本文選題：腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑 + 心肌成纖維細胞�。� 參考：《山東大學(xué)》2013年碩士論文

【摘要】：背景： 高血壓為最常見的慢性病,在其發(fā)生、發(fā)展的進程中,可導(dǎo)致心血管系統(tǒng)、腦、腎臟等多器官并發(fā)癥,嚴(yán)重威脅著人類健康。心肌纖維化(myocardial fibrosis,MF)作為高血壓病心肌損害的主要病理基礎(chǔ),表現(xiàn)為心肌細胞外基質(zhì)(extracellular matrix,ECM)膠原的過度沉積和肌成纖維細胞(cardiac fibroblasts,CFs)數(shù)目的增多,進而影響心室壁動度及心室的舒張及收縮功能,從而導(dǎo)致嚴(yán)重心律失常、心功能不全及心源性猝死等嚴(yán)重并發(fā)癥。因此,高血壓心肌纖維化的發(fā)生機制和防治措施已成為近年來國內(nèi)外研究熱點。 近來研究發(fā)現(xiàn)MF是一個復(fù)雜的病理過程受免疫系統(tǒng)、腎素-血管緊張素-醛固酮系統(tǒng)(RAAS)和多種細胞因子調(diào)控。而炎癥因子作為參與調(diào)控MF的重要因素在其發(fā)生、發(fā)展進程中發(fā)揮重要作用。 在諸多的細胞因子中,TNF超家族作為一個有多重生物學(xué)效應(yīng)的促炎反應(yīng)因子,與多種疾病發(fā)生、發(fā)展過程密切相關(guān)。TNF是一組具有多樣生物學(xué)效應(yīng)的促炎反應(yīng)細胞因子,參與多種疾病的發(fā)生、發(fā)展。如：TNF-a表達升高可導(dǎo)致心功能不全及心肌病并且TNF-a還參與糖尿病大鼠心肌炎癥反應(yīng)及纖維化。作為TNF超家族中的新成員腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑(TWEAK),于1997年發(fā)現(xiàn)其主要由淋巴樣細胞表達,在其合成之初為Ⅱ型膜結(jié)合蛋白,隨后很快被furin酶水解成具有生物學(xué)活性的可溶性的小片段。Fnl4主要由非淋巴樣細胞,如上皮細胞、內(nèi)皮細胞、間充質(zhì)細胞中表達,作為TWEAK特異性相關(guān)受體為人們所熟知,是Ⅰ型跨膜蛋白。TWEAK和Fn14廣泛表達于各種組織及細胞內(nèi),在胰腺、腸、心臟、大腦、肺、卵巢及骨骼肌中均有TWEAKmRNA的表達,在肝臟及腎臟中亦少量表達。近年TWEAK及其特異性受體Fn14在創(chuàng)傷修復(fù)及組織再生方面介導(dǎo)多種炎癥反應(yīng)受到越來越多的關(guān)注,TWEAK作為一種促炎及促血管生成的細胞因子介導(dǎo)多種細胞的生長、增殖、凋亡,促炎性反應(yīng)和血管生成等作用。研究發(fā)現(xiàn)TWEAK與其受體Fn14結(jié)合主要通過核轉(zhuǎn)錄因子(nuclear factor-KB, NF-κB)、絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)、蛋白激酶B (protein kinase B,PKB)途徑等多個下游信號通路的瀑布樣反應(yīng)發(fā)揮作用。隨著研究深入,TWEAK與其受體Fn14促進AS、MF及介導(dǎo)心肌重塑等在心血管系統(tǒng)過程中所發(fā)揮的的作用越來越得到重視。TWEAK/Fn14能促進內(nèi)皮功能不全、增強炎癥反應(yīng)、促進平滑肌細胞增殖及遷移、上調(diào)MMPs表達、促進細胞死亡,參與動脈粥樣硬化斑塊進展,促使其不穩(wěn)定,進而導(dǎo)致急性冠脈綜合征的發(fā)生。在ST段抬高型急性心肌梗死病人時血清可溶性TWEAK (sTWEAK)顯著升高,其升高程度與病人的短期預(yù)后密切相關(guān)。在心肌纖維化方面,研究發(fā)現(xiàn)在自發(fā)性高血壓大鼠(spontaneous hypertension rat, SHR)心肌中Fn14表達明顯增強,Fn14可能參與SHR心肌纖維化的過程,培哚普利可能通過抑制其表達減輕心肌纖維化。進而在體外細胞水平發(fā)現(xiàn)TWEAK/Fn14明顯促進CFs中NF-κB和MMP9mRNA及蛋白表達,而以PDTC抑制NF-κB通路后,CFs增殖降低且MMP9表達減少。進而揭示TWEAK通過激活經(jīng)典的NF-κB通路能促CFs增殖和膠原合成,參與心肌纖維化的發(fā)生、發(fā)展過程發(fā)現(xiàn)。Chen等研究發(fā)現(xiàn)TWEAK通過激活經(jīng)典的NF-κB通路,促進大鼠CFs增殖和Ⅰ/Ⅲ膠原合成,促進心肌纖維化進展。然而,在心肌纖維化方面相關(guān)研究發(fā)現(xiàn),TWEAK作為一種具有多種活性的炎癥因子,除能激活經(jīng)典的NF-κB通路外,還可通過介導(dǎo)Fn14激活非經(jīng)典的NF-κB通路以及MAPK通路,然而相關(guān)研究未見報道。