TWEAK通过P38MAPK途径促进大鼠心肌成纤维细胞Ⅰ型胶原和MMP-1表达

发布时间：2018-04-24 21:50

  本文选题：肿瘤坏死因子样凋亡微弱诱导剂 + 心肌成纤维细胞　； 参考：《山东大学》2013年硕士论文

【摘要】：背景： 高血压为最常见的慢性病,在其发生、发展的进程中,可导致心血管系统、脑、肾脏等多器官并发症,严重威胁着人类健康。心肌纤维化(myocardial fibrosis,MF)作为高血压病心肌损害的主要病理基础,表现为心肌细胞外基质(extracellular matrix,ECM)胶原的过度沉积和肌成纤维细胞(cardiac fibroblasts,CFs)数目的增多,进而影响心室壁动度及心室的舒张及收缩功能,从而导致严重心律失常、心功能不全及心源性猝死等严重并发症。因此,高血压心肌纤维化的发生机制和防治措施已成为近年来国内外研究热点。 近来研究发现MF是一个复杂的病理过程受免疫系统、肾素-血管紧张素-醛固酮系统(RAAS)和多种细胞因子调控。而炎症因子作为参与调控MF的重要因素在其发生、发展进程中发挥重要作用。 在诸多的细胞因子中,TNF超家族作为一个有多重生物学效应的促炎反应因子,与多种疾病发生、发展过程密切相关。TNF是一组具有多样生物学效应的促炎反应细胞因子,参与多种疾病的发生、发展。如：TNF-a表达升高可导致心功能不全及心肌病并且TNF-a还参与糖尿病大鼠心肌炎症反应及纤维化。作为TNF超家族中的新成员肿瘤坏死因子样凋亡微弱诱导剂(TWEAK),于1997年发现其主要由淋巴样细胞表达,在其合成之初为Ⅱ型膜结合蛋白,随后很快被furin酶水解成具有生物学活性的可溶性的小片段。Fnl4主要由非淋巴样细胞,如上皮细胞、内皮细胞、间充质细胞中表达,作为TWEAK特异性相关受体为人们所熟知,是Ⅰ型跨膜蛋白。TWEAK和Fn14广泛表达于各种组织及细胞内,在胰腺、肠、心脏、大脑、肺、卵巢及骨骼肌中均有TWEAKmRNA的表达,在肝脏及肾脏中亦少量表达。近年TWEAK及其特异性受体Fn14在创伤修复及组织再生方面介导多种炎症反应受到越来越多的关注,TWEAK作为一种促炎及促血管生成的细胞因子介导多种细胞的生长、增殖、凋亡,促炎性反应和血管生成等作用。研究发现TWEAK与其受体Fn14结合主要通过核转录因子(nuclear factor-KB, NF-κB)、丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)、蛋白激酶B (protein kinase B,PKB)途径等多个下游信号通路的瀑布样反应发挥作用。随着研究深入,TWEAK与其受体Fn14促进AS、MF及介导心肌重塑等在心血管系统过程中所发挥的的作用越来越得到重视。TWEAK/Fn14能促进内皮功能不全、增强炎症反应、促进平滑肌细胞增殖及迁移、上调MMPs表达、促进细胞死亡,参与动脉粥样硬化斑块进展,促使其不稳定,进而导致急性冠脉综合征的发生。在ST段抬高型急性心肌梗死病人时血清可溶性TWEAK (sTWEAK)显著升高,其升高程度与病人的短期预后密切相关。在心肌纤维化方面,研究发现在自发性高血压大鼠(spontaneous hypertension rat, SHR)心肌中Fn14表达明显增强,Fn14可能参与SHR心肌纤维化的过程,培哚普利可能通过抑制其表达减轻心肌纤维化。进而在体外细胞水平发现TWEAK/Fn14明显促进CFs中NF-κB和MMP9mRNA及蛋白表达,而以PDTC抑制NF-κB通路后,CFs增殖降低且MMP9表达减少。进而揭示TWEAK通过激活经典的NF-κB通路能促CFs增殖和胶原合成,参与心肌纤维化的发生、发展过程发现。Chen等研究发现TWEAK通过激活经典的NF-κB通路,促进大鼠CFs增殖和Ⅰ/Ⅲ胶原合成,促进心肌纤维化进展。然而,在心肌纤维化方面相关研究发现,TWEAK作为一种具有多种活性的炎症因子,除能激活经典的NF-κB通路外,还可通过介导Fn14激活非经典的NF-κB通路以及MAPK通路,然而相关研究未见报道。因此,本文通过观察TWEAK介导P38丝裂原活化蛋白激酶(P38mitogen-activated protein kinases, P38MAPK)通路对大鼠心肌成纤维细胞中胶原代谢影响,及MMP-1表达的影响,以进一步明确TWEAK参与心肌纤维化的机制。 目的： 探讨肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)影响心肌成纤维细胞(CFs)中Ⅰ型胶原和基质金属蛋白酶1(matrix metalloproteinase-1, MMP-1)表达的作用机制。 方法： 1.采用胰蛋白酶消化法及时间差法培养并纯化新生Wistar大鼠心肌成纤维细胞。并采用倒置显微镜及波形蛋白免疫荧光法观察并鉴定心肌成纤维细胞。 2.加入重组人TWEAK(rhTWEAK)及P38MAPK抑制剂(SB203580)干预。 将实验分为①对照组：不加干预因素；②TWEAK组：加入100ng/mLTWEAK;③TWEAK+SB203580组：加入100ng/mLTWEAK+SB203580。 3.各实验组分别采用MTT法检测CFs增殖；western blot法检测Ⅰ型胶原蛋白及磷酸化P38MAPK(P-P38MAPK)蛋白表达；qRT-PCR法检测Ⅰ型胶原mRNA表达；ELASA法检测CFs细胞培养液中MMP-1的浓度。 