本文選題：乙型肝炎病毒(HBV)　切入點(diǎn)：siRNA　出處：《揚(yáng)州大學(xué)》2011年碩士論文

【摘要】：乙型肝炎病毒(Hepatitis B Virus, HBV)感染嚴(yán)重影響人類的健康和生命安全,目前對慢性HBV感染者和攜帶者無特效治療方法。HBV基因組較小,易發(fā)生突變,限制了靶向病毒的抗病毒藥物研究。為了研究和開發(fā)新型抗HBV藥物,我們設(shè)計(jì)了一種新型雙功能siRNA (5'-ppp-siRNA,3p-siRNA)核酸藥物,一方面通過刺激宿主細(xì)胞模式識(shí)別受體誘導(dǎo)宿主抗病毒I型干擾素的表達(dá),同時(shí)通過RNAi作用特異性沉默HBV基因表達(dá)從而抑制病毒復(fù)制。 3p-siRNA結(jié)合一個(gè)核酶復(fù)合物從而形成RNA誘導(dǎo)沉默復(fù)合物(RNA-induced silencing complex, RISC),由RISC介導(dǎo)切割靶mRNA分子中與3p-siRNA反義鏈互補(bǔ)的區(qū)域,從而達(dá)到沉默特異性基因表達(dá)作用；同時(shí),3p-siRNA可以活化維甲酸誘導(dǎo)基因I(retionic-acid-inducible genel, RIG-I),使RIG-I與位于線粒體外膜的銜接蛋白干擾素啟動(dòng)子刺激因子-1(IFN-βpromoter stimulator-1, IPS-1)/CARDIF(CARD adaptor inducing IFN-β)/MAVS (mitochondrial antiviral signaling)/VISA (virus-induced signaling adapter)相互作用,下傳信號,激活核轉(zhuǎn)錄因子-κB (nuclear factor-KB, NF-κB)或干擾素調(diào)節(jié)因子(interferon regulatory factor-3, IRF3)等,誘導(dǎo)I型IFN和促炎因子的表達(dá)和分泌,從而實(shí)現(xiàn)抑制HBV的目的。 本研究首先通過體外轉(zhuǎn)錄生成3p-siRNA。通過細(xì)胞毒性檢測發(fā)現(xiàn),在100nM,200nM時(shí)3p-siRNA對HepG2.2.15細(xì)胞無顯著毒性,但是在500nM時(shí)3p-siRNA對HepG2.2.15細(xì)胞具有一定毒性。研究發(fā)現(xiàn),將IFN-β熒光素酶報(bào)告基因與RIG-I共同轉(zhuǎn)染HepG2.2.15細(xì)胞,同時(shí)轉(zhuǎn)入海腎蟲熒光素酶報(bào)告基因作為內(nèi)參,轉(zhuǎn)染24小時(shí)后將3p-siRNA轉(zhuǎn)染細(xì)胞,16小時(shí)后檢測IFN-p熒光素酶活性,發(fā)現(xiàn)未轉(zhuǎn)染RIG-I時(shí)3p-siRNA只能微弱地誘導(dǎo)IFN表達(dá),但是共轉(zhuǎn)RIG-I后3p-siRNA顯著地誘導(dǎo)IFN表達(dá),說明在HepG2.2.15細(xì)胞中3p-siRNA具有RIG-I依賴的激活宿主抗病毒天然免疫反應(yīng)。然而,我們用CIAP去除3p-siRNA5’末端的5’-PPP基團(tuán)后,將其與IFN-p熒光素酶報(bào)告基因和RIG-I共同轉(zhuǎn)染HepG2.2.15細(xì)胞后,檢測IFN-β熒光素酶活性發(fā)現(xiàn)經(jīng)CIAP處理的3p-siRNA并未激活RIG-I介導(dǎo)的IFN通路,說明3p-siRNA誘導(dǎo)IFN表達(dá)主要是依賴于其5’末端的5'-ppp基團(tuán)。此外,3p-siRNA對干擾素刺激調(diào)控元件(ISRE)也具有RIG-I依賴的激活效應(yīng),在未共轉(zhuǎn)RIG-I時(shí)3p-siRNA微弱地誘導(dǎo)ISRE表達(dá),然而共轉(zhuǎn)RIG-I后3p-siRNA明顯地誘導(dǎo)ISRE表達(dá)。 抗病毒活性測定發(fā)現(xiàn),在HepG2.2.15細(xì)胞中,與siRNA抗HBV活性相比,3p-siRNA表現(xiàn)出更明顯的抗病毒活性。3p-siRNA轉(zhuǎn)染48小時(shí)后對HBsAg、HBeAg以及HBV DNA和HBV mRNA都有明顯的抑制效果。3p-siRNA對HBV具有持續(xù)的抑制效應(yīng),當(dāng)轉(zhuǎn)染3p-siRNA后48小時(shí)、96小時(shí)、144小時(shí),3p-siRNA對HBsAg和HBeAg的抑制率分別是55%和41.8%、37%和24.3%、33.3%和14.8%；當(dāng)共轉(zhuǎn)染RIG-I和3p-siRNA時(shí),3p-siRNA對HBsAg和HBeAg的抑制率分別是66.3%和53%、55.5%和49.7%、45.6%和26.5%。與只轉(zhuǎn)染3p-siRNA相比,共轉(zhuǎn)染RIG-I時(shí)3p-siRNA對HBV表現(xiàn)出更明顯的抑制活性。以不同劑量(50nM、100nM、200nM)3p-siRNA轉(zhuǎn)染HepG2.2.15細(xì)胞,發(fā)現(xiàn)3p-siRNA對HBV的抑制作用呈明顯劑量依賴性。 綜上所述,本研究成功設(shè)計(jì)了一種新型雙功能抗HBV核酸藥物3p-siRNA,一方面具有能通過RNAi特異識(shí)別并切割HBV mRNA靶分子從而抑制HBV特性；同時(shí),可以激活宿主細(xì)胞RIG-I依賴的抗病毒天然免疫反應(yīng),從而高效阻止HBV復(fù)制。抗HBV雙功能3p-siRNA核酸藥物的發(fā)現(xiàn)為研究開發(fā)新型抗HBV藥物研究具有重要應(yīng)用價(jià)值。
[Abstract]:Hepatitis B Virus ( HBV ) infection seriously affects human health and life safety . There is no special treatment method for chronic HBV infection and carrier . In order to study and develop a new anti - HBV drug , we designed a novel bifunctional siRNA ( 5 ' - ppp - siRNA , 3P - siRNA ) nucleic acid medicine . In order to study and develop a new anti - HBV drug , we designed a novel bifunctional siRNA ( 5 ' - ppp - siRNA , 3P - siRNA ) nucleic acid medicine . On the one hand , we designed a novel bifunctional siRNA ( 5 ' - ppp - siRNA , 3P - siRNA ) nucleic acid medicine . On the one hand , we designed a novel bifunctional siRNA ( 5 ' - ppp - siRNA , 3P - siRNA ) nucleic acid medicine , while silencing HBV gene expression by RNAi action , thus inhibiting viral replication .