因此,本文通過觀察TWEAK介導(dǎo)P38絲裂原活化蛋白激酶(P38mitogen-activated protein kinases, P38MAPK)通路對大鼠心肌成纖維細胞中膠原代謝影響,及MMP-1表達的影響,以進一步明確TWEAK參與心肌纖維化的機制。 目的： 探討腫瘤壞死因子樣凋亡微弱誘導(dǎo)劑(TWEAK)影響心肌成纖維細胞(CFs)中Ⅰ型膠原和基質(zhì)金屬蛋白酶1(matrix metalloproteinase-1, MMP-1)表達的作用機制。 方法： 1.采用胰蛋白酶消化法及時間差法培養(yǎng)并純化新生Wistar大鼠心肌成纖維細胞。并采用倒置顯微鏡及波形蛋白免疫熒光法觀察并鑒定心肌成纖維細胞。 2.加入重組人TWEAK(rhTWEAK)及P38MAPK抑制劑(SB203580)干預(yù)。 將實驗分為①對照組：不加干預(yù)因素；②TWEAK組：加入100ng/mLTWEAK;③TWEAK+SB203580組：加入100ng/mLTWEAK+SB203580。 3.各實驗組分別采用MTT法檢測CFs增殖；western blot法檢測Ⅰ型膠原蛋白及磷酸化P38MAPK(P-P38MAPK)蛋白表達；qRT-PCR法檢測Ⅰ型膠原mRNA表達；ELASA法檢測CFs細胞培養(yǎng)液中MMP-1的濃度。 結(jié)果： 1.CFs的形態(tài)觀察及鑒定 培養(yǎng)48h后,CFs已長滿或接近長滿單層培養(yǎng)瓶,長滿或接近長滿單層。于倒置顯微鏡下觀察,CFs多呈梭形,無自發(fā)性搏動。其細胞核較大,胞漿透明。采用波形蛋白免疫熒光鑒定,CFs的波形蛋白呈現(xiàn)陽性反應(yīng),其胞漿內(nèi)圍繞胞核呈現(xiàn)綠色網(wǎng)絡(luò)狀物質(zhì)。 2.干預(yù)因素對CFs增殖的影響 rhTWEAK干預(yù)心肌成纖維細胞48h后,對照組(0.302±0.019),TWEAK組(0.493±0.042), TWEAK+SB203580組(0.362±0.039)三者間相比較,TWEAK組較對照組可顯著促進CFs增殖,差異有統(tǒng)計學(xué)意義(P0.01)；而TWEAK+SB203580組較對照組比較也可促進心肌成纖維細胞增殖(P0.05)。SB203580干預(yù)組及未干預(yù)組比較,CFs增殖程度降低,差異具有統(tǒng)計學(xué)意義(P0.05)。 3.各組對Ⅰ型膠原mRNA和Ⅰ型膠原蛋白的表達 以上各實驗組終止干預(yù)后,測定以上各組中Ⅰ型膠原mRNA表達分別為TWEAK組(6.080±0.676),TWEAK+SB203580組(3.400±0.810)。與對照組比較,TWEAK組及TWEAK+SB203580組中Ⅰ型膠原mRNA表達水平明顯增多,差異間具有統(tǒng)計學(xué)意義(P0.05)。在特異性阻斷P38MAPK通路后,其Ⅰ型膠原mRNA表達水平較TWEAK組降低,二者間差異有統(tǒng)計學(xué)意義(P0.05),見圖3a。與對照組比較,二者Ⅰ型膠原蛋白表達明顯升高差異(P0.05)。同TWEAK組相比,TWEAK+SB203580組中Ⅰ型膠原蛋白較其減少42.6%,差異有統(tǒng)計學(xué)意義(P0.05)。 4.各組CFs中P-P38MAPK蛋白的表達 各實驗組間相比,TWEAK組較對照組及TWEAK+SB203580組,可明顯促進CFs中P-P38MAPK蛋白表達(P0.01)。TWEAK+SB203580組與對照組相比,TWEAK+SB203580組部分抑制P-P38MAPK蛋白表達(P0.05)。 5.各組CFs中MMP-1的表達 各實驗組終止干預(yù)后,應(yīng)用ELISA法測定各組心肌成纖維細胞中MMP-1濃度,TWEAK組較其他兩組明顯上調(diào)CFs中MMP-1表達,差異有統(tǒng)計學(xué)意義(P0.05)。TWEAK+SB203580組與TWEAK組相比,部分抑制心肌成纖維細胞培養(yǎng)液中MMP-1的表達,差異有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論： 1.TWEAK/Fn14可顯著促進大鼠心肌成纖維細胞增殖,上調(diào)細胞中Ⅰ型膠原mRNA及Ⅰ型膠原蛋白和P-P38MAPK蛋白表達,并促進炎癥因子MMP-1表達。 2.應(yīng)用SB203580后,可部分抑制TWEAK促進心肌成纖維細胞增殖程度,并部分抑制細胞中Ⅰ型膠原mRNA、I型膠原蛋白和P-P38MAPK蛋白及MMP-1表達。 3.本實驗通過進一步研究發(fā)現(xiàn),除經(jīng)典的NF-κB途徑外,TWEAK通過P38MAPK通路介導(dǎo)CFs的增殖及ECM代謝異常,從而進一步闡明TWEAK/Fnl4參與MF的機制。從而為全面揭示心肌纖維化的研究提供新的途徑和方法,然而MAPK級聯(lián)是一個錯綜復(fù)雜的相互交錯的網(wǎng)絡(luò)系統(tǒng),本實驗只是通過局部抑制某一通路的方法來研究,并且對于JNK、ER及其余MAPK等信號轉(zhuǎn)導(dǎo)途徑未做進一步深入研究。
[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錛，

本文編號：1798394