本文選題：肝纖維化　切入點(diǎn)：miR-484　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景肝纖維化是指當(dāng)肝臟受到各類有害因素的損傷時(shí),細(xì)胞外基質(zhì)(extracellular matrix,ECM)沉積而導(dǎo)致肝臟正常結(jié)構(gòu)和功能發(fā)生改變的病理過(guò)程。多種慢性肝病均可經(jīng)此過(guò)程并最終進(jìn)展為預(yù)后極差的肝硬化和肝癌。所以,針對(duì)肝纖維化的早期診斷和治療成為研究的重中之重。而目前為止,肝穿刺仍為診斷肝纖維化的金標(biāo)準(zhǔn),但因其有創(chuàng)性而得不到廣泛、及時(shí)實(shí)施,在一定程度上耽誤了患者的早期診治。雖已有肝纖維化無(wú)創(chuàng)性早期診斷的方法,但均不十分理想,新的更有效的手段亟待被發(fā)現(xiàn),這就有必要對(duì)肝纖維化發(fā)生發(fā)展的分子機(jī)制行進(jìn)一步深入研究。miRNA(micro RNA)是一段大小約20~25個(gè)核苷酸序列的小分子片段,其在真核細(xì)胞中廣泛存在,可通過(guò)負(fù)向調(diào)控其靶基因參與多種疾病的發(fā)生和進(jìn)展,且在內(nèi)分泌、心血管和腫瘤等多種研究領(lǐng)域受到廣泛關(guān)注。目前亦有多種研究表明miRNA與肝纖維化發(fā)生密切相關(guān),其可通過(guò)影響肝星狀細(xì)胞(hepatic stellated cell,HSC)的活化、增殖、凋亡、遷移等細(xì)胞學(xué)功能,在肝纖維發(fā)生及進(jìn)展過(guò)程中起促進(jìn)或抑制作用。本課題組多年來(lái)一直從事miRNAs與肝纖維化發(fā)生發(fā)展的分子機(jī)制研究,前期工作發(fā)現(xiàn):miR-484在DMN誘導(dǎo)的大鼠肝纖維化模型中呈顯著性差異表達(dá),且相關(guān)文章已經(jīng)發(fā)表。本研究為進(jìn)一步探討miR-484是否對(duì)HSC細(xì)胞功能學(xué)產(chǎn)生影響,通過(guò)提取原代HSC,檢測(cè)到miR-484在HSC自然活化過(guò)程中表達(dá)呈上升趨勢(shì)。并通過(guò)生物信息學(xué)和雙熒光素酶報(bào)告基因?qū)嶒?yàn)篩選并驗(yàn)證得知HIPK1(homeodomain interacting protein kinase 1,同源結(jié)構(gòu)域交互蛋白激酶1)為miR-484的靶基因。已有研究表明,HIPK1可調(diào)控細(xì)胞增殖、分化、凋亡等多種生物過(guò)程。本研究中同樣發(fā)現(xiàn)miR-484可通過(guò)靶向HIPK1促進(jìn)HSC活化并抑制其凋亡,且在此過(guò)程中伴有Wnt/β-catenin信號(hào)通路的激活,若在疾病早期對(duì)miR-484的表達(dá)進(jìn)行抑制,或許可阻遏肝纖維化進(jìn)程。研究目的本研究在前期工作的基礎(chǔ)上,旨在進(jìn)一步探討miR-484參與肝纖維化形成的具體分子機(jī)制。共包括三個(gè)部分:(一)檢測(cè)miR-484在大鼠原代HSC自然活化過(guò)程中的表達(dá)變化;(二)miR-484靶基因的篩選及驗(yàn)證;(三)miR-484對(duì)HSC活化和凋亡的信號(hào)通路機(jī)制研究。研究方法一、檢測(cè)miR-484在大鼠原代HSC自然活化過(guò)程中的表達(dá)變化(一)提取大鼠原代hsc,以自發(fā)熒光實(shí)驗(yàn)鑒定所提細(xì)胞的純度;將培養(yǎng)2天內(nèi)的細(xì)胞作為靜止態(tài),培養(yǎng)至14天的細(xì)胞作為完全活化態(tài),并用免疫熒光實(shí)驗(yàn)鑒定細(xì)胞狀態(tài);(二)qrt-pcr檢測(cè)原代細(xì)胞不同時(shí)期mir-484的表達(dá)水平。二、mir-484靶基因的篩選及驗(yàn)證(一)以mir-484為關(guān)鍵詞,在microrna.org、mirbase、targetscan等網(wǎng)站進(jìn)行了靶基因預(yù)測(cè),并對(duì)感興趣的靶基因進(jìn)行g(shù)eneontology(go)分析,發(fā)掘其可能參與的功能;(二)雙熒光素酶報(bào)告基因驗(yàn)證mir-484與hipk1之間是否存在直接靶向關(guān)系。(三)利用hsc-t6細(xì)胞株進(jìn)行轉(zhuǎn)染實(shí)驗(yàn),將mir-484inhibitor/mimics及其陰性對(duì)照轉(zhuǎn)染入細(xì)胞48h后,提取細(xì)胞總蛋白。利用westernblot技術(shù)檢測(cè)hipk1是否被mir-484負(fù)向調(diào)控;三、mir-484對(duì)hsc活化和凋亡的信號(hào)通路機(jī)制研究(一)在hsc-t6細(xì)胞株中轉(zhuǎn)染mir-484inhibitor后,qrt-pcr和westernblot檢測(cè)不同分組之間肝纖維化相關(guān)指標(biāo)α-平滑肌肌動(dòng)蛋白(α-smoothmuscleactin,α-sma)和?型膠原(type?collagen,col?)表達(dá)變化;(二)在hsc-t6細(xì)胞株中轉(zhuǎn)染mir-484inhibitor后,利用qrt-pcr和westernblot技術(shù)檢測(cè)wnt-3a、wnt-5a、β-catenin表達(dá)是否發(fā)生相應(yīng)改變,以檢測(cè)mir-484對(duì)hsc細(xì)胞功能的調(diào)控,是否通過(guò)wnt/β-catenin信號(hào)通路實(shí)現(xiàn);(三)annexinv-fitc/pi雙染法用以檢測(cè)不同分組之間細(xì)胞凋亡的差異。四、統(tǒng)計(jì)學(xué)方法:計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差表示;兩組數(shù)據(jù)之間比較采用t檢驗(yàn);統(tǒng)計(jì)軟件采用spss21.0;統(tǒng)計(jì)學(xué)顯著性定義為p0.05。