本文選題：短發(fā)卡RNA　切入點(diǎn)：小RNA　出處：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)感染引起的丙型病毒性肝炎及誘發(fā)的肝硬化、肝癌等疾病已嚴(yán)重威脅到人類健康,缺乏相應(yīng)的疫苗和抗病毒藥物的局限性是控制HCV感染和治療相關(guān)疾病的主要障礙。HCV主要依賴于宿主因子完成病毒的入侵、復(fù)制、組裝和釋放,因此找出與HCV相互作用的宿主因子有助于闡明HCV的感染過(guò)程和宿主應(yīng)答的分子機(jī)制,同時(shí)對(duì)發(fā)現(xiàn)新的抗病毒治療的藥物靶標(biāo)和研制有效的HCV疫苗有著重要的理論和實(shí)際意義。HCV感染能誘導(dǎo)宿主細(xì)胞發(fā)生自噬,HCV復(fù)制的起始依賴于自噬相關(guān)蛋白,而且HCV誘導(dǎo)的自噬通過(guò)抑制宿主細(xì)胞天然免疫應(yīng)答進(jìn)而促進(jìn)HCV的復(fù)制。但是HCV利用自噬進(jìn)行自身復(fù)制的具體分子機(jī)制目前仍不清楚。本課題組在篩選與HCV誘導(dǎo)自噬相關(guān)的宿主因子時(shí),發(fā)現(xiàn)荷電多囊體蛋白4B(charged multivesicular body protein 4B, CHMP4B)表達(dá)升高。CHMP4B作為轉(zhuǎn)運(yùn)必需內(nèi)涵體分選復(fù)合物-Ⅲ(endosomal sorting complex required for transport-Ⅲ, ESCRT-Ⅲ)的重要組成部分,參與細(xì)胞分裂,調(diào)節(jié)自噬,同時(shí)還參與HCV病毒顆粒的釋放。但是,CHMP4B調(diào)節(jié)自噬和影響HCV病毒顆粒釋放的具體分子機(jī)制不明。因此,本研究擬解決的第一個(gè)問(wèn)題是：CHMP4B在HCV感染中發(fā)揮的作用是什么?本研究利用慢病毒介導(dǎo)的shRNA敲低CHMP4B,通過(guò)Western Blot和Real-time PCR檢測(cè)發(fā)現(xiàn),瞬時(shí)敲低CHMP4B導(dǎo)致宿主細(xì)胞內(nèi)HCV蛋白水平下降而HCV RNA水平不變。為進(jìn)一步研究CHMP4B通過(guò)與哪些宿主蛋白相互作用從而影響HCV蛋白表達(dá),我們擬利用串聯(lián)親和純化(tandem affinity purification, TAP)技術(shù)篩選HCV感染時(shí)與CHMP4B相互作用的宿主蛋白。為此,我們利用慢病毒介導(dǎo)的shRNA構(gòu)建了CHMP4B穩(wěn)定敲低的Huh7.5細(xì)胞系,擬通過(guò)外源表達(dá)與內(nèi)源表達(dá)水平相當(dāng)?shù)膸Ъ兓瘶?biāo)簽的CHMP4B進(jìn)行串聯(lián)親和純化以獲得與CHMP4B相互作用蛋白。然而,我們通過(guò)Western Blot和Real-time PCR發(fā)現(xiàn),穩(wěn)定轉(zhuǎn)導(dǎo)CHMP4B shRNA和無(wú)靶標(biāo)對(duì)照shRNA時(shí)Huh7.5細(xì)胞胞內(nèi)HCV蛋白和RNA水平均明顯下降,而瞬時(shí)轉(zhuǎn)導(dǎo)無(wú)靶標(biāo)對(duì)照shRNA胞內(nèi)HCV蛋白和RNA水平無(wú)明顯變化,提示shRNA穩(wěn)定轉(zhuǎn)導(dǎo)可能誘導(dǎo)產(chǎn)生脫靶效應(yīng)抑制HCV的增殖。因此,本研究針對(duì)shRNA穩(wěn)定轉(zhuǎn)導(dǎo)誘導(dǎo)的這種非特異性脫靶效應(yīng)的機(jī)制進(jìn)行了深入研究。為了驗(yàn)證脫靶效應(yīng)是不是由于激活天然免疫應(yīng)答所致,首先,我們通過(guò)雙熒光素酶報(bào)告系統(tǒng)檢測(cè)HCV感染后shRNA穩(wěn)定轉(zhuǎn)導(dǎo)細(xì)胞中IFN-β啟動(dòng)子的活性,發(fā)現(xiàn)IFN-β啟動(dòng)子活性無(wú)明顯變化,提示脫靶效應(yīng)并非通過(guò)激活I(lǐng)型干擾素通路誘導(dǎo)產(chǎn)生；其次,我們通過(guò)Real-time PCR方法檢測(cè)shRNA穩(wěn)定轉(zhuǎn)導(dǎo)細(xì)胞上清處理Huh7.5細(xì)胞后胞內(nèi)HCVRNA的變化,發(fā)現(xiàn)胞內(nèi)HCV RNA的含量無(wú)明顯變化,提示脫靶效應(yīng)也不依賴于抗病毒因子的產(chǎn)生。瞬時(shí)轉(zhuǎn)導(dǎo)時(shí)shRNA高水平表達(dá)會(huì)通過(guò)干擾宿主miRNA從而抑制HCV復(fù)制,提示shRNA的穩(wěn)定轉(zhuǎn)導(dǎo)可能也干擾miRNA的表達(dá)從而抑制HCV復(fù)制,因此本研究用miRNA芯片檢測(cè)了shRNA穩(wěn)定轉(zhuǎn)導(dǎo)細(xì)胞的miRNA表達(dá)譜。結(jié)果發(fā)現(xiàn)宿主細(xì)胞的miRNA表達(dá)譜發(fā)生顯著變化,提示脫靶效應(yīng)的產(chǎn)生可能與miRNA表達(dá)譜的變化相關(guān)。對(duì)miRNA表達(dá)譜進(jìn)一步分析表明,低水平的shRNA穩(wěn)定表達(dá)即可影響宿主miRNA的表達(dá),進(jìn)而影響HCV增殖。另外,shRNA整合位點(diǎn)也可能影響miRNA表達(dá)譜的變化,進(jìn)而影響HCV的增殖。miRNA表達(dá)譜中有60個(gè)miRNA表達(dá)水平有兩倍以上的變化,其中三個(gè)miRNA文獻(xiàn)已報(bào)道參與HCV感染的調(diào)控：miR-196a、miR-491和miR-149。為進(jìn)一步驗(yàn)證是不是miRNA表達(dá)譜的變化影響了HCV的增殖,除上述3個(gè)miRNA卜,本研究還挑選了變化倍數(shù)較大且與上述miRNA變化趨勢(shì)一致的4個(gè)miRNA:miR-1246、miR-193、miR-744和miR-769,通過(guò)Real-time PCR檢測(cè)轉(zhuǎn)染相應(yīng)miRNA mimic和inhibitor后HCV增殖的情況。結(jié)果發(fā)現(xiàn),過(guò)表達(dá)miR-196a或敲低miR-491均抑制HCV的增殖,與文獻(xiàn)報(bào)道一致,miR-149對(duì)HCV增殖無(wú)影響,也與文獻(xiàn)報(bào)道其參與調(diào)控HCV入侵一致；過(guò)表達(dá)miR一1246抑制HCV增殖,而miR-744、miR-193和miR-769對(duì)HCV增殖無(wú)影響。上述結(jié)果提示,慢病毒介導(dǎo)的shRNA通過(guò)干擾miRNA表達(dá)譜非特異性抑制HCV的增殖。另外,我們新發(fā)現(xiàn)miR-1246參與調(diào)控HCV增殖,其具體分子機(jī)制仍待進(jìn)-步研究。綜上所述,我們的研究一方面提高了對(duì)慢病毒shRNA穩(wěn)定轉(zhuǎn)導(dǎo)產(chǎn)生非特異性脫靶效應(yīng)的認(rèn)識(shí),另一方面本研究鑒定出的受shRNA調(diào)節(jié)的miRNA可能會(huì)為HCV感染提供新的治療策略。
