【作者】 郜玉峰；

【导师】 沈继龙；

【作者基本信息】 安徽医科大学 ， 临床药理学， 2008， 博士

【摘要】 乙型肝炎病毒(hepatitis B virus, HBV)感染是全球重要的公共卫生问题。目前全球慢性HBV感染人数己超过3.5亿,是导致肝硬化、重型肝炎及肝细胞癌(hepatocellular carcinoma, HCC)的主要原因。在中国,有大约10%的人口存在慢性HBV感染,直到目前,尚无有效的药物能完全达到理想的抗病毒效果或者清除病毒。因此,寻找一种新的治疗策略治疗慢性HBV感染是必要而且紧迫的。RNA干扰(RNA interference, RNAi)是一项新兴的基因沉默技术,主要通过siRNA和microRNA(miRNA)两种小RNA分子来发挥作用。siRNA介导的RNAi沉默基因表达是通过小片段双链RNA以序列特异性的方式与靶基因的mRNA相结合来实现的。但越来越多的研究发现,许多病毒在治疗选择压力下可以通过靶基因序列的突变或产生抑制因子来逃避RNAi,针对病毒的RNAi治疗效果因为病毒的变异而降低疗效,甚至失去治疗作用。miRNA作用方式很大程度上和siRNA介导的RNAi途径重叠。与siRNA相比,miRNA仅仅需要和靶基因部分的结合即可以发挥作用,因而可以在很大程度上避免因为病毒变异导致的RNAi治疗失败。这将对容易发生变异的病毒(如HCV, HBV, HIV)病的治疗更有优势。miRNA功能的研究可能有助于提出治疗病毒感染性疾病的新方法。另一种有效的病毒RNAi治疗途径将可能是针对病毒感染和复制所需要的宿主细胞因素进行RNA干扰研究。HBV在体内感染细胞和体内病毒复制需要宿主基因的参与,因此,以其为靶位进行RNA干扰降低宿主基因表达,可能会导致病毒复制水平的降低。以宿主基因为靶位RNAi治疗的优点是它不依赖于病毒的基因组,因而能避免病毒通过突变逃避RNAi的缺点。但目前国内外尚未见利用microRNA介导的RNA干扰技术对HBV和HBV复制所需的宿主基因进行基因治疗的研究报道。最近大量研究表明,通过借助于miRNA表达框架,可以实现将靶向特定基因的外源性人工合成的miRNA(artificial microRNA, amiRNA)导入细胞内,达到抑制靶基因的效果。因此,本实验研究分别构建了靶向HBV不同位点和靶向La蛋白基因和ASGPR1基因等宿主基因的外源性amiRNA表达载体,瞬时和稳定转染HepG2.2.15细胞,观察外源性amiRNA介导的RNA干扰能否阻断HBV和HBV复制相关的宿主基因表达,通过测定靶基因的抑制程度和HBV蛋白以及HBV DNA降低的程度,从而了解靶向HBV和相关宿主基因的外源性amiRNA表达对HBV复制水平的影响。目的:通过利用miRNA表达框架构建能够特异性靶向HBV和HBV感染和复制相关宿主基因的amiRNA表达载体,转染到HepG2.2.15细胞中,观察外源性amiRNA介导的RNA干扰对HBV复制水平的影响。方法:构建13个靶向HBV不同位点、La蛋白基因和ASGPR1基因的外源性amiRNA表达载体,测序证实插入序列的正确性。然后通过脂质体转染技术瞬时和稳定转染HepG2.2.15细胞,半定量RT-PCR方法观察靶基因mRNA的抑制程度;Western blotting方法观察La蛋白和ASGPR1蛋白的抑制程度;测定培养上清中的HBsAg、HBeAg浓度和HBV DNA的水平,观察靶向HBV、La蛋白基因和ASGPR1基因的外源性amiRNA对HBV复制和表达水平的影响。结果:1.瞬时转染amiRNA-HBV对HBsAg和HBeAg的抑制作用将构建的7个amiRNA表达质粒转染HepG2.2.15细胞,流式细胞仪检测转染效率在50%~60%左右。在这种情况下,HBsAg和HBeAg测定结果显示:7个质粒均有不同程度的抑制作用,其中有3个质粒对HBsAg和HBeAg的表达具有显著的抑制作用。抑制效应在转染后48h抑制效果开始较明显,72h病毒抑制达到高峰,96h后抑制作用开始渐减弱。其中以amiRNA-HBV-S608对HBsAg的抑制作用最为明显,amiRNA-HBV-S608在转染后的48h、72h和96h三个时间点对HBsAg的平均抑制率分别为29.5%,49.8%和46.3%, P<0.01;amiRNA-P2227对HBeAg的抑制最显著,amiRNA-HBV-P2227在转染后的48h、72h和96h三个时间点对HBeAg的平均抑制率分别为36.4%,45.2%和40.3%, P<0.01。而阴性对照组对蛋白表达的影响与空白对照组相比无显著性差异, P>0.05。2.瞬时转染amiRNA-HBV对HBV DNA复制的抑制作用对瞬时转染各个时间点培养上清中的HBV DNA水平采用实时荧光定量PCR检测结果表明:与HBsAg和HBeAg抑制的作用相似,7个质粒对培养上清中的HBV DNA也均有不同程度的抑制作用,而阴性对照组载体与空白转染组相比对HBV DNA抑制无显著性差异, P>0.05。其中amiRNA-HBV-S608和amiRNA-HBV-P2227的抑制效果最为明显,但整体抑制率低于对HBsAg和HBeAg的抑制率。amiRNA-HBV-S608在转染后48h、72h和96h对HBV DNA的平均抑制率为27.8%,39.0%和36.5%,P<0.01。amiRNA-HBV-P2227在转染后48h、72h和96h对HBV DNA的平均抑制率为35.7%,48.2%和39.5%,P<0.01。3.稳定转染amiRNA-HBV对HBsAg和HBeAg的抑制作用检测结果显示,与2.2.15细胞相比,转染了阴性质粒得到的稳定株的HBsAg和HBeAg的分泌没有显著性变化,P>0.05,而转染的三个干扰HBV表达的质粒均显示了显著的抑制效果。其中amiRNA-HBV-P2227组对HBsAg和HBeAg的抑制率分别为76.2%和74.9%,P<0.01。amiRNA-HBV-S608组对HBsAg和HBeAg的抑制率分别为81.3%和58.2%,P<0.01。amiRNA-HBV-X402组对HBsAg和HBeAg的平均抑制率分别为70.2%和70.5%,P<0.01。4.稳定转染amiRNA-HBV对HBV DNA复制水平的影响在稳定转染的细胞株中,与瞬时转染相比,对HBV DNA的抑制率更高。荧光定量PCR (FQ-PCR)检测结果显示,amiRNA-HBV能够高效、特异地抑制HBV DNA的复制。amiRNA-HBV-S608组对培养上清中HBV DNA的平均抑制率为70.1%,P<0.01amiRNA-HBV-P2227对培养上清中HBV DNA的抑制率最高,达76.1%,P<0.01。amiRNA -HBV-X402的平均抑制率为67.9%,P<0.01。5.靶向宿主基因La蛋白的外源性amiRNA对HBV复制和表达水平的影响实验结果显示:转染amiRNA-La质粒后,HepG2.2.15细胞的细胞增殖和细胞形态与对照比较无显著差异;在La mRNA和蛋白水平上,结果显示3种amiRNA均能明显降低La mRNA和蛋白的表达,amiRNA-La-1130抑制效应最强,在转染后72h,La mRNA和蛋白的水平分别下降了58.6%和50.6%,P<0.01;但在病毒水平3种amiRNA-La质粒中仅有1个质粒能显著抑制相应细胞株中HBsAg和HBeAg的分泌,且抑制作用不强,其中以amiRNA-La-1130抑制作用最好,它对培养72h的细胞上清中的HBsAg和HBeAg平均抑制率分别为23.3%和25.8%,P<0.01,HBV DNA的平均抑制率为22.3%,P<0.01。