研究結(jié)果一、mir-484在大鼠原代hsc自然活化過(guò)程中的表達(dá)變化(一)自發(fā)熒光實(shí)驗(yàn)鑒定結(jié)果提示所提原代hsc的純度可達(dá)90%以上,2天內(nèi)靜止態(tài)的細(xì)胞呈橢圓形,脂質(zhì)含量豐富,在328nm波長(zhǎng)紫外光照射下,可發(fā)射出黃綠色熒光;培養(yǎng)至14天達(dá)到完全活化狀態(tài),細(xì)胞延展生長(zhǎng),呈星狀;免疫熒光實(shí)驗(yàn)顯示活化態(tài)細(xì)胞中α-sma表達(dá)量明顯升高;(二)qrt-pcr結(jié)果提示,相對(duì)于靜止態(tài),活化態(tài)hsc中mir-484表達(dá)呈顯著性升高(p0.05)。二、mir-484靶基因的篩選與驗(yàn)證(一)生物信息學(xué)分析結(jié)果提示,hipk1與mir-484在3’utr區(qū)域存在兩個(gè)結(jié)合位點(diǎn);(二)雙熒光素酶報(bào)告基因驗(yàn)證miR-484與HIPK1之間確實(shí)存在靶向關(guān)系;(三)Western Blot結(jié)果表明,在HSC-T6細(xì)胞株中轉(zhuǎn)染miR-484 inhibitor后,HIPK1在蛋白水平表達(dá)明顯上調(diào);而轉(zhuǎn)染miR-484 mimics后,HIPK1在蛋白水平表達(dá)明顯下調(diào)。三、miR-484對(duì)HSC活化和凋亡的信號(hào)通路機(jī)制研究(一)相對(duì)于陰性對(duì)照組,轉(zhuǎn)染miR-484 inhibitor后48h,HSC-T6細(xì)胞株中α-SMA和col?在mRNA和蛋白水平表達(dá)均明顯下降(P0.05);而空白對(duì)照組與陰性對(duì)照組表達(dá)則無(wú)明顯差異(P0.05);(二)轉(zhuǎn)染miR-484 inhibitor后,Wnt/β-catenin信號(hào)通路相關(guān)分子Wnt-3a、Wnt-5a、β-catenin表達(dá)下降(P0.05);(三)人為下調(diào)miR-484后,流式細(xì)胞儀檢測(cè)得HSC-T6細(xì)胞凋亡率明顯升高(P0.05)。結(jié)論一、miR-484在原代HSC自然活化過(guò)程中表達(dá)升高;二、HIPK1是miR-484的直接靶基因;三、miR-484靶向HIPK1促進(jìn)HSC活化并抑制其凋亡,使得細(xì)胞外基質(zhì)合成增多,促進(jìn)肝纖維化形成及進(jìn)展;四、miR-484靶向HIPK1對(duì)HSC細(xì)胞功能的調(diào)控,可能是通過(guò)Wnt/β-catenin信號(hào)通路實(shí)現(xiàn)的。
[Abstract]:The research background of liver fibrosis is when the liver caused by various harmful factors of injury, extracellular matrix (extracellular matrix ECM) deposition caused the pathological process of the normal structure and function of liver changes. Chronic liver diseases may pass through this process and ultimately progress to prognosis of cirrhosis and liver cancer. So, according to becometop Study on the early diagnosis and treatment of liver fibrosis. Liver biopsy is still so far, the gold standard for diagnosis of liver fibrosis, but because it is invasive and not widely implemented in a timely manner, to a certain extent delayed the early diagnosis and treatment of patients. Although liver fibrosis non-invasive methods for early diagnosis. But they are not very ideal, a new and more effective instrument to be found, it is necessary for the development of liver fibrosis. The molecular mechanism for the further research on.MiRNA (micro RNA) is a size of about 20~25 Small molecular fragment nucleotide sequences, which exists widely in eukaryotic cells, occurrence and progress can be involved in a variety of diseases to regulate the target gene by negative, and in many research fields of endocrine, cardiovascular and cancer received extensive attention. At present, there are many studies show that miRNA and liver fibrosis is closely related to the the effect of hepatic stellate cells (hepatic stellated cell, HSC) activation, proliferation, apoptosis, migration and other cellular functions, in the process and progress of liver fibrosis in promoting or inhibiting effect. Molecular mechanism of the group has been engaged in miRNAs and liver fibrosis occurrence and development of this topic, previous work found that miR-484 