[Abstract]:Hepatitis C virus (Hepatitis C, Virus, HCV) infection caused by hepatitis C virus and induced liver cirrhosis, liver cancer and other diseases have been a serious threat to human health, the lack of limitation of vaccines and antiviral drugs is the main obstacle to the corresponding control of HCV infection and treatment of diseases related to.HCV mainly depends on the completion of virus invasion to host factor replication, assembly and release, the molecular mechanism of the infection process and host response of the host factor therefore find out the interaction between HCV and HCV will help to clarify, but the discovery of new drug targets of antiviral therapy and the development of effective HCV vaccine has important theoretical and practical significance of.HCV infection can induce cell autophagy initiation HCV replication is dependent on autophagy related protein and HCV induced autophagy by inhibiting host cell innate immune response and promote the replication of HCV. But HCV by self Bite of the molecular mechanism of self replication is not clear. This research group induced autophagy associated host factor in screening and HCV, found that charged multivesicular body protein 4B (charged multivesicular body protein 4B, CHMP4B) increased the expression of.CHMP4B as the essential connotation of complex transport body separation - III (endosomal sorting complex required for transport- third, ESCRT- III) an important part involved in cell division, is also involved in the regulation of autophagy, HCV virus particle release. However, CHMP4B regulates autophagy and influence the molecular mechanism of HCV virus particle release is unknown. Therefore, this study intends to solve the first problem is: what is the CHMP4B play in HCV infection? This study using lentivirus mediated shRNA knockdown of CHMP4B by Western, Blot and Real-time PCR assay showed that the transient knockdown of CHMP4B resulted in host cells in HCV eggs The white level was decreased and HCV RNA level unchanged. For the further study of CHMP4B through which host proteins interact to influence the expression of HCV protein, we proposed using tandem affinity purification (tandem affinity, purification, TAP) screening of HCV infection of host proteins interacting with CHMP4B. Therefore, we used lentivirus mediated shRNA constructs CHMP4B stable cell line on Huh7.5 low, quasi and endogenous expression of tandem affinity purification to obtain the interaction between CHMP4B and protein purification tag CHMP4B equivalent level by exogenous expression. However, I have passed Western Blot and Real-time PCR found that stable transduction of CHMP4B shRNA and no shRNA target control level of HCV protein and RNA Huh7.5 cells were significantly decreased, while the instantaneous control level of HCV protein transduction without target and RNA shRNA cells had no obvious change, suggesting that shRNA may lead to stable Miss effect induced by inhibit