【作者】 李敏然；

【导师】 孙殿兴； 李兵顺；

【作者基本信息】 河北医科大学 ， 内科学， 2007， 硕士

【摘要】 目的:慢性乙型病毒性肝炎是我国的常见病,迄今尚无满意疗法。基因疗法为乙型肝炎提供了新的治疗策略。RNA干扰(RNA interference , RNAi)技术是指内源性或外源性双链RNA(dsRNA)介导的,细胞内mRNA发生特异性降解,并导致靶基因的表达沉默,产生相应的功能表型缺失的现象。RNAi针对乙型肝炎病毒(Hepatitis B virus , HBV)基因具有强效的沉默功能,但尚缺乏理想的向细胞内转运的工具,本研究应用腺病毒载体转导HBV载体表达发夹状RNA(short hairpin RNA , shRNA ),在HBV转基因小鼠体内利用野生型HBV为之提供包装,分泌抗HBV的HBV样颗粒,实现导向型及放大效应,有望为发展特异性的抗HBV基因治疗提供新的技术平台。方法:应用AdMaxTM腺病毒载体系统,将HBV载体表达双靶位shRNA的质粒- pdp3ssx2m-203H1sh6-1715U6sh8插入腺病毒载体,构建了重组腺病毒Ad-CH-sh68及对照载体Ad-CH-RFP2,转染293细胞,大量扩增后用氯化铯密度梯度纯化法纯化,测重组腺病毒滴度。后将重组腺病毒感染HBV转基因小鼠,30只HBV转基因小鼠随机分为3组:实验组、阴性对照组和空白对照组,每组分别为10只。实验组小鼠经尾静脉注射Ad-CH-sh68,阴性对照组经尾静脉注射Ad-CH-RFP2,空白对照组注射生理盐水。感染重组腺病毒Ad-CH-RFP2后4、12、20天观察小鼠体内红色荧光蛋白(Red Fluorescent Protein, RFP)的表达。在感染0、4、8、12、16、20天分别观察shRNA对HBV的抑制效应。应用MEIA进行病毒蛋白定量检测,免疫组织化学检测HBV特定蛋白的表达,荧光定量PCR检测HBV-DNA,还同时检测了组织病理学变化。将治疗组和阴性对照组小鼠血清中提取HBV-DNA,做PCR扩增观察体内是否形成重组HBV。结果:1成功设计并构建了重组腺病毒Ad-CH-sh68和对照载体Ad-CH-RFP2,在293细胞内大量扩增后,氯化铯密度梯度纯化法纯化后测的浓度分别为1.23×1013、4.73×1012病毒颗粒(viral particles, vp)/mL。2重组腺病毒经尾静脉感染HBV转基因小鼠后,小鼠一般情况良好,在实验期间均未出现死亡。实验组和阴性对照组小鼠肝脏病理HE染色结果显示感染腺病毒后12天肝细胞出现轻度肿胀,局部有淋巴细胞浸润,后恢复正常。3阴性对照组小鼠在感染Ad-CH-RFP2后4天肝脏为粉红色,冰冻切片观察RFP表达量结果显示重组腺病毒能有效感染HBV转基因小鼠,感染后4天大部分的肝细胞表达RFP,随时间推移荧光表达量下降。4 shRNA抑制HBV转基因小鼠体内HBV-DNA的复制:三组小鼠血清提取DNA后,经荧光定量PCR检测,结果显示shRNA对血清内的HBV-DNA有显著的抑制作用,重组腺病毒AD-HBV-sh68感染后4天开始下降,感染后20天对HBV-DNA的抑制高达约2log10数量级,效果优于对体内HBV特定蛋白表达的抑制。与空白对照组相比差别有显著意义(P<0.001),与阴性对照组相比差别也有显著意义(P<0.001),阴性对照组与空白对照组相比无意义。治疗组20天与0天相比差别有意义(p<0.001),阴性对照组、空白对照组均无意义(p>0.05)。5 shRNA对HBV转基因小鼠体内表面抗原(hepatitis B surface antigen,HBsAg)的抑制作用:通过对三组小鼠血清中HBsAg定量的检测,观察shRNA对HBV基因表达的抑制效果。注射重组腺病毒AD-HBV-sh68后,在治疗不同时间定量检测小鼠血清中的HBsAg,结果发现在感染后4天开始下降,至20天降于一低水平表达,抑制率为67.39%。腺病毒的转染率和HBsAg半衰期较长对抑制率也有影响。与空白对照组相比差别有显著意义(P<0.001),与阴性对照组相比差别也有显著意义(P<0.001),阴性对照组与空白对照组相比无意义。治疗组20天与0天相比差别有意义(p<0.001),阴性对照组、空白对照组均无意义(p>0.05)。6 HBV转基因小鼠肝脏内表面抗原(HBsAg)和核心抗原(hepatitis B core antigen ,HBcAg)免疫组织化学:HBsAg与HBcAg主要呈核浆型分布,少部分在胞浆内表达。空白对照组HBV转基因小鼠肝细胞胞核内有大量棕褐色颗粒(HBsAg),阴性对照组小鼠内棕色颗粒数较空白对照组略有下降,治疗组小鼠在感染重组腺病毒4、12天时棕色颗粒数所占比例明显降低,感染20天体内只有少量棕色颗粒表达。HBcAg在三组的表达情况类似于HBsAg,治疗组在感染20天只有很少的阳性颗粒表达,阴性对照组较空白对照组略有下降。7 HBV转基因小鼠体内重组HBV形成:HBV转基因小鼠血清PCR结果证明了HBV载体在野生型HBV辅助下能够形成重组HBV,且随着载体质粒比例的增大,重组载体也增加。结论:本研究应用腺病毒载体转导HBV载体表达shRNA,经尾静脉注射感染HBV转基因小鼠,实验结果表明shRNA能有效且较长时间的抑制小鼠体内HBV基因的复制和表达。尽管把HBV载体插入腺病毒载体,利用腺病毒感染HBV转基因小鼠,能有效的将shRNA转入小鼠肝脏细胞中,但走向临床,仍需不断的全方面研究。总之,本研究结果为RNAi技术治疗HBV的慢性感染提供了重要的实验数据,为进一步深入研究奠定了基础。更多还原

【Abstract】 Objective: Chronic infection with the hepatitis B virus(HBV) is a major problem of public health and a common disease in our country, and currently available therapies have limited efficacy. Gene therapy, which has become one of the most attractive antiviral strategies, is bringing a promising light. RNA interference(RNAi), is a newly technique in which exogenous, double-stranded RNAs(dsRNAs) that are complimentary to known mRNA’s, are introduced into a cell to specifically destroy that particular mRNA, thereby