was significantly the difference in rat liver fibrosis model induced by DMN expression, and related articles have been published. This study to further investigate the effect of miR-484 on HSC cell function, provided by The primary HSC, detected miR-484 activation increased expression in the process of HSC. And through bioinformatics and dual luciferase reporter assay to screen and verify that the HIPK1 (homeodomain interacting protein kinase 1 homeodomain interacting protein kinase 1) is the target gene of miR-484. Studies have shown that HIPK1 can regulate cell the proliferation, differentiation, apoptosis and other biological processes. This study also found that miR-484 can promote HSC activation and inhibit its apoptosis by targeting HIPK1 activation, and in the process with Wnt/ beta -catenin signaling pathway, if early in the disease on the expression of miR-484 was inhibited, or permit repression of hepatic fibrosis. This study on the basis of previous work, to further investigate the molecular mechanism of miR-484 involved in the formation of liver fibrosis. Consists of three parts: (a) the detection of miR-484 in primary rat HSC natural expression changes in the process; (two) screening and identification of target genes of miR-484; (three) research on the mechanism of HSC signaling pathway activation and apoptosis of miR-484. The method of detection of miR-484 activation, natural expression change in the process of primary rat HSC (1) extract in primary rat HSC, the experimental identification of the autofluorescence of cell purity; the culture within 2 days of the cell as a stationary state, after 14 days of culture the cells as a fully activated state, and cells were identified by immunofluorescence; (two) the expression of qRT-PCR was detected in different periods of mir-484 primary cells. Two, screening and verification mir-484 target gene (1) with mir-484 as key words in microrna.org, miRBase, targetscan and other sites of target genes, and target genes of interest were geneontology (go) analysis, explore the possible involvement of function; (two) dual luciferase substrate Because mir-484 and hipk1 verify whether there is a direct relationship between the target. (three) using HSC-T6 cell transfection experiments, mir-484inhibitor/mimics and negative control were transfected into 48h cells after extraction of total cellular protein. Westernblot was used to detect whether hipk1 is mir-484 negative regulation; three, study on the mechanism of HSC signaling pathway activation and apoptosis mir-484 (a) in HSC-T6 cells after transfection of mir-484inhibitor, qRT-PCR and Westernblot detection between the different groups related to liver fibrosis of alpha smooth muscle actin (alpha -smoothmuscleactin, alpha -sma) and collagen (type?? collagen, col?) expression; (two) in HSC-T6 cells after transfection of mir-484inhibitor, detected by Wnt-3a, qRT-PCR and Westernblot