microRNA-200a抑制TGF-β1诱导肝星状细胞增殖作用的机制研究
发布时间：2018-08-20 18:48
【摘要】：肝纤维化（hepatic fibrosis，HF）通常是由体内外多种刺激因素，如酒精、肝毒物、酶催化剂及过敏原性物质等共同作用于肝脏所引起的细胞外基质（extracellular matrix，ECM）过度沉积的病变过程。肝星状细胞（hepatic stellatescell，HSC）的活化作为肝纤维化形成和发展的中心环节，可受到多条信号通路及细胞因子的影响，如Wnt/β-catenin通路、转化生长因子β1（TGF-β1）介导的TGF-β/Smad通路等。此外，除了受到传统信号通路的影响外，近年来发现，HSC的活化还可受到microRNA（miRNA）的调控。miRNA作为继转录因子外，参与基因转录调控的另一类新兴因子，在细胞的发育、分化、代谢及细胞信号通路转导等一系列生命活动中均产生了重要作用。其中，miR-200a在癌症及纤维化疾病中逐渐显示出的多重效应引起了研究人员的普遍关注。但miR-200a是否以及如何参与TGFβ和Wnt/β-catenin通路在HSC的活化则鲜有报道。本课题通过研究miR-200a在大鼠病理性肝纤维化组织及活化的HSC中的表达变化，在分子水平上，深刻探讨其对TGFβ和Wnt/β-catenin通路的影响，以揭示miR-200a介导HSC活化的新的作用机制。 本研究按照正常组10只、CCl4模型组15只的标准将雄性Sprague Dawley大鼠分为两组。自实验开始后，模型组大鼠给予1ml/kg50%CCl4植物油溶液皮下注射建立肝纤维化模型，每周2次，总计12周；正常组给予同剂量油溶液皮下注射。12周造模结束后，取肝组织进行HE及Masson胶原染色检测，并采用免疫组化法测出α-SMA在大鼠肝组织中的表达变化。通过实时定量PCR检测CCl4模型组大鼠肝纤维化组织及在不同浓度、时间点TGF-β1刺激的HSC中miR-200a的表达。通过脂质体LipofectamineTM2000转染miR-200a mimics（模拟物）至肝星状细胞内以观察miR-200a对TGF-β1诱导的HSC活化的影响，采用荧光倒置显微镜观察细胞的转染效率。对于体外培养的大鼠HSC，转染不同浓度的miR-200a mimics（60nM，80nM，100nM），4-6h后用5ng/ml TGF-β1刺激细胞24h或48h，采用四甲基偶氮唑盐（MTT）法，流式细胞术观察miR-200a对TGF-β1诱导的HSC的增殖、周期、凋亡的影响。运用生物数据库预测出miR-200a的潜在性功能靶点：转化生长因子β2（TGF-β2）和β-链蛋白（β-catenin）。利用双荧光素酶报告基因技术验证这一预测并采用PCR及Western blots等技术，分别检测TGF-β1诱导活化的HSC中α-SMA, TGF-β2, β-catenin mRNA及蛋白水平的表达。 结果发现，在体内实验中，与正常组相比，miR-200a在CCl4诱导的肝纤维化组织中，其表达是降低的。在体外实验中，用不同浓度（0，5，10，15ng/ml）的TGF-β1，以及在不同时间点（0，6，24，48h）用同一浓度的TGF-β1（5ng/ml）分别刺激HSC，miR-200a的表达呈现出剂量-时间依赖性下调趋势。与阴性对照组相比，不同浓度的miR-200a mimics（60nM，80nM，100nM）瞬时转染进入HSC，可以显著降低TGF-β1诱导的α-SMA的表达水平。过表达miR-200a可以明显抑制TGF-β1刺激的HSC的增殖及活化，但并未增加细胞的凋亡。此外，采用双荧光素酶报告系统证实了TGF-β2和β-catenin是miR-200a的功能性靶点。在TGF-β1刺激活化的HSC中，miR-200a可以显著降低TGF-β2的mRNA及蛋白水平表达，但对于其另一个靶点β-catenin，miR-200a只能降低其蛋白水平表达，对其mRNA水平则不产生影响。以上研究提示，miR-200a对TGF-β2及β-catenin二者的作用方式存在差异，miR-200a可通过影响不同靶蛋白（TGF-β2，β-catenin）的表达，参与调控TGF-β及Wnt/β-catenin通路，为探索肝纤维化中细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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2
本文编号：2194648
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2
【参考文献】
相关期刊论文 前1条
1 Roel NUSSE;Wnt signaling in disease and in development[J];Cell Research;2005年01期
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