结果： 1.CFs的形态观察及鉴定 培养48h后,CFs已长满或接近长满单层培养瓶,长满或接近长满单层。于倒置显微镜下观察,CFs多呈梭形,无自发性搏动。其细胞核较大,胞浆透明。采用波形蛋白免疫荧光鉴定,CFs的波形蛋白呈现阳性反应,其胞浆内围绕胞核呈现绿色网络状物质。 2.干预因素对CFs增殖的影响 rhTWEAK干预心肌成纤维细胞48h后,对照组(0.302±0.019),TWEAK组(0.493±0.042), TWEAK+SB203580组(0.362±0.039)三者间相比较,TWEAK组较对照组可显著促进CFs增殖,差异有统计学意义(P0.01)；而TWEAK+SB203580组较对照组比较也可促进心肌成纤维细胞增殖(P0.05)。SB203580干预组及未干预组比较,CFs增殖程度降低,差异具有统计学意义(P0.05)。 3.各组对Ⅰ型胶原mRNA和Ⅰ型胶原蛋白的表达 以上各实验组终止干预后,测定以上各组中Ⅰ型胶原mRNA表达分别为TWEAK组(6.080±0.676),TWEAK+SB203580组(3.400±0.810)。与对照组比较,TWEAK组及TWEAK+SB203580组中Ⅰ型胶原mRNA表达水平明显增多,差异间具有统计学意义(P0.05)。在特异性阻断P38MAPK通路后,其Ⅰ型胶原mRNA表达水平较TWEAK组降低,二者间差异有统计学意义(P0.05),见图3a。与对照组比较,二者Ⅰ型胶原蛋白表达明显升高差异(P0.05)。同TWEAK组相比,TWEAK+SB203580组中Ⅰ型胶原蛋白较其减少42.6%,差异有统计学意义(P0.05)。 4.各组CFs中P-P38MAPK蛋白的表达 各实验组间相比,TWEAK组较对照组及TWEAK+SB203580组,可明显促进CFs中P-P38MAPK蛋白表达(P0.01)。TWEAK+SB203580组与对照组相比,TWEAK+SB203580组部分抑制P-P38MAPK蛋白表达(P0.05)。 5.各组CFs中MMP-1的表达 各实验组终止干预后,应用ELISA法测定各组心肌成纤维细胞中MMP-1浓度,TWEAK组较其他两组明显上调CFs中MMP-1表达,差异有统计学意义(P0.05)。TWEAK+SB203580组与TWEAK组相比,部分抑制心肌成纤维细胞培养液中MMP-1的表达,差异有统计学意义(P0.05)。 结论： 1.TWEAK/Fn14可显著促进大鼠心肌成纤维细胞增殖,上调细胞中Ⅰ型胶原mRNA及Ⅰ型胶原蛋白和P-P38MAPK蛋白表达,并促进炎症因子MMP-1表达。 2.应用SB203580后,可部分抑制TWEAK促进心肌成纤维细胞增殖程度,并部分抑制细胞中Ⅰ型胶原mRNA、I型胶原蛋白和P-P38MAPK蛋白及MMP-1表达。 3.本实验通过进一步研究发现,除经典的NF-κB途径外,TWEAK通过P38MAPK通路介导CFs的增殖及ECM代谢异常,从而进一步阐明TWEAK/Fnl4参与MF的机制。从而为全面揭示心肌纤维化的研究提供新的途径和方法,然而MAPK级联是一个错综复杂的相互交错的网络系统,本实验只是通过局部抑制某一通路的方法来研究,并且对于JNK、ER及其余MAPK等信号转导途径未做进一步深入研究。
[Abstract]:Background:
Hypertension is the most common chronic disease. In the course of its occurrence and development, it can lead to multiple organ complications such as cardiovascular system, brain, kidney and other organ complications, which seriously threaten human health. Myocardial fibrosis (MF) is the main pathological basis of myocardial damage in hypertension, which is expressed as the extracellular matrix of extracellular (extracellular matrix, ECM). The excessive deposition of collagen and the increase in the number of cardiac fibroblasts (CFs), which affect ventricular wall movement and ventricular diastolic and systolic function, lead to severe arrhythmias, cardiac insufficiency and sudden cardiac death. Therefore, the mechanism and preventive measures of high blood pressure myocardial fibrosis have become a result. In recent years, the research focuses at home and abroad.
