發(fā)布時間：2018-05-12 09:20

  本文選題：MiR-145 + 肝纖維化　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：肝纖維化是指一種或多種慢性致病因子致使肝細(xì)胞彌漫性損傷,進(jìn)而使肝星狀細(xì)胞(hepatic stellate cell,HSC)活化增殖并導(dǎo)致細(xì)胞外基質(zhì)(extracellular matrix,ECM)大量沉積的一種病理過程。HSC的活化增殖是肝纖維化發(fā)生發(fā)展的中心環(huán)節(jié),抑制HSC的活化增殖可減緩或阻止肝纖維化的發(fā)生。研究發(fā)現(xiàn)在機體多種組織或器官的纖維化疾病進(jìn)程中,都發(fā)現(xiàn)有microRNA(miRNA)的調(diào)控作用。Mi RNA能調(diào)控多種促肝纖維化或抑肝纖維化相關(guān)因子,反過來,肝纖維化的發(fā)生又能使多種mi RNA表達(dá)發(fā)生變化。近年也有研究發(fā)現(xiàn)miR-145與多種纖維化的發(fā)生發(fā)展密切相關(guān),但是miR-145在肝纖維化以及肝星狀細(xì)胞活化過程中的作用鮮有報道,這需要進(jìn)一步的研究。本實驗主要觀察miR-145在肝纖維化發(fā)生過程中和HSC活化過程中的表達(dá)變化,并進(jìn)一步探討miR-145對轉(zhuǎn)化生長因子β1(transforming growth factor-β1,TGF-β1)誘導(dǎo)的HSC活化增殖的影響及其可能的作用分子機制。研究內(nèi)容主要概況如下:(1)ZEB2和miR-145在肝纖維化過程中的表達(dá)體內(nèi)我們采用四氯化碳(carbon tetrachloride,CCl4)皮下注射4周建立小鼠肝纖維化模型,取肝組織進(jìn)行HE和Masson染色,同時檢測α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)和I型膠原(Collagen type1,Colα1)的蛋白表達(dá)鑒定模型構(gòu)建成功。通過qPCR技術(shù)檢測肝組織和原代HSC中ZEB2和miR-145的表達(dá),同時采用免疫熒光雙染技術(shù)觀察ZEB2是否表達(dá)于肝星狀細(xì)胞中。體外我們采用不同濃度TGF-β1分別對大鼠肝星狀細(xì)胞HSC-T6和人肝星狀細(xì)胞lx-2進(jìn)行刺激,24h后檢測ZEB2和mir-145的表達(dá)變化。結(jié)果發(fā)現(xiàn)在ccl4誘導(dǎo)的肝纖維化小鼠的肝組織和原代HSC中以及體外TGF-β1處理的HSC中,mir-145的表達(dá)均是降低的,而ZEB2表達(dá)水平升高。ZEB2和α-sma雙重染色顯示,ZEB2主要表達(dá)于α-sma陽性的細(xì)胞即HSC中。(2)mir-145對TGF-β1誘導(dǎo)的HSC-T6細(xì)胞活化增殖的影響在HSC-T6中,采用mir-145mimics和inhibitor建立過表達(dá)和沉默mir-145細(xì)胞模型,同時采用TGF-β1(10ng/ml,24h)進(jìn)行刺激,westernblot檢測處理后的HSC-T6細(xì)胞中colα1和α-sma的蛋白表達(dá),流式細(xì)胞術(shù)檢測細(xì)胞周期情況。結(jié)果顯示:過表達(dá)mir-145能抑制colα1和α-sma的蛋白表達(dá),同時使TGF-β1引起的HSC-T6增殖明顯減少;沉默mir-145,使TGF-β1誘導(dǎo)的HSC-T6增殖增多,同時colα1和α-sma的蛋白表達(dá)水平進(jìn)一步升高。(3)ZEB2對TGF-β1誘導(dǎo)的HSC-T6細(xì)胞活化增殖的影響在HSC-T6細(xì)胞中,采用ZEB2小干擾rna(ZEB2sirna)建立沉默ZEB2細(xì)胞模型,同時采用TGF-β1(10ng/ml,24h)進(jìn)行刺激,westernblot檢測處理后colα1和α-sma蛋白表達(dá),流式細(xì)胞術(shù)檢測細(xì)胞周期情況。結(jié)果顯示:沉默ZEB2能抑制colα1和α-sma的蛋白表達(dá),同時使TGF-β1誘導(dǎo)的HSC-T6增殖明顯減少。(4)mir-145與ZEB2之間靶向關(guān)系的研究使用生物信息學(xué)軟件分析了解到mir-145同ZEB2之間存在可能的靶向關(guān)系。在HSC-T6細(xì)胞中,我們采用mir-145mimics和inhibitor建立過表達(dá)和沉默細(xì)胞模型,westernblot檢測ZEB2mrna和蛋白表達(dá)情況。結(jié)果顯示:過表達(dá)mir-145能抑制ZEB2mrna和蛋白表達(dá),而沉默mir-145能升高ZEB2mrna和蛋白表達(dá)。進(jìn)一步,本課題組采用雙熒光素(螢火蟲和海腎)酶報告基因?qū)嶒灤_認(rèn)兩者的靶向關(guān)系,發(fā)現(xiàn)共轉(zhuǎn)染mir-145mimics和ZEB2的3’端非編碼區(qū)(3’untranslatedregion,3’-UTR)質(zhì)粒能抑制HSC-T6細(xì)胞中ZEB2 3’-UTR的相對熒光值表達(dá)。(5)miR-145和ZEB2對Wnt/β-catenin信號通路的影響在HSC-T6中,采用miR-145 mimics、inhibitor和ZEB2 siRNA進(jìn)行轉(zhuǎn)染以后,采用Western blot技術(shù)檢測β-catenin及其下游靶蛋白cyclinD1和c-Myc的表達(dá)情況。結(jié)果顯示:過表達(dá)miR-145和沉默ZEB2能抑制β-catenin及其相關(guān)靶蛋白的表達(dá),而沉默miR-145則能促進(jìn)β-catenin及其相關(guān)靶蛋白的表達(dá)。上述結(jié)果提示,miR-145可能通過靶向ZEB2來負(fù)調(diào)控Wnt/β-catenin信號通路進(jìn)而影響HSC-T6細(xì)胞的活化增殖。
[Abstract]:Liver fibrosis refers to one or more chronic pathogenic factors that cause diffuse damage to the liver cells, and then the hepatic stellate cell (HSC) is activated and proliferated and the extracellular matrix (extracellular matrix, ECM) is deposited in a large number of pathological processes. The activation and proliferation of.HSC is the central link in the development of liver fibrosis, and the inhibition of HSC is the central part of the liver fibrosis. It is found that in the process of fibrosis of various tissues or organs of the body, the regulatory role of microRNA (miRNA),.Mi RNA, can regulate a variety of liver fibrosis or liver fibrosis related factors. In turn, the occurrence of liver fibrosis can also cause a variety of MI RNA expression. Recent studies have also found that miR-145 is closely related to the development of multiple fibrosis, but the role of miR-145 in liver fibrosis and the activation of hepatic stellate cells is rarely reported. This need further study. This experiment mainly observed the expression of miR-145 during the activation of Cheng Zhonghe HSC in liver fibrosis, and the results were also observed. One step is to investigate the effect of miR-145 on the activation and proliferation of HSC induced by transforming growth factor beta 