6.靶向宿主基因ASGPR1的外源性amiRNA对HBV复制和表达水平的影响实验结果显示:转染amiRNA-ASGPR1质粒后,HepG2.2.15细胞的细胞增殖以及细胞生长速度与对照无显著差异,提示导入ASGPR1蛋白特异性amiRNA表达载体对HepG2.2.15细胞的生长无明显影响;在mRNA和蛋白水平上,RT-PCR结果证实3种amiRNA均能明显降低ASGPR1 mRNA和蛋白的表达;amiRNA-ASGPR1-610抑制效应最强,在转染后72h,ASGPR1 mRNA和蛋白的水平分别下降了57.3%和49.8%,P<0.01;在病毒水平3种amiRNA均能明显抑制相应细胞株中HBsAg和HBeAg的分泌,其中以amiRNA-ASGPR1-772抑制作用最强,它对培养72h的细胞上清中的HBsAg和HBeAg抑制率分别为31.3%和33.6%, P<0.01,HBV DNA的抑制率为29.7%,P<0.01。结论1.外源性amiRNA介导的RNA干扰能够高效特异性的阻断HBV的复制和表达。2.外源性amiRNA介导的RNA干扰可以高效阻断La和ASGPR1宿主靶基因的表达。3.宿主基因La和ASGPR1的表达水平与HBV的复制和表达有密切关系,进一步证实两者在HBV的感染和复制中起着关键作用。与HBV感染和复制相关的宿主基因可以作为HBV基因治疗的一个候选靶位。4.在提高转染效率的情况下,外源性amiRNA能够作为一种新的抗HBV的基因治疗手段。更多还原

【Abstract】 Chronic hepatitis B virus (HBV) infection remains an important world-wide public health problem. The number of persons with chronic HBV infection has exceeded 350 millions all over the world. It is one of the leading causes of liver cirrhosis and hepatocellular carcinoma(HCC). China is the highly affected area in which 10 percent of populations are infected with HBV. Current treatment strategies of HBV have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects.RNA interference (RNAi) is a new developmental gene silencing technology. It is a natural process by which double-stranded RNA directs sequence-specific post-transcriptional silencing of homologous genes. RNAi can be triggered by small RNA molecules such as siRNA and miRNA. However, siRNA mediated RNAi silences gene expression in a sequence-specific manner through the actions of small pieces of double-stranded RNAs. Many viruses escape RNAi-mediated suppression by counteracting the RNAi machinery through mutation of the targeted region, by encoding viral suppressors, or both. MicroRNAs (miRNAs) are endogenously expressed small ssRNA sequences of ~22 nucleotide in length, which naturally direct gene silencing through components shared with the RNAi pathway. The mature miRNAs can regulate gene expression by mRNA cleavage or translational repression. Compared with siRNA, miRNA still can play a translational repression role when miRNA complement partly with the target gene. Because of the flexibility of miRNA in binding with partially complementary mRNA targets, miRNA can serve as an anti-virus drug or vaccine to achieve a breakthrough in the treatments of viral mutations. The study of amiRNA functions will be helpful for improving HBV therapy.Given these circumstances, an alternative approach to antiviral siRNA therapy is to target one or more cellular genes making products that help to support virus replication. HBV infection and replication in vivo need the participation of host cellular genes. It is possible that RNAi targeting host cellular genes can decrease the replication of HBV. The advantage of RNAi targeting host genes is not depended on virus gene, so it can escape virus mutation in RNAi.Recently a large number of studies show that amiRNA, which targeted specific gene, can be introduced into cells through microRNA expression framework, and can inhibit the expression of target