本文選題：ANO1　切入點(diǎn)：TGF-β/smad3信號(hào)通路　出處：《南京醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景心肌梗死后心肌纖維化嚴(yán)重影響氧傳輸、電信號(hào)傳導(dǎo)、心臟舒張及收縮功能等,所以有效防治心肌梗死后心肌纖維化的發(fā)生發(fā)展顯得至關(guān)重要。然而,即使經(jīng)過數(shù)十年的不懈研究,截至目前為止仍未找到針對(duì)心肌纖維化的有效治療方法。Anoctamin 1(ANO1)于2008年首次被證實(shí)為經(jīng)典鈣激活氯離子通道(CaCC)的分子基礎(chǔ),自此引領(lǐng)了世界范圍內(nèi)對(duì)ANO1研究的熱潮。之后的眾多研究表明,ANO1參與了眾多細(xì)胞的增殖、遷移和分化等生理病理進(jìn)程,這從而奠定了我們假設(shè)ANO1參與了心肌梗死后心肌纖維化的基礎(chǔ)。目的研究ANO1對(duì)心肌梗死后心肌纖維化是否有抑制作用,并探討其作用機(jī)制。方法和內(nèi)容b)乳大鼠心肌成纖維細(xì)胞的分離和培養(yǎng)從新生1-3天Sprague-Dawley(SD)乳大鼠心臟提取和分離心肌細(xì)胞與心肌成纖維細(xì)胞,并于DMEM高糖培養(yǎng)基中培養(yǎng),心肌細(xì)胞以及第2-3代心肌成纖維細(xì)胞留供后續(xù)實(shí)驗(yàn)使用。c)構(gòu)建編碼ANO1的重組腺病毒載體及感染乳大鼠心肌成纖維細(xì)胞ANO1重組腺病毒載體由上海吉?jiǎng)P基因化學(xué)技術(shù)有限公司構(gòu)建的;本課題組檢測(cè)了 ANO1重組腺病毒載體感染ANO1的效率。d)心肌成纖維細(xì)胞的缺氧實(shí)驗(yàn)將培養(yǎng)的第2-3代心肌成纖維細(xì)胞進(jìn)行缺氧處理;繼而通過MTT檢測(cè)細(xì)胞增殖,qRT-PCR檢測(cè)ANO1基因表達(dá),蛋白免疫印跡檢測(cè)缺氧后相關(guān)蛋白的表達(dá)。e)心肌梗死動(dòng)物模型的構(gòu)建以及病毒載體的轉(zhuǎn)染采用8-10周齡C57B1/6J雄性小鼠進(jìn)行冠狀動(dòng)脈前降支結(jié)扎從而構(gòu)建心肌梗死模型;并于小鼠心肌中注入重組病毒載體從而完成ANO1重組腺病毒載體于小鼠體內(nèi)的轉(zhuǎn)染。f)過表達(dá)ANO1對(duì)心肌成纖維細(xì)胞缺氧后纖維化指標(biāo)的影響心肌成纖維細(xì)胞過表達(dá)ANO1后進(jìn)行缺氧處理,繼之通過MTT檢測(cè)細(xì)胞增殖,qRT-PCR檢測(cè)ANO1基因表達(dá),蛋白免疫印跡檢測(cè)纖維化相關(guān)蛋白的表達(dá)。g)過表達(dá)ANO1對(duì)小鼠心肌梗死后纖維化指標(biāo)的影響構(gòu)建ANO1小鼠模型后,結(jié)扎小鼠冠狀動(dòng)脈前降支構(gòu)建心梗模型;繼之通過免疫組化、蛋白免疫印跡檢測(cè)相關(guān)纖維化指標(biāo)。h)明確TGF-β/smad3通路在ANO1抑制心肌梗死后心肌纖維化中的作用;通過蛋白免疫印跡實(shí)驗(yàn)檢測(cè)缺氧后及過表達(dá)ANO1后TGF-β、p-smad3及相關(guān)纖維化指標(biāo)的蛋白表達(dá)。i)統(tǒng)計(jì)學(xué)處理所有數(shù)據(jù)通過SPSS 17.0系統(tǒng)進(jìn)行分析,并使用GraphPad Prism軟件進(jìn)行數(shù)據(jù)的圖像呈現(xiàn)。所有的實(shí)驗(yàn)都重復(fù)至少三次。數(shù)據(jù)進(jìn)行了方差分析和t檢驗(yàn),P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果1.ANO1表達(dá)于心肌成纖維細(xì)胞通過qRT-PCR、蛋白免疫印跡和細(xì)胞免疫熒光檢測(cè)心肌細(xì)胞與心肌成纖維細(xì)胞中ANO1表達(dá),得出ANO1較高水平的表達(dá)于心肌成纖維細(xì)胞中。2.心肌成纖維細(xì)胞缺氧后纖維化指標(biāo)與ANO1表達(dá)均升高1)MTT結(jié)果顯示:心肌成纖維細(xì)胞在缺氧0,6,8,10,16和24小時(shí)呈逐漸增殖趨勢(shì);2)蛋白免疫印跡結(jié)果顯示:a-SMA的表達(dá)于心肌成纖維細(xì)胞缺氧0,6,8,10,16和24小時(shí)呈逐漸增加趨勢(shì);ANO1蛋白表達(dá)也隨缺氧時(shí)間的增加而呈增加趨勢(shì),并于缺氧16小時(shí)時(shí)達(dá)到表達(dá)高峰;3)qRT-PCR結(jié)果顯示:ANO1 mRNA表達(dá)隨心肌成纖維細(xì)胞缺氧時(shí)間的增加而增加,并于缺氧16小時(shí)達(dá)到表達(dá)高峰。3.心肌梗死后ANO1表達(dá)增加蛋白免疫印跡結(jié)果顯示與假手術(shù)組相比心肌梗死后ANO1的表達(dá)增加。4.過表達(dá)ANO1降低了缺氧后心肌纖維化指標(biāo)1)首先qRT-PCR和蛋白免疫印跡結(jié)果證實(shí)心肌成纖維細(xì)胞過表達(dá)ANO1;2)MTT結(jié)果顯示:過表達(dá)ANO1心肌成纖維細(xì)胞于缺氧后細(xì)胞的增殖明顯降低;3)蛋白免疫印跡結(jié)果顯示:過表達(dá)ANO1心肌成纖維細(xì)胞于缺氧后其a-SMA和collagen I蛋白表達(dá)明顯降低;5.ANO1降低了心肌梗死后心肌纖維化程度1)Masson染色結(jié)果表明:過表達(dá)ANO1心肌組織于心肌梗死后其纖維化程度明顯降低;2)蛋白免疫印跡和免疫熒光結(jié)果顯示:過表達(dá)ANO1心肌組織于心肌梗死后其a-SMA蛋白表達(dá)明顯降低;6.過表達(dá)ANO1抑制了缺氧對(duì)TGF-β/smad3通路的激活作用1)蛋白免疫印跡結(jié)果顯示:心肌成纖維細(xì)胞于缺氧0,6,8,10,16和24小時(shí)TGF-β、p-smad3蛋白表達(dá)逐漸增加,且其增加趨勢(shì)與ANO1表達(dá)相一致;2)心肌成纖維細(xì)胞過表達(dá)ANO1,其細(xì)胞TGF-β、p-smad3表達(dá)于缺氧后明顯降低(與陰性對(duì)照空載腺病毒轉(zhuǎn)染組相比)。結(jié)論ANO1通過抑制TGF-β/smad3信號(hào)通路活性從而抑制心肌梗死后心肌纖維化。