1 (transforming growth factor- beta 1, TGF- beta 1) and its possible molecular mechanism. The main contents are as follows: (1) we use carbon tetrachloride (carbon tetrachloride, CCl4) subcutaneous injection for the expression of ZEB2 and miR-145 in the process of liver fibrosis. The liver fibrosis model of mice was established for 4 weeks. The liver tissue was stained with HE and Masson, and the protein expression identification model of alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA) and I collagen (Collagen Type1, Col a 1) was successfully constructed. The immunofluorescence double staining technique was used to observe whether ZEB2 was expressed in hepatic stellate cells. In vitro, different concentrations of TGF- beta 1 were used to stimulate the rat hepatic stellate cells HSC-T6 and human hepatic stellate cells lx-2. After 24h, the expression of ZEB2 and miR-145 was detected. The results were found in the liver and primary HSC induced by CCl4 in mice. And in the HSC treated with TGF- beta 1 in vitro, the expression of miR-145 was reduced, while ZEB2 expression level increased by.ZEB2 and alpha -sma double staining showed that ZEB2 was mainly expressed in alpha -sma positive cells, HSC. (2) miR-145 has an effect on the activation and proliferation of HSC-T6 cells induced by TGF- beta 1. The silent miR-145 cell model was stimulated by TGF- beta 1 (10ng/ml, 24h), and Westernblot was used to detect the protein expression of col alpha 1 and alpha -sma in the HSC-T6 cells treated with Westernblot. Flow cytometry was used to detect the cell cycle. The results showed that overexpression of miR-145 could inhibit the expression of col a 1 and alpha -sma, and the proliferation of TGF- beta 1 was significantly reduced. MiR-145, silencing miR-145, increased the proliferation of HSC-T6 induced by TGF- beta, while the protein expression level of col alpha 1 and alpha -sma increased further. (3) the effect of ZEB2 on the activation and proliferation of HSC-T6 cells induced by TGF- beta 1 in HSC-T6 cells, using ZEB2 small interference RNA (ZEB2sirna) to establish a silent cell model, and stimulated by beta 1. The expression of col alpha 1 and alpha -sma protein was detected by Westernblot, and cell cycle was detected by flow cytometry. The results showed that silent ZEB2 could inhibit the protein expression of col alpha 1 and alpha -sma, and the proliferation of HSC-T6 induced by TGF- beta 1 decreased significantly. (4) the study of the relationship between miR-145 and ZEB2 using the bioinformatics software analysis and understanding of mir-14 There is a possible targeting relationship between 5 and ZEB2. In HSC-T6 cells, we use mir-145mimics and inhibitor to establish the over expression and silencing cell model, and Westernblot to detect the expression of ZEB2mrna and protein. The results show that overexpression of miR-145 can inhibit the expression of ZEB2mrna and protein, while silence miR-145 can increase the expression of ZEB2mrna and protein. Step, we identified the target relationship between the two fluorescein (fluorescein and sea kidney) enzyme reporter gene experiment, and found that the 3 'terminal non coding region (3' untranslatedregion, 3 '-UTR) plasmids of CO transfected mir-145mimics and ZEB2 could inhibit the relative fluorescence value expression of ZEB2 3' -UTR in HSC-T6 cells. (5) miR-145 and ZEB2 pairs Wnt/ beta -catenin letter After transfection in HSC-T6, miR-145 mimics, inhibitor and ZEB2 siRNA were used to detect the expression of cyclinD1 and c-Myc of beta -catenin and its downstream target protein by Western blot technique. The results showed that the expression of beta and related target proteins could be inhibited by overexpression of miR-145 and silencing. It can promote the expression of beta -catenin and its related target proteins. These results suggest that miR-145 may negatively regulate the Wnt/ beta -catenin signaling pathway by targeting ZEB2 and then affect the activation and proliferation of HSC-T6 cells.