the proliferation of HCV. Therefore, the study of mechanism for this non-specific off target effects induced by shRNA stable transduction is studied. In order to verify the effect of distance is not due to the result of the activation of the innate immune response, first of all, we through dual luciferase reporter system for detection of HCV infection after shRNA stable cells IFN- beta promoter activity, IFN- beta promoter activity had no obvious change, that is not miss effect induced by activation of type I interferon pathway; secondly, we through the change of Real-time PCR method for detection of shRNA stably transduced cell supernatant after treatment of Huh7.5 cells with intracellular HCVRNA, found that the content of intracellular HCV RNA had no obvious change. That Miss effect does not depend on antiviral cytokines. ShRNA high level expression of instantaneous transduction through interference with the host miRNA inhibits HCV complex System, expression of stable transduction of shRNA may also interfere with miRNA to inhibit the replication of HCV, based on the miRNA chip shRNA stable transduction cell miRNA expression spectrum. The results showed that the expression changes of host cells of miRNA, suggesting that Miss effect may be related to the change of spectrum and the expression of miRNA expression further. According to the analysis of the miRNA, shRNA stable low level expression can influence the expression of host miRNA, thereby affecting the proliferation of HCV. In addition, the integration sites of shRNA may also the expression of miRNA, thereby affecting HCV proliferation.MiRNA expression profiles in 60 miRNA expression level changes by more than two times, of which three miRNA it is reported that participate in the regulation of HCV infection: miR-196a, miR-491 and miR-149. for further validation is not miRNA expression changes affect the proliferation of HCV, in addition to the above 3 miRNA, and the The study also picked up 4 miRNA:miR-1246, and the changes of multiple large miRNA changes with the miR-193, miR-744 and miR-769, by HCV Real-time PCR was used to detect the proliferation of miRNA mimic and inhibitor. The results showed that miR-196a overexpression or knockdown of miR-491 inhibited the proliferation of HCV, consistent with the literature, miR-149 the impact of the proliferation of HCV, and also reported to participate in the regulation of HCV invasion; overexpression of miR 1246 inhibit the proliferation of HCV, miR-744, miR-193 and miR-769 had no effect on the proliferation of HCV. The results indicate that the lentivirus mediated shRNA spectrum of nonspecific inhibition of HCV proliferation by interfering miRNA expression. In addition, our new miR-1246 is involved in the regulation of HCV proliferation and its molecular mechanism remains to be further studied. To sum up, on the one hand, we improve the non specificity of shRNA lentiviral transduction stable target The understanding of the effect, on the other hand, the shRNA - regulated miRNA in this study may provide a new therapeutic strategy for HCV infection.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373.21