[Abstract]:Background myocardial infarction after myocardial fibrosis affects oxygen transport, signal transduction, cardiac diastolic and systolic function, so the effective prevention and treatment of myocardial infarction after myocardial fibrosis is crucial. However, even after decades of unremitting study, so far,.Anoctamin has yet to find the effective method for treatment of myocardial fibrosis in 1 (ANO1) for the first time in 2008 was confirmed as the classical calcium activated chloride channel (CaCC) on the molecular basis, since leading the upsurge of ANO1 research in the world. After many studies show that ANO1 is involved in numerous cell proliferation, migration and differentiation of physiological and pathological processes, so as to lay a foundation that we assume that ANO1 is involved in myocardial infarction after the myocardial fibrosis. Objective to study the effect of ANO1 on myocardial fibrosis after myocardial infarction is inhibited, and explore its mechanism. Methods and contents of B ) in neonatal rat myocardial fibroblasts were isolated and cultured from postnatal day 1-3 Sprague-Dawley (SD) rat heart extraction and separation of myocardial cells and cardiac fibroblasts, and cultured in DMEM high glucose, myocardial cells and the 2-3 generation of cardiac fibroblasts left for subsequent experiments using the.C construct encoding ANO1) the myocardial recombinant adenovirus vector and infect neonatal rat fibroblast ANO1 recombinant adenovirus vector was constructed by Shanghai genechem Co. Ltd; the detection efficiency of the.D infected ANO1 ANO1 recombinant adenovirus vector) into myocardial hypoxia experiment fibroblasts cultured in the 2-3 generation of cardiac fibroblasts by hypoxia treatment; then the cell proliferation was detected by MTT, expression of ANO1 gene qRT-PCR, expression of.E related protein determined by Western blot after hypoxia) myocardial infarction animal model construction and disease The transfection of virus vector 8-10 weeks old male C57B1/6J mice of anterior descending coronary artery was ligated to construct the model of myocardial infarction; and injection of recombinant virus vector in mouse myocardium in order to complete the ANO1 recombinant adenovirus vector in vivo transfection of.F) overexpression of ANO1 on myocardial fibrosis effect of fiber cells after hypoxia in cardiac fibroblasts over expression of ANO1 cells after hypoxia treatment, followed by MTT cell proliferation assay, to detect the expression of ANO1 gene qRT-PCR and protein expression of.G related fibrosis determined by Western blot the expression construct) mouse model of ANO1 ANO1 effect on fibrosis of mice after myocardial infarction in mice after ligation of the anterior descending coronary artery following myocardial infarction model construction; by immunohistochemistry, Western blot detection of.H related fibrosis) clear TGF- beta /smad3 pathway in the inhibition of ANO1 after myocardial infarction heart muscle fiber The role of vitamin; by Western blotting assay after hypoxia and after overexpression of ANO1 beta TGF-, p-smad3 expression and fibrosis protein.I) statistical analysis all the data were analyzed by SPSS 17 system, and the data of the image is rendered using the GraphPad Prism software. All experiments were repeated at least three times. The data were