【作者】 杨兵；

【导师】 张莉萍；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2011， 硕士

【摘要】 乙型肝炎病毒感染是一个全球性的公共卫生问题,慢性乙型肝炎常常发展为肝硬化、肝癌甚至肝衰竭。肝细胞肝癌(HCC)患者中乙型肝炎病毒(HBV)感染率高达50%～80%,其中HBX蛋白在HBV致癌过程中发挥了重要作用。目前治疗HBV感染主要采用干扰素及核酸类似物,但存在副作用大、病毒变异逃逸等问题,因此,新的治疗手段和新药开发具有重要意义。RNA干扰(RNA interference,RNAi)作为一种转录后基因沉默手段已经在肿瘤、慢性疾病及感染性疾病的治疗中显示出巨大潜力及应用前景;目前认为siRNA临床应用中最主要的瓶颈在于其稳定性较低并缺乏有效的靶向转运策略。常用于转运siRNA的系统主要有纳米载体(nanocarrier)和分子连接体(molecular conjugate)两类。分子连接体多采用靶向性配体及穿透性多肽策略。HBV表面蛋白包含大蛋白(L)、中蛋白(M)、小蛋白(S),三者均具有S结构域,M蛋白较S蛋白多出55个氨基酸的preS2区域,而最外面的L蛋白较M蛋白又多出108或119个氨基酸的preS1区域。HBV侵入肝细胞的过程尚未彻底清楚,其特异性受体尚无明确结论。但相关的研究均表明preS1可能是病毒与细胞膜上特异性受体结合并携带病毒基因组进入细胞内的配体;而preS1与受体结合的区域主要在其N端,其中21-47aa多肽片断被证实起关键性作用。目的:本实验旨在成功构建HepG2-HBX稳定表达株的基础上,验证preS1连接体特异转运siRNA进入细胞内进行RNA干扰的能力,为乙型肝炎病毒及肝癌的治疗及预防提供新的探索。而目前国内外尚无相关实验报道。方法:本课题以HBV病毒中的X基因为RNA干扰对象;用脂质体将含有HBX序列的真核表达质粒pCDNA3.1(+)/HBX-Flag转染入HepG2细胞系,通过G418筛选后分别经RT-PCR和免疫组化,对HBX的稳定表达进行验证;化学合成靶向HBX的siRNA(即siHBX),用脂质体转染进入HepG2-HBX细胞株,分别以RT-PCR和实时荧光定量RT-PCR检测HBX mRNA表达水平变化,Western Blot检测HBX蛋白表达水平差异。在验证HBX稳定表达及siHBX有效性的基础上,化学合成pres1分子连接体,pres1分子连接体分别与肝细胞系与siRNA进行结合能力试验,然后以preS1分子连接体作为转运载体转染siHBX入HepG2-HBX后经实时荧光RT-PCR及Western Blot检测siHBX对HBX基因的抑制效果。结果:转染筛选试验结果显示HBX转染成功;进一步的RT-PCR和免疫组化结果提示19号阳性克隆高水平稳定表达HBX基因。siHBX作用HepG2-HBX细胞系后36h和72h后对HBX mRNA的抑制率分别为85.37%±3.02%和58.05%±4.00%,而非特异性对照siRNA对HBX mRNA的抑制率分别2.11%±0.04%和1.02%±0.05%;免疫印迹实验及细胞流式检测验显示siHBX能够抑制HBX蛋白及细胞周期进展。进一步凝胶电泳迁移试验(EMSA)证实preS1分子连接体能够与siRNA进行有效结合(摩尔比10:1),同时可以携带siRNA进入肝细胞内高效抑制HepG2-HBX细胞株内HBX的表达。与空白组相比,非特异阴性对照组抑制率为11.94±6.3%,而阳性对照组(脂质体)及实验组(preS1分子连接体)的抑制率分别为85.24±2.6%和77.80±6.10%。结论:上述结果显示本实验组成功构建了HepG2-HBX细胞株,同时证实了siHBX的有效性及特异性;preS1分子连接体可作为一种新型siRNA转运载体,携带siHBX进入肝癌细胞内发挥RNA干扰机制,为乙型肝炎病毒相关性肝脏疾病的治疗提供了新的途径。更多还原

