【摘要】：肝纖維化（hepatic fibrosis，HF）通常是由體內(nèi)外多種刺激因素，如酒精、肝毒物、酶催化劑及過敏原性物質(zhì)等共同作用于肝臟所引起的細(xì)胞外基質(zhì)（extracellular matrix，ECM）過度沉積的病變過程。肝星狀細(xì)胞（hepatic stellatescell，HSC）的活化作為肝纖維化形成和發(fā)展的中心環(huán)節(jié)，可受到多條信號通路及細(xì)胞因子的影響，如Wnt/β-catenin通路、轉(zhuǎn)化生長因子β1（TGF-β1）介導(dǎo)的TGF-β/Smad通路等。此外，除了受到傳統(tǒng)信號通路的影響外，近年來發(fā)現(xiàn)，HSC的活化還可受到microRNA（miRNA）的調(diào)控。miRNA作為繼轉(zhuǎn)錄因子外，參與基因轉(zhuǎn)錄調(diào)控的另一類新興因子，在細(xì)胞的發(fā)育、分化、代謝及細(xì)胞信號通路轉(zhuǎn)導(dǎo)等一系列生命活動中均產(chǎn)生了重要作用。其中，miR-200a在癌癥及纖維化疾病中逐漸顯示出的多重效應(yīng)引起了研究人員的普遍關(guān)注。但miR-200a是否以及如何參與TGFβ和Wnt/β-catenin通路在HSC的活化則鮮有報道。本課題通過研究miR-200a在大鼠病理性肝纖維化組織及活化的HSC中的表達(dá)變化，在分子水平上，深刻探討其對TGFβ和Wnt/β-catenin通路的影響，以揭示miR-200a介導(dǎo)HSC活化的新的作用機(jī)制。 本研究按照正常組10只、CCl4模型組15只的標(biāo)準(zhǔn)將雄性Sprague Dawley大鼠分為兩組。自實(shí)驗開始后，模型組大鼠給予1ml/kg50%CCl4植物油溶液皮下注射建立肝纖維化模型，每周2次，總計12周；正常組給予同劑量油溶液皮下注射。12周造模結(jié)束后，取肝組織進(jìn)行HE及Masson膠原染色檢測，并采用免疫組化法測出α-SMA在大鼠肝組織中的表達(dá)變化。通過實(shí)時定量PCR檢測CCl4模型組大鼠肝纖維化組織及在不同濃度、時間點(diǎn)TGF-β1刺激的HSC中miR-200a的表達(dá)。通過脂質(zhì)體LipofectamineTM2000轉(zhuǎn)染miR-200a mimics（模擬物）至肝星狀細(xì)胞內(nèi)以觀察miR-200a對TGF-β1誘導(dǎo)的HSC活化的影響，采用熒光倒置顯微鏡觀察細(xì)胞的轉(zhuǎn)染效率。對于體外培養(yǎng)的大鼠HSC，轉(zhuǎn)染不同濃度的miR-200a mimics（60nM，80nM，100nM），4-6h后用5ng/ml TGF-β1刺激細(xì)胞24h或48h，采用四甲基偶氮唑鹽（MTT）法，流式細(xì)胞術(shù)觀察miR-200a對TGF-β1誘導(dǎo)的HSC的增殖、周期、凋亡的影響。運(yùn)用生物數(shù)據(jù)庫預(yù)測出miR-200a的潛在性功能靶點(diǎn)：轉(zhuǎn)化生長因子β2（TGF-β2）和β-鏈蛋白（β-catenin）。利用雙熒光素酶報告基因技術(shù)驗證這一預(yù)測并采用PCR及Western blots等技術(shù)，分別檢測TGF-β1誘導(dǎo)活化的HSC中α-SMA, TGF-β2, β-catenin mRNA及蛋白水平的表達(dá)。 結(jié)果發(fā)現(xiàn)，在體內(nèi)實(shí)驗中，與正常組相比，miR-200a在CCl4誘導(dǎo)的肝纖維化組織中，其表達(dá)是降低的。在體外實(shí)驗中，用不同濃度（0，5，10，15ng/ml）的TGF-β1，以及在不同時間點(diǎn)（0，6，24，48h）用同一濃度的TGF-β1（5ng/ml）分別刺激HSC，miR-200a的表達(dá)呈現(xiàn)出劑量-時間依賴性下調(diào)趨勢。與陰性對照組相比，不同濃度的miR-200a mimics（60nM，80nM，100nM）瞬時轉(zhuǎn)染進(jìn)入HSC，可以顯著降低TGF-β1誘導(dǎo)的α-SMA的表達(dá)水平。過表達(dá)miR-200a可以明顯抑制TGF-β1刺激的HSC的增殖及活化，但并未增加細(xì)胞的凋亡。此外，采用雙熒光素酶報告系統(tǒng)證實(shí)了TGF-β2和β-catenin是miR-200a的功能性靶點(diǎn)。在TGF-β1刺激活化的HSC中，miR-200a可以顯著降低TGF-β2的mRNA及蛋白水平表達(dá)，但對于其另一個靶點(diǎn)β-catenin，miR-200a只能降低其蛋白水平表達(dá)，對其mRNA水平則不產(chǎn)生影響。以上研究提示，miR-200a對TGF-β2及β-catenin二者的作用方式存在差異，miR-200a可通過影響不同靶蛋白（TGF-β2，β-catenin）的表達(dá)，參與調(diào)控TGF-β及Wnt/β-catenin通路，為探索肝纖維化中細(xì)胞的增殖和活化提供了新的研究思路。
[Abstract]:Hepatic fibrosis (HF) is a pathological process of excessive deposition of extracellular matrix (ECM) caused by various stimuli in vivo and in vitro, such as alcohol, hepatotoxins, enzyme catalysts and allergens. Activation of hepatic stellate cells (HSC) acts as hepatic fibers. In addition to the traditional signaling pathways, the activation of HSC has been found to be regulated by microRNA (microRNA) in recent years. In addition to transcription factors, another emerging class of factors involved in gene transcription regulation plays an important role in a series of life activities, such as cell development, differentiation, metabolism and cell signaling pathway transduction. It is rarely reported whether or how microRNAs participate in the activation of TGFbeta and Wnt/beta-catenin pathways in HSC. In order to reveal the novel activation of HSC mediated by microRNAs-200a, we studied the expression of microRNAs in rat hepatic fibrosis tissues and activated HSC, and explored the effects of microRNAs-200a on TGFbeta and Wnt/beta-catenin pathways at the molecular level. Mechanism of action.