diminishing or abolishing gene expression. RNAi turned out to be a powerful tool for HBV gene silencing. But the gene deliver systems are still a formidable obstacle. In this study, we tested a new strategy by using adenovirus vector transducing HBV vector expressing shRNA. When used in vivo, the wild type HBV will help the HBV vector to form recombinant HBV virion, then oriented to infect hepatocytes and to amplify the anti-HBV effects. Because of this, large dosage of adenovirus is no needed. This will overcome the limitation of adenovirus for its cytotoxic effects and induction of immune reaction. It will provide a promising therapeutic strategy toward the goal of long-term control of HBV infection.Methods: The system of AdMaxTM was used to complete the construction of recombinant adenovirus. Firstly, the shuttle plasmid was constructed, HBV-2shRNA (pdp3ssx2m -203H1sh6-1715U6sh8) or HBV-RFP (pDP3LJ3-1472-RFP2) was inserted into digested pDC312; Secondly, the shuttle plasmids were cotransfected with rescue plasmid pBHG1ox△E1,3Cre into the adenoviral packaging 293 cells to acquire the recombinant adenovirus Ad-CH-sh68 and control adenoviral vector Ad-CH-RFP2 , then amplified the virus。After identified, the desired Ad vectors were purified by CsCl2 density gradient ultracentrifuge and titrated. 30 mice were randomly divided into there groups: the experimental group、the negative control group and the blank control group, 10 mice for each, which received Ad-CH-sh68、Ad-CH-RFP2 or the same dose of saline via tail vein injection. PCR was used to test the recombinant HBV formation by Ad-HBV vector in HBV transgenic mice. Mouse livers were taken out for preparation of frozen section after infection at 4d, 12d, 20d. The expression of RFP was observed on fluorescent microscope. The inhibition effect of shRNA on the replication and expression of HBV in HBV transgenic mice was observed at different time-points 0d, 4d, 8d, 12d, 16d, 20d. The MEIA and immunohistochemistry were carried out to detect the changes of viral proteins. The fluorescence quantitation PCR was used to observe the HBV-DNA respectively. We also observed the alteration of pathology in liver tissue by HE staining.Results:1 The recombinant adenoviral vector of HBV-shRNA gene- Ad-CH-sh68 and the control recombinant adenovirus Ad-CH-RFP2 were constructed and amplified in 293 cells successfully. After purified by density gradient ultracentrifuge and titrated, the titers of Ad-CH-sh68 and Ad-CH-RFP2 were 1.23×1013 vp/mL and 4.73×1012 vp/mL , respectively.2 After Ad-CH-sh68 or Ad-CH-RFP2 was delivered into HBV transgenic mice using the tail vein injection method , no mice was died. The HE staining showed slight pathological changes such as hepatocyte swelling and lymphocyte infiltration in the liver of mice infected by Ad-CH-sh68 and Ad-CH-RFP.3 By day 4 post-infection, the color of the negative control group mice’s livers