genes. Therefore, this experimental study constructed amiRNA expression plasmids which targeted different HBV sites,ASGPR1 gene and La protein genes to transiently and stable transfected HepG2.2.15 cells. We observed whether amiRNA-mediated RNA interference could block replication of HBV and HBV-related host gene expression by measuring the inhibition effect of the target gene, HBV DNA, and protein decrease level, so that we could understand whether amiRNA mediated RNAi targeting HBV and related host gene can be used in HBV gene therapy. Objective: The amiRNA vectors which targeted HBV different sites,ASGPR1 gene and La protein genes were constructed by untilizing microRNA expression framework, then were transfected into HepG2.2.15 cells. The aims of this study were to observe whether amiRNA-mediated RNA interference could decrease the replication level of HBV.Method: Thirteen amiRNA expression vectors were constructed based on the this study, the inserted DNA sequences of recombinant vectors mentioned above were all correct by DNA sequencing. The plasmids were transient and stably transfected by lipofectemine 2000 reagment. semi-quantitative RT-PCR was utilized to observe suppression level of the target gene mRNA; Western blot was utilized to observe suppression level of the target gene proteins. The MEIA was carried out to detect quantitatively HBsAg and HBeAg, the fluorescent quantitation PCR was utilized to observe the changes of HBV DNA copies.Results:1. The inhibition of HBsAg and HBeAg by transient transfected amiRNA-HBV.The seven amiRNA expression plasmid were transfected into HepG2.2.15 cells. The transfection efficiency were 50%~60% by FCM analysis. Under such circumstances, the assay results showed: the three of seven plasmid had obvious effect from 48h post-transfection, the inhibition of the HBsAg and HBeAg at 72h reached a peak, inhibitory effect gradually weakened at 96h post-transfection. The strongest inhibitor among the seven vectors, compared with mock group, the secretion of HBsAg in the supernatant was inhibited by 30.5%,49.8% and 46.3% respectively at 48h, 72h and 96h after amiRNA-HBV-S608 plasmids transfection, P<0.01, the secretion of HBeAg in the supernatant was inhibited by 25.5%,39.9% and 37.4% respectively at 48h, 72h and 96h after amiRNA-HBV-P2227 plasmids transfection, P<0.01. However, the change of negative control group was not obvious, P>0.05. 2. The inhibition of HBV DNA level by transient transfected amiRNA-HBVReal-time fluorescence quantitative PCR was performed to detrmine HBV DNA level at different time points. Similarly with the decrease degree of HBsAg and HBeAg, seven amiRNA-HBV plasmids resulted in different inhibition degree. Quantitative assay revealed that the greatest reduction was seen in the amiRNA-HBV-P2227 transfection group. The copies of HBV DNA in cells treated with amiRNA-HBV-P2227 was reduced by35.7%,48.2% and 39.5% at 48h, 72h and 96h respectively compared to mock controls, P<0.01.3. The inhibition of HBsAg and HBeAg level by stable transfected amiRNA-HBVHBsAg and HBeAg in stable transfected cells culture medium were assayed. The HBsAg levels of all