【Abstract】 Hepatitis B Virus(HBV) infection is a global pubulic healthy problem, chronic infection has a variable course after several decades, resulting in hepatic cirrhosis、liver cancer even to hepatic failure. HBV infection ratio in HCC patient is up to 50%-80%. Hepatitis B virus X protein has been suggested to play an important role in hepaocarinogenesis. Now licensed therapies that have been employed to eliminate HBV are some interferons and nucleic acid analogues, but there are some problems such as side effects, virus mutation escape. Therefore, the development of novel approaches to inhibit HBV replication is requiered.As a new therapeutic strategy, RNA interference may be commonly used in many fields to treat human tumors、virus infections or metabolic diseases . However, the key hurdles of RNA interference are the low stability of siRNA and the uneffective delivery system. At present, the siRNA delivery systems mainly inculde nanocarrier and molecular conjugate, and the molecuar conjugate used the CPPs and CTLs strategy.The surface proteins of HBV include large proteins(L)、middle proteins(M) and small proteins(S). They all have the domains of S. Compared to S protein, M protein has another pres2 domain which consists of 55 amimo acids. Compared to M protein, the most outside of L protein has another pres1 domain which consists of 108 or 118 amimo acids. At present, it is not completely clear how HBV invades into liver cells, and there is no definite conclusion about the host specific receptors.Recent studies indicate that preS1 might be the ligand which binds to specific membrane receptors, thereby leading to HBV entering into liver cell. The principal binding domain of preS1 is N teminal, and the 21th to the 47th amino acids are suggested to play a critical role.Objective: This study aims to construct a HepG2-HBX cell line that stably expressing the hepatitis B virus protein X (HBX) and to explore the specific silencing effects of siRNA which is carried by preS1 molecular conjugate on the HBX gene, and finally to establish an experimental basis for subsequent therapeutic and preventive research in HBV infection. There is no related study reports about these described above.Methods: In this study, the role of HBV X gene was investigated by RNAi. The pCDNA3.1(+)/HBX-Flag plasmid containing the HBX sequence was transfected into HepG2 cell lines with lipofectamine 2000, and the HepG2-HBX cell line stably expressing the HBX gene was screened out with G418; RT-PCR and immunohistochemistry were employed to validate the expression of the HBX gene; the siHBX fragment targeting the HBX gene was chemically synthesized and transfected in to the HepG2-HBX cell line, and RT-PCR, real time quantitative RT-PCR and Western blotting were respectively performed to evaluate the the expression of HBX at both mRNA and protein levels in order to validate the specific silencing effects of siHBX on the HBX gene. Moreover, the pres1 molecule conjugate was chemically synthesized, and we tested the ability of the preS1 to bind to HepG2-HBX and the siRNA. The siRNA was then transfected into the HepG2-HBX cell line by preS1. Quantitative RT-PCR and Western blotting were respectively employed to validate the inhibition effects of siHBX on the expression of the HBX gene.Results: G418 tests showed that the pCDNA3.1(+)/HBX-Flag plasmid containing the HBX sequence was transfected into the HepG2, RT-PCR and immunohistochemistry validated that HBX19 highly expressed HBX gene. Compared to the empty control, the inhibition rate of the nagative group was 2.11%±0.04% and 1.02%±0.05% after 36h and 72h, the lipofectamine were 85.37±2.6% and 58.05%±4.00%, respectively. Moreover, HBX protein and the cell cycle progression were repressed analyzed by Western Blot and Flow cytomety, respectively.Further EMSA study showed that preS1 could effectively bind to siRNA (10mol:1mol), and siRNA was then transfected into the HepG2-HBX cell line by preS1 to inhibit the expression of the HBX gene. Compared to empty control, the inhibition rate of the nagative group was 11.97±6.3%, while the preS1 molecule conjugate and the lipofectamine were 85.24±2.6% and 77.80±6.1%, respectively.Conclusions: These results indicated that HepG2-HBX cell line stably expressing the HBX gene was successfully constructed and suggested the effctiveness and specificty of the siHBX to the HBX gene. Moreover, preS1 molecule conjugate could be used as a novel siRNA carrier to inhibit the expression of HBX gene in liver cancer cells, laying an experimental basis for the subsequent therapeutic research in HBV infection-related liver diseases.更多还原