was pink. RFP expression was visually observed on fluorescent microscope, most of cells expressed RFP on day 4.The expression of RFP declined on days 12 and 20 post-infection.4 shRNA-mediated inhibition of HBV-DNA in vivo:The fluorescence quantitative PCR(FQ-PCR) analysis was perfor- med with total serum DNA from the three groups mice. On day 4, the average level of HBV-DNA in the mice receiving Ad-CH-sh68 had began decreased. The result of FQ-PCR proved that the shRNA could depress the HBV-DNA by 2log10 in serum of transgenic mice on 20 days after injection and effect of inhibition is better than that of viral proteins. There were significance between the Ad-CH-sh68 and Ad-CH-RFP treated mice (p<0.001), as also the saline treated mice . Ad-CH-sh68 could inhibit the HBV-DNA significantly on day 20(p<0.001).5 shRNA-mediated inhibition of HBsAg in vivo:Level of HBsAg was measured in the serum of mice as a downstream indicator of HBV gene expression. By day 4 postinfection, mice receiving AD-HBV-sh68 had began to declined. HBsAg levels continued to decline in those mice until day 20. The maximal inhibition of HBsAg in serum of HBV transgenic mice was 67.39% on 20 days after injection. The result may be related with transfection efficiency of Ad-CH-sh68 and longer half-time of HBsAg in vivo. The irrelevant RFP or saline exhibited no effect on HBsAg levels, demonstrating the specificity of the shRNA effect(P<0.001),Ad-CH-sh68 could inhibit the HBsAg significantly on day 20(p<0.001).6 Effects of siRNA on HBsAg and HBcAg expression in the liver of mice: Immunohistochemical staining for 3.5-kb–derived gene product HBsAg and HBcAg in liver from Ad-CH-sh68-infected mice allowed us to directly observe HBV clearance from hepatocyte. HBsAg-positive hepatocytes scattered in the cytoplasm, and HBcAg-positive hepatocytes scattered also in the cytoplasm. Like serum antigen levels, staining for HBsAg or HBcAg was greatly reduced in the hepatocytes of Ad-CH-sh68-infected mice on day 20. Compared with the control group, HBsAg- or HBcAg-positive cells were significantly reduced in the liver of Ad-CH-sh68 treated mice. In contrast, the mice treated with Ad-CH-RFP or saline exhibited no inhibitory effect on the antigens.7 Recombinant HBV virions could formed in the transgenic mice when present with wild-type HBV.Conclusion: In this study, we have demonstrated that adenovirus-mediated delivery of HBV-shRNA to the liver of mice is an extremely effective and long-lasting approach to suppress ongoing viral gene expression and replication in vivo. Although this approach should prove to be very useful as a research tool for transient gene suppression in vivo, alternative delivery options for clinical use need to be examined. Nonetheless, the data presented here provide the justification needed to pursue the challenge of developing RNAi as an effective treatment for chronic HBV infection.更多还原