【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】：2017
【分類號】：R575.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前8條

1 潘志恒;成家茂;李永偉;何宏文;謝瑤;何義華;;大黃蟄蟲丸對大鼠肝星狀細(xì)胞活化增殖的影響[J];中山大學(xué)學(xué)報(醫(yī)學(xué)科學(xué)版);2009年03期

2 許靖霞,蔡仙德,王立新,譚劍萍;抗CD_3McAb誘導(dǎo)外周血單個核細(xì)胞活化增殖的觀察[J];南京鐵道醫(yī)學(xué)院學(xué)報;1998年04期

3 劉佳佳;阪根剛;恒松德五郎;;IL—2、IL—4和 IL—6對 T 細(xì)胞活化增殖的影響途徑比較[J];中國免疫學(xué)雜志;1992年03期

4 張萃;;LPS等絲裂原所致THP-1細(xì)胞活化增殖的優(yōu)化因素[J];山西醫(yī)科大學(xué)學(xué)報;2010年03期

5 李鳴，，楊敬，沈關(guān)心，張茜，劉忠北，劉慎沛，葉維新;淋巴細(xì)胞經(jīng)TCR－CD3活化增殖作用的分析[J];免疫學(xué)雜志;1995年02期

6 林長樂;曾耀英;曾祥鳳;趙令齋;黃秀艷;葉雪儀;;鷹嘴豆芽素A對小鼠T淋巴細(xì)胞體外活化增殖和細(xì)胞周期的影響[J];暨南大學(xué)學(xué)報(醫(yī)學(xué)版);2007年02期

7 劉琳琳;;IL-21重組載體的構(gòu)建及其誘導(dǎo)T細(xì)胞活化增殖的研究[J];中華臨床醫(yī)師雜志(電子版);2014年17期

8 陶輝;黃成;楊晶晶;馬陶陶;張磊;卞爾保;李政通;章慧;呂雄文;李俊;;RNAi介導(dǎo)MeCP2基因沉默對HSC-T6細(xì)胞活化增殖的影響[J];中國藥理學(xué)通報;2012年03期

相關(guān)會議論文 前2條

1 平潔;汪暉;;吲哚-3-原醇對肝星狀細(xì)胞活化增殖的抑制作用及其分子機制[A];中國藥理學(xué)會第九次全國會員代表大會暨全國藥理學(xué)術(shù)會議論文集[C];2007年

2 欒希英;張光波;段巧艷;胡玉敏;於葛華;段祥;張學(xué)光;;人骨髓間充質(zhì)干細(xì)胞對外周血T細(xì)胞活化增殖的調(diào)節(jié)作用及其機制[A];中國免疫學(xué)會第五屆全國代表大會暨學(xué)術(shù)會議論文摘要[C];2006年

相關(guān)碩士學(xué)位論文 前1條

1 楊晶晶;MeCP2調(diào)控PTCH1表達(dá)對大鼠HSC活化增殖的影響[D];安徽醫(yī)科大學(xué);2014年

本文編號：1878037