【作者】 王盛；

【导师】 于长明； 童贻刚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2010， 硕士

【摘要】 丙型肝炎病毒(Hepatitis C Virus,HCV)是引起人类丙型肝炎的病原体。全球HCV的感染率约为3%,近1.7亿人感染HCV[1]。我国约为5000万人,并且年发病人数呈高速上升趋势,2009年发病人数比2003年上升了534%,达到131849人。HCV感染易慢性化[2],其慢性化率可高达70%,部分慢性丙型肝炎患者最终可发展为肝纤维化、肝硬化,甚至肝细胞癌。目前临床上主要采用干扰素和利巴韦林联合治疗丙型肝炎,但该疗法并非对所有慢性丙型肝炎患者均有效,约50%患者不能产生持续病毒学应答(SVR),其中I型HCV感染患者SVR率最低[3, 4]。因此,亟需寻找新的抗HCV药物靶点和研制新的抗HCV药物。长期以来,由于一直缺乏有效的HCV体外培养模型和小动物模型严重阻碍了HCV致病机制的研究、抗HCV药物体内作用效果的评价和保护性疫苗的研发。因此探索建立有效的HCV模型,包括HCV体外培养细胞模型和HCV小动物模型,具有十分重要的意义。就目前国内外的研究成果来看,HCV体外培养细胞模型主要采用的是2005年日本学者Wakita等人[5]建立的体外转录模型,该模型存在操作繁琐、稳定性差等缺陷,限制了它的应用。HCV小动物模型进展比较缓慢,比较成功的是人肝嵌合uPA-SCID小鼠模型[6],但是由于该小鼠出生时肝脏损伤严重,致使成活率较低,而用于移植的人肝又不易获得,这使该模型局限于少数实验室的应用,无法满足日趋紧迫的HCV研究的需要。正是基于以上的原因,本研究建立了一种基于核酶自剪切机制的稳定分泌HCV细胞模型,并用核酶自剪切载体对HCV小鼠模型进行了探索性研究。具体研究内容和结果如下:1、HCV蛋白多克隆抗体的制备。根据实验的需要,原核表达六个HCV不同的蛋白片段,以表达纯化的蛋白