the mechanism of myocardial fibrosis and the role of myocardial fibrosis in rats SF cardiac structure and function to improve the effectiveness of clinical treatment for the provision of myocardial fibrosis useful information.The main results of this study are as follows:the rats were injected 15 mg/kg body weight Iso and appeared ischemic ECG changes, serum enzymes at different time points in different levels of increased morphological observation also found that injection of Iso after 2 h, myocardial cells that appear point-like degeneration and necrosis, with the injection of Iso time, degeneration and necrosis of an area of gradual change, and 1 w after the injection of clear boundaries, multiple scattered necrotic foci, 3 w after the lesions of fibrosis significantly. Note the use of a one-time multi-point injection 15 mg / kg body weight can be successfully constructed Iso rat model of myocardial fibrosis, myocardial necrosis, the incidence of myocardial fibrosis mechanisms and experimental studies, such as drug screening provides a new model. Iso injection, the myocardial tissue TGF-β1, CTGF, AT1R, COX2 and tTG mRNA expression peaked at 24 h, the protein expression with Iso injection time and gradually increase to 3 w protein expression in the highest, SF can be reduced Desmoplastic material above the gene and protein expression, to reduce myocardial fibrosis in myocardial ECM major components of the deposition; Rho A and RockI of gene expression and protein expression of TGF-β1, CTGF, such as parallel expression, suggesting that Rho / Rock signaling pathway may be a variety of substances Desmoplastic activation, activation of the signaling pathway promotes the occurrence and development of myocardial fibrosis;The main conclusions of this study are as follows:1. Using a one-time multi-point injection 15 mg / kg ideal body weight Iso-induced rat model MF.2. Desmoplastic material (TGF-β1, CTGF, AT1R, COX2 and tTG) in the myocardial fibrosis play an important role in the occurrence; SF can be reduced myocardial fibrosis in promoting myocardial fibrosis genetic material and protein expression; SF can reduce heart rat cardiac muscle fibrosis of the major components of ECM deposition.3. Rho/Rock signaling pathway may be a variety of substances Desmoplastic activation, Rho / Rock signaling pathway activation to promote the development of myocardial fibrosis.4. In vitro studies further confirmed, SF can reduce the CFB culture supernatant caused by Iso increased secretion of ColⅠ, down in the culture supernatant material promoting fibrosis gene and protein expression.These results are provided for clinical prevention and treatment of MF is more comprehensive, the new experimental evidence. The main innovation of this study is: to prove the use of a one-time multi-point injection Iso can induce myocardial fibrosis ideal model; Desmoplastic substances may activate Rho / Rock signaling pathway, thereby promoting the occurrence and development of myocardial fibrosis; SF can Iso-induced reduction of myocardial fibrosis in myocardial fibrosis and promote the material expression of gene and protein to reduce the major components of ECM deposition, the ability to improve myocardial metabolism.更多还原