the three cell lines integrated with the pcDNA6.2-HBV-amiRNA vector were significantly reduced compared with HepG2.2.15 cells, and the greatest reduction on amiRNA-S608 is 81.3%, P<0.01. HBeAg was also reduced in stable transfected cells, and the greatest reduction on amiRNA-S608 was 74.9%, P<0.01. No significant reduction was measured on the cells stably transfected with negative control compared with 2.2.15 cells, P>0.05.4. The inhibition of HBV DNA level by stable transfected amiRNA-HBVFor stable transfected cell clones, compared with negative control vector, amiRNA-mediated RNAi resulted in a higher reduced level of HBV DNA in three plasmids transfected cells than transient transfection. Among them, amiR-HBV-P2227 and amiRNA-HBV-X402 was much more efficient with 76.3% and 67.9% inhibitory rate, P<0.01.5. The inhibition of HBV replication and expression level by amiRNA targeted La gene.The results showed that: compared with mock control, HepG2.2.15 cell proliferation and cell growth rate had no significant difference. In La mRNA and protein level, the results showed that three amiRNA can significantly reduce the La mRNA and protein expression, the greatest reduction on amiRNA-La-1130 transfected group, the expression of La mRNA and protein levels were down-regulation by 58.6% and 50.6% after 72 h, P<0.01. In the virus level, the one of three amiRNA-La plasmids could obviously inhibit the secretion of HBsAg and HBeAg, the greatest reduction on amiRNA-La-1130 transfected group, the expression of HBsAg and HBeAg were down-regulation by 23.3% and 25.8% at 72 h, P<0.01, HBV DNA were down-regulation by 22.3% after 72 h, P<0.01.6. The inhibition of HBV replication and expression level by amiRNA targeted ASGPR1 gene.The results showed that: compared with mock control, HepG2.2.15 cell proliferation and cell growth rate had no significant difference. In La mRNA and protein level, the results showed that three amiRNA can significantly reduce the La mRNA and protein expression, the greatest reduction on amiRNA-ASGPR1-610 transfected group, the expression of La mRNA and protein levels were down-regulation by 57.3% and 49.8% at 72 h, P<0.01. In the virus level, the two of three amiRNA-ASGPR1 plasmids could obviously inhibit the secretion of HBsAg and HBeAg, the greatest reduction on amiRNA-ASGPR1-610 transfected group, the expression levels of HBsAg and HBeAg were down-regulation by 31.3% and 33.6% after 72 h, P<0.01, HBV DNA level was down-regulation by 29.7% at 72 h, P<0.01.Conclusion:1. Artificial microRNA-mediated RNA interference can be high effectively block HBV expression in specific manners.2. Artificial microRNA-mediated RNA interference can block La and ASGPR1 host target gene expression. 3. Host La and ASGPR1 gene expression level is closely related with HBV replication and expression, furtherly confirmed that the two gene play a key role in the HBV infection and replication. The relevant host genes of HBV infection and replication could be an optional targets for HBV gene therapy.4. Improving the efficiency of transfection, artificial microRNA could be act as a new means of HBV gene therapy更多还原