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王玫;李思光;羅玉萍;;microRNA識(shí)別和鑒定方法[J];細(xì)胞生物學(xué)雜志;2007年04期

2 王晶;朱榮勝;宋萬(wàn)坤;張聞博;劉春燕;胡國(guó)華;陳慶山;;microRNA的實(shí)驗(yàn)和生物信息學(xué)開(kāi)發(fā)方法[J];生物技術(shù)通報(bào);2008年S1期

3 田明;費(fèi)菁;胡曉湘;李寧;劉娣;孟慶勇;;microRNA-1,-133,-206在鼠肌肉中的表達(dá)譜[J];中國(guó)畜牧獸醫(yī);2009年08期

4 LEE Younghee;LUSSIER Yves A;;Identification of common microRNA-mRNA regulatory biomodules in human epithelial cancer[J];Chinese Science Bulletin;2010年31期

5 王芳;余佳;張俊武;;小RNA(MicroRNA)研究方法[J];中國(guó)生物化學(xué)與分子生物學(xué)報(bào);2006年10期

6 鄭玉姝;趙樸;趙宏坤;;microRNA及其應(yīng)用前景[J];生物技術(shù)通訊;2007年01期

7 王春梅;李慶章;;microRNA在小鼠乳腺不同發(fā)育時(shí)期差異表達(dá)譜及作用(英文)[J];遺傳學(xué)報(bào);2007年11期

8 鄒文輝;;新奇的調(diào)控分子——microRNA[J];安徽農(nóng)業(yè)科學(xué);2008年34期

9 ;MIRE: A GRAPHICAL R PACKAGE FOR MICRORNARELATED ANALYSIS[J];Chinese Medical Sciences Journal;2008年04期

10 張坤山;李思光;羅玉萍;;調(diào)控過(guò)氧化物酶體生物合成和增殖的microRNA的計(jì)算機(jī)分析[J];細(xì)胞生物學(xué)雜志;2009年02期

相關(guān)會(huì)議論文 前10條

1 荊清;袁文俊;秦永文;;microRNA的基因調(diào)控新功能[A];中國(guó)生理學(xué)會(huì)第五屆全國(guó)心血管、呼吸和腎臟生理學(xué)學(xué)術(shù)會(huì)議論文摘要匯編[C];2005年

2 靳新;駱志剛;王金華;管乃洋;;microRNA靶標(biāo)研究進(jìn)展[A];中國(guó)遺傳學(xué)會(huì)功能基因組學(xué)研討會(huì)論文集[C];2006年

3 ;Comparasion of Inhibitory Effects on Survivin Gene Expression in Prostate Cancer by Vector-based microRNA and siRNA[A];2008年全國(guó)生物化學(xué)與分子生物學(xué)學(xué)術(shù)大會(huì)論文摘要[C];2008年

4 朱丹霞;徐衛(wèi);繆扣榮;方成;朱華淵;董華潔;王冬梅;范磊;喬純;李建勇;;慢性淋巴細(xì)胞白血病microRNA異常表達(dá)研究[A];第13屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要[C];2011年

5 Yangchao Chen;;Small molecule modulators of microRNA-34a with anti-cancer activities[A];2013醫(yī)學(xué)前沿論壇暨第十三屆全國(guó)腫瘤藥理與化療學(xué)術(shù)會(huì)議論文集[C];2013年

6 金希;厲有名;;單純性脂變與非酒精性脂肪性肝炎間差異microRNA表達(dá)譜研究[A];2009香港-北京-杭州內(nèi)科論壇暨2009年浙江省內(nèi)科學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2009年

7 賈新正;盧肖男;聶慶華;張細(xì)權(quán);;雞部分microRNA的同源預(yù)測(cè)與克隆驗(yàn)證[A];中國(guó)動(dòng)物遺傳育種研究進(jìn)展——第十五次全國(guó)動(dòng)物遺傳育種學(xué)術(shù)討論會(huì)論文集[C];2009年

8 李炯;段德民;鄭克孝;;新型非標(biāo)記高通量microRNA芯片技術(shù)[A];第一屆全國(guó)生物物理化學(xué)會(huì)議暨生物物理化學(xué)發(fā)展戰(zhàn)略研討會(huì)論文摘要集[C];2010年

9 徐小濤;陸曉;孫婧;束永前;;肺癌側(cè)群細(xì)胞microRNA表達(dá)譜檢測(cè)及初步分析[A];2010’全國(guó)腫瘤分子標(biāo)志及應(yīng)用學(xué)術(shù)研討會(huì)暨第五屆中國(guó)中青年腫瘤專家論壇論文匯編[C];2010年