Wnt-5a, beta -catenin expression is changed, in order to detect mir-484 on HSC cell function regulation, whether through wnt/ beta -cate NIN signal pathway; (three) annexinv-fitc/pi double staining method used to detect the differences between the different groups of apoptosis. Four, statistical methods: measurement data with standard deviation; between the two groups of data were compared using t test using spss21.0 statistical software;; statistical significance is defined as the results of p0.05., mir-484 expression change in the process of primary rat HSC (1) autofluorescence experiment results suggest that the primary identification of the purity of HSC can reach more than 90%, within 2 days of resting cells were oval, lipid content at the wavelength of 328nm under UV light can emit yellow green fluorescence; cultured for 14 days to complete the activation of cell spreading, growth, are star shaped; immunofluorescence experiments showed increased expression of activation of alpha -sma cells; (two) the results of qRT-PCR showed that, relative to a stationary state, the activation of mir-484 expression in HSC state Was significantly increased (P0.05. Two), screening and identification of mir-484 target genes (1) bioinformatics analysis showed that hipk1 and mir-484 in the presence of the 3 'UTR region of two binding sites; (two) there is relationship between the targeted dual luciferase reporter gene verification of miR-484 and HIPK1; (three) Western Blot the results showed that in HSC-T6 cells transfected with miR-484 inhibitor, the expression of HIPK1 is upregulated at the protein level; and miR-484 mimics after transfection, the expression of HIPK1 at protein level was significantly reduced. Three. Study on the mechanism of HSC signaling pathway activation and apoptosis of miR-484 (a) compared with the negative control group, transfected with miR-484 inhibitor 48h, alpha -SMA Col and HSC-T6 cell lines? Decreased expression at mRNA and protein level (P0.05); and the blank control group and negative control group was no significant difference (P0.05); (two) miR-484 inhibitor after transfection, Wnt/ beta -catenin signal Pathway related molecules Wnt-3a, Wnt-5a, -catenin beta expression decreased (P0.05); (three) artificially lowered after miR-484, flow cytometry was used to detect HSC-T6 cell apoptosis rate increased significantly (P0.05). Conclusion the increased expression of miR-484 in a process of activation of primary HSC; two, HIPK1 is a direct target gene of miR-484; three, miR-484 targeting HIPK1 to promote HSC activation and apoptosis, the increased synthesis of extracellular matrix, and promote the progress of hepatic fibrosis; four, miR-484 targeting HIPK1 on the regulation of HSC cell function, may be achieved through Wnt/ beta -catenin signaling pathway.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2

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