Recent studies have found that MF is a complex pathological process that is regulated by the immune system, the renin angiotensin aldosterone system (RAAS) and a variety of cytokines, and inflammatory factors play an important role in the development process as an important factor involved in the regulation of MF.
Among many cytokines, TNF superfamily is a proinflammatory response factor with multiple biological effects, which is closely related to a variety of diseases. The development process is closely related to.TNF, a group of proinflammatory cytokines with various biological effects, participating in the occurrence and development of various diseases. For example, the increase of TNF-a expression can lead to cardiac insufficiency and Cardiomyopathy and TNF-a also participate in the reaction and fibrosis of myocarditis in diabetic rats. As a new member of the TNF superfamily, a new member of the tumor necrosis factor like apoptosis weak inducer (TWEAK), it was found to be mainly expressed by lymphoid cells in 1997. At the beginning of its synthesis, the type II membrane was combined with egg white, and then quickly hydrolyzed to biology by the furin enzyme. The active soluble small fragment.Fnl4 is mainly expressed in non lymphoid cells, such as epithelial cells, endothelial cells, and mesenchymal cells. As a specific receptor for TWEAK, it is known that type I transmembrane protein.TWEAK and Fn14 are widely expressed in various tissues and cells, in the pancreas, intestines, heart, brain, lung, ovary and skeletal muscle. The expression of TWEAKmRNA is also expressed in a small amount in the liver and kidney. In recent years, TWEAK and its specific receptor, Fn14, have attracted more and more attention in the field of trauma repair and tissue regeneration. TWEAK, as a proinflammatory and angiogenic cytokine, mediates the growth, proliferation, apoptosis, proinflammatory response and proinflammatory response of many cells. It is found that the combination of TWEAK and its receptor Fn14 plays a role mainly through the waterfall like response of several downstream signaling pathways, such as the nuclear factor (nuclear factor-KB, NF- kappa B), the mitogen activated protein kinase (mitogen-activated protein kinase, MAPK), the protein kinase B pathway, and so on. The role of TWEAK and its receptor Fn14 in promoting AS, MF and myocardial remodeling in the cardiovascular system is becoming more and more important..TWEAK/Fn14 can promote endothelial dysfunction, enhance inflammatory response, promote the proliferation and migration of smooth muscle cells, increase the expression of MMPs, promote cell death, and participate in the progression of atherosclerotic plaque. Acute coronary syndrome is caused by instability, which leads to the occurrence of acute coronary syndrome. The serum soluble TWEAK (sTWEAK) increases significantly in patients with ST segment elevation acute myocardial infarction, which is closely related to the short-term prognosis of the patients. In the field of myocardial fibrosis, the study was found in self hypertensive rats (spontaneous hypertension rat, SHR). The expression of Fn14 in myocardium was obviously enhanced, and Fn14 might participate in the process of SHR fibrosis. Perindopril may reduce the expression of myocardial fibrosis by inhibiting its expression. Then, the expression of NF- kappa B and MMP9mRNA and protein in CFs was obviously promoted at the cell level in vitro, and the CFs proliferation decreased and the expression decreased after the PDTC inhibition of NF- kappa B pathway. Furthermore, it is revealed that TWEAK can promote the proliferation of CFs and collagen synthesis by activating the classical NF- kappa B pathway and participate in the development of myocardial fibrosis. The development process found that.Chen and other studies found that TWEAK promotes the proliferation of CFs and collagen synthesis of I / III by activating the classical NF- kappa B pathway and promotes the progress of myocardial fibrosis. However, it is related to myocardial fibrosis. As an inflammatory factor with multiple activity, TWEAK can activate the classical NF- kappa B pathway and activate the non classical NF- kappa B pathway and MAPK pathway by mediating Fn14. However, the related studies have not been reported. Therefore, this paper has observed TWEAK mediated P38 mitogen activated protein kinase (P38mitogen-activated protein Ki). The effect of nases, P38MAPK) pathway on collagen metabolism in rat myocardial fibroblasts and the effect of MMP-1 expression, in order to further clarify the mechanism of TWEAK involvement in myocardial fibrosis.
Objective:
To investigate the effect of tumor necrosis factor like apoptosis weak inducer (TWEAK) on the expression of type I collagen and matrix metalloproteinase 1 (matrix metalloproteinase-1, MMP-1) in myocardial fibroblasts (CFs).
Method锛，

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