10 王俊峰;李巍;吳小江;阮康成;;大鼠附睪microRNA表達(dá)譜的研究[A];第十一次中國(guó)生物物理學(xué)術(shù)大會(huì)暨第九屆全國(guó)會(huì)員代表大會(huì)摘要集[C];2009年

相關(guān)重要報(bào)紙文章 前2條

1 陳英云 喬蕤琳;哈醫(yī)大成功研發(fā)國(guó)內(nèi)首例microRNA轉(zhuǎn)基因及敲減小鼠模型[N];黑龍江經(jīng)濟(jì)報(bào);2010年

2 記者 陳青;2厘米以下肝癌檢出率88%[N];文匯報(bào);2011年

相關(guān)博士學(xué)位論文 前10條

1 崔洪亮;microRNA的腫瘤表達(dá)研究及協(xié)同調(diào)控網(wǎng)絡(luò)分析[D];中南大學(xué);2014年

2 馬建華;高粱低磷低氮形態(tài)生理特征及低氮響應(yīng)的microRNA研究[D];山西農(nóng)業(yè)大學(xué);2014年

3 李虔楨;microRNA相關(guān)基因遺傳多態(tài)性與冠心病臨床關(guān)聯(lián)及預(yù)后研究[D];福建醫(yī)科大學(xué);2015年

4 王耀輝;血清microRNA作為乳腺癌診斷標(biāo)志物的研究[D];復(fù)旦大學(xué);2014年

5 周霽子;環(huán)境因素對(duì)心臟畸形胎兒影響的表觀遣傳學(xué)機(jī)制[D];復(fù)旦大學(xué);2013年

6 張麗;microRNA通過(guò)調(diào)控小膠質(zhì)細(xì)胞炎性反應(yīng)參與血管性認(rèn)知障礙發(fā)生發(fā)展[D];復(fù)旦大學(xué);2014年

7 查若鵬;肝癌轉(zhuǎn)移相關(guān)microRNA的鑒定及其分子機(jī)制研究[D];復(fù)旦大學(xué);2013年

8 方硯田;結(jié)腸癌干細(xì)胞分選、差異microRNA表達(dá)譜檢測(cè)以及miR-449b-CCND1、E2F3通路在結(jié)腸癌干細(xì)胞自我更新的機(jī)制研究[D];復(fù)旦大學(xué);2014年

9 辛成齊;椰棗microRNA鑒定及其在果實(shí)發(fā)育過(guò)程中的表達(dá)譜研究[D];中國(guó)科學(xué)院北京基因組研究所;2015年

10 李棟;先天性心臟病血漿microRNA表達(dá)譜及與GATA4靶序列單核苷酸多態(tài)性的關(guān)聯(lián)研究[D];山東大學(xué);2015年

相關(guān)碩士學(xué)位論文 前10條

1 高智紅;應(yīng)用多樣性增量方法識(shí)別人類基因組microRNA前體序列[D];內(nèi)蒙古大學(xué);2010年

2 王娜娜;microRNA進(jìn)化關(guān)系及編碼特性研究[D];內(nèi)蒙古大學(xué);2007年

3 王玫;小麥microRNA的鑒定與分析[D];南昌大學(xué);2007年

4 秦保東;原發(fā)性膽汁性肝硬化microRNA表達(dá)譜的檢測(cè)及其功能研究[D];第二軍醫(yī)大學(xué);2013年

5 吳曉妍;基于納米復(fù)合材料及生物放大技術(shù)構(gòu)建的電化學(xué)microRNA傳感器的研究[D];西南大學(xué);2015年

6 周景輝;2型糖尿病microRNA表達(dá)譜及其信號(hào)通路研究[D];華南理工大學(xué);2015年

7 姜溪;血漿microRNA-486、microRNA-499在肺癌中的表達(dá)及臨床價(jià)值的研究[D];河北大學(xué);2015年

8 林杉;營(yíng)養(yǎng)誘導(dǎo)舍飼綿羊非繁殖季節(jié)發(fā)情相關(guān)microRNA篩選及其靶基因功能驗(yàn)證[D];石河子大學(xué);2015年

9 林妹妹;microRNA-192 和 microRNA-21 在 IBD 患者中的表達(dá)情況[D];福建醫(yī)科大學(xué);2015年

10 徐嬌陽(yáng);循環(huán)microRNA用于低氧性肺動(dòng)脈高壓早期診斷的實(shí)驗(yàn)研究[D];石河子大學(xué);2015年

，

本文編號(hào)：1715307