3 - siRNA is combined with a ribozyme complex to form an RNA - induced silencing complex ( RISC ) , and a region complementary to an anti - sense strand of the 3P - siRNA in the target mRNA molecule is cut by RISC , so that silencing specific gene expression is achieved ;
At the same time , the expression and secretion of type I IFN and pro - inflammatory factors can be induced by interacting with the promoter stimulation factor - 1 ( IFN - & beta promoter isoform - 1 , IPS - 1 ) / CARDIF ( CARD - inducing IFN - 尾 ) / MAVS ( interferon - induced signaling adapter ) and interferon regulatory factor - 3 , IRF3 , etc . , which can induce the expression and secretion of type I IFN and pro - inflammatory factor , thus achieving the purpose of inhibiting HBV .

IFN - 尾 luciferase reporter gene was co - transfected into HepG2.2 . 15 cells at 100 nM and 200 nM .

The antiviral activity assay showed that , in HepG2.2 . 15 cells , compared with the anti - HBV activity of siRNA , there was a significant inhibitory effect on HBsAg , HBeAg and HBV DNA and HBV mRNA after transfection for 48 hours . The inhibition rates for HBsAg , HBeAg and HBV DNA and HBV mRNA were 55 % and 41.8 % , 37 % and 24 . 3 % , 33.3 % and 14.8 % respectively .
When we co - transfected the IL3 - siRNA , the inhibition rates for HBsAg and HBeAg were 66.3 % and 53 % , 55.5 % and 49.7 % , 45.6 % and 26.5 % , respectively .

In conclusion , the present study successfully designed a novel double - functional anti - HBV nucleic acid drug 3 - siRNA , on the one hand , has the ability to specifically identify and cut HBV mRNA target molecules through RNAi , thereby inhibiting HBV characteristics ;
At the same time , the anti - HBV replication can be effectively prevented by activating the antiviral natural immune reaction which is dependent on the cell of the host cells , and the detection of the anti - HBV double - function 3P - siRNA nucleic acid drug has important application value for the research and development of a novel anti - HBV drug research .

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R373

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 縱書芳;陳勇;王莉娟;劉興祥;徐云芳;;靶向乙型肝炎病毒X區(qū)的siRNAs對HepG2.2.15細(xì)胞HBV復(fù)制的抑制作用[J];實(shí)用肝臟病雜志;2010年04期

相關(guān)博士學(xué)位論文 前1條

1 唐彤宇;基于microRNA的RNA干擾抗乙型肝炎病毒的實(shí)驗(yàn)研究[D];吉林大學(xué);2009年

相關(guān)碩士學(xué)位論文 前1條

1 關(guān)彥彥;DMSO、地塞米松對于HBV感染HepCHLine-4細(xì)胞能力的影響[D];山東大學(xué);2011年

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本文編號：1698947