In this study, male Sprague Dawley rats were divided into two groups according to the criteria of 10 normal rats and 15 CCl4 model rats. Hepatic tissue was stained with HE and Masson collagen, and the expression of alpha-SMA was detected by immunohistochemistry. The expression of microRNA-200a was detected by real-time quantitative PCR in hepatic fibrosis tissues of CCl4 model rats and in HSC stimulated by TGF-beta 1 at different concentrations and time points. MicroRNA-200a was transfected by liposome Lipofectamine TM2000. Mimics (mimics) were transfected into hepatic stellate cells to observe the effect of microRNAs-200a on the activation of HSC induced by TGF-beta 1. The transfection efficiency of HSC was observed by fluorescence inverted microscope. MTT assay and flow cytometry were used to observe the effects of microRNAs-200a on proliferation, cycle and apoptosis of TGF-beta 1-induced HSC. The potential functional targets of microRNAs-200a, transforming growth factor-beta 2 (TGF-beta 2) and beta-catenin (beta-catenin), were predicted using a biological database. This prediction was validated by dual luciferase reporter gene technique and confirmed by PCR and We. The expressions of alpha-SMA, TGF-beta 2, and beta catenin mRNA and protein in HSC activated by TGF-beta 1 were detected by stern blots.
The results showed that the expression of microRNA-200a in CCl4-induced hepatic fibrosis was lower in vivo than in the normal group. In vitro, the expression of HSC and microRNA-200a was stimulated by different concentrations of TGF-beta 1 (0,5,10,15 ng/ml) and the same concentration of TGF-beta 1 (5 ng/ml) at different time points (0,6,24,48 h) respectively. Compared with the negative control group, transient transfection of different concentrations of microRNAs (60nM, 80nM, 100nM) into HSC significantly decreased the expression of TGF-beta 1-induced alpha-SMA. Overexpression of microRNAs could significantly inhibit the proliferation and activation of HSC stimulated by TGF-beta 1, but did not increase cell apoptosis. The Luciferase Reporting System confirmed that TGF-beta 2 and beta catenin were functional targets of microRNAs.In activated HSC stimulated by TGF-beta 1, microRNAs-200a significantly decreased the expression of TGF-beta 2 mRNA and protein levels, but for another target beta catenin, microRNAs-200a only decreased the expression of protein levels, but did not affect their mRNA levels. It is suggested that the effects of microRNAs on TGF-beta 2 and beta catenin are different. MicroRNAs can regulate the expression of TGF-beta 2 and Wnt/beta catenin pathways by affecting the expression of different target proteins (TGF-beta 2, beta catenin). This provides a new research idea for exploring the proliferation and activation of cells in hepatic fibrosis.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Roel NUSSE;Wnt signaling in disease and in development[J];Cell Research;2005年01期

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