双功能siRNA对乙型肝炎病毒（HBV）的抑制作用

发布时间：2018-04-02 05:17

本文选题：乙型肝炎病毒(HBV)　切入点：siRNA　出处：《扬州大学》2011年硕士论文

【摘要】：乙型肝炎病毒(Hepatitis B Virus, HBV)感染严重影响人类的健康和生命安全,目前对慢性HBV感染者和携带者无特效治疗方法。HBV基因组较小,易发生突变,限制了靶向病毒的抗病毒药物研究。为了研究和开发新型抗HBV药物,我们设计了一种新型双功能siRNA (5'-ppp-siRNA,3p-siRNA)核酸药物,一方面通过刺激宿主细胞模式识别受体诱导宿主抗病毒I型干扰素的表达,同时通过RNAi作用特异性沉默HBV基因表达从而抑制病毒复制。 3p-siRNA结合一个核酶复合物从而形成RNA诱导沉默复合物(RNA-induced silencing complex, RISC),由RISC介导切割靶mRNA分子中与3p-siRNA反义链互补的区域,从而达到沉默特异性基因表达作用；同时,3p-siRNA可以活化维甲酸诱导基因I(retionic-acid-inducible genel, RIG-I),使RIG-I与位于线粒体外膜的衔接蛋白干扰素启动子刺激因子-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,下传信号,激活核转录因子-κB (nuclear factor-KB, NF-κB)或干扰素调节因子(interferon regulatory factor-3, IRF3)等,诱导I型IFN和促炎因子的表达和分泌,从而实现抑制HBV的目的。本研究首先通过体外转录生成3p-siRNA。通过细胞毒性检测发现,在100nM,200nM时3p-siRNA对HepG2.2.15细胞无显著毒性,但是在500nM时3p-siRNA对HepG2.2.15细胞具有一定毒性。研究发现,将IFN-β荧光素酶报告基因与RIG-I共同转染HepG2.2.15细胞,同时转入海肾虫荧光素酶报告基因作为内参,转染24小时后将3p-siRNA转染细胞,16小时后检测IFN-p荧光素酶活性,发现未转染RIG-I时3p-siRNA只能微弱地诱导IFN表达,但是共转RIG-I后3p-siRNA显著地诱导IFN表达,说明在HepG2.2.15细胞中3p-siRNA具有RIG-I依赖的激活宿主抗病毒天然免疫反应。然而,我们用CIAP去除3p-siRNA5’末端的5’-PPP基团后,将其与IFN-p荧光素酶报告基因和RIG-I共同转染HepG2.2.15细胞后,检测IFN-β荧光素酶活性发现经CIAP处理的3p-siRNA并未激活RIG-I介导的IFN通路,说明3p-siRNA诱导IFN表达主要是依赖于其5’末端的5'-ppp基团。此外,3p-siRNA对干扰素刺激调控元件(ISRE)也具有RIG-I依赖的激活效应,在未共转RIG-I时3p-siRNA微弱地诱导ISRE表达,然而共转RIG-I后3p-siRNA明显地诱导ISRE表达。抗病毒活性测定发现,在HepG2.2.15细胞中,与siRNA抗HBV活性相比,3p-siRNA表现出更明显的抗病毒活性。3p-siRNA转染48小时后对HBsAg、HBeAg以及HBV DNA和HBV mRNA都有明显的抑制效果。3p-siRNA对HBV具有持续的抑制效应,当转染3p-siRNA后48小时、96小时、144小时,3p-siRNA对HBsAg和HBeAg的抑制率分别是55%和41.8%、37%和24.3%、33.3%和14.8%；当共转染RIG-I和3p-siRNA时,3p-siRNA对HBsAg和HBeAg的抑制率分别是66.3%和53%、55.5%和49.7%、45.6%和26.5%。与只转染3p-siRNA相比,共转染RIG-I时3p-siRNA对HBV表现出更明显的抑制活性。以不同剂量(50nM、100nM、200nM)3p-siRNA转染HepG2.2.15细胞,发现3p-siRNA对HBV的抑制作用呈明显剂量依赖性。综上所述,本研究成功设计了一种新型双功能抗HBV核酸药物3p-siRNA,一方面具有能通过RNAi特异识别并切割HBV mRNA靶分子从而抑制HBV特性；同时,可以激活宿主细胞RIG-I依赖的抗病毒天然免疫反应,从而高效阻止HBV复制。抗HBV双功能3p-siRNA核酸药物的发现为研究开发新型抗HBV药物研究具有重要应用价值。
[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 .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【共引文献】