analyzed by analysis of variance and t test, P0.05 was statistically significant. Results the expression of 1.ANO1 in cardiac fibroblasts by qRT-PCR, Western blotting and immunofluorescence detection of myocardial cells and ANO1 expression in myofibroblasts, expression of ANO1 reached high level in myocardial.2. into cardiac fibrosis index and ANO1 expression were increased in 1 fiber cells after hypoxia cells) MTT showed that cardiac fibroblasts in hypoxia 0,6,8,10,16 and 24 hours showed a gradual growth trend; 2) Western blot The results showed that the expression of a-SMA in cardiac fibroblasts and 0,6,8,10,16 hypoxia 24 hours increased gradually; the expression of ANO1 protein also increased with the increase of hypoxia was increased, and reached the peak of expression in hypoxia 16 hours; 3) qRT-PCR showed that ANO1 mRNA expression in heart muscle cells to increase the fiber of hypoxic time increased, and reached the peak expression of.3. after myocardial infarction and increase the expression of ANO1 protein by Western blot results showed that compared with the sham operation group, overexpression of ANO1 reduces myocardial fibrosis after hypoxia 1 ANO1 increased expression of.4. after myocardial infarction in the first 16 hours of hypoxia) qRT-PCR and Western blot results showed that cardiac fibroblasts overexpressing ANO1; 2 MTT) results showed that overexpression of ANO1 in cardiac fibroblasts after hypoxia cell proliferation decreased significantly; 3) Western blot results showed that overexpression of ANO1 in cardiac fibroblasts The expression of a-SMA and collagen in cell I protein decreased significantly after hypoxia; 5.ANO1 reduces myocardial fibrosis after myocardial infarction 1) Masson staining results showed that: the expression of ANO1 in myocardial tissue after myocardial infarction, the degree of fibrosis decreased significantly; 2) Western blotting and immunofluorescence results showed that overexpression of ANO1 in myocardial tissue was significantly lower in the protein expression of a-SMA after myocardial infarction; 6. overexpression of ANO1 inhibits the activation of hypoxia on TGF- beta /smad3 pathway 1) Western blot results showed that cardiac fibroblasts in hypoxia 0,6,8,10,16 and 24 hours TGF- beta, p-smad3 protein expression gradually increased, and the increasing trend is consistent with the expression of ANO1; 2) myocardial overexpression of ANO1 fibroblasts, the cells of TGF- beta, p-smad3 expression decreased significantly after hypoxia (compared with negative control) empty adenovirus transfection group. Conclusion ANO1 through inhibition of TGF- beta /smad3 signal. The activity of the road inhibits myocardial fibrosis after myocardial infarction.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R542.22

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相關(guān)期刊論文 前1條

1 Dietmar Benke;Khaled Zemoura;Patrick J Maier;;Modulation of cell surface GABA B receptors by desensitization,trafficking and regulated degradation[J];World Journal of Biological Chemistry;2012年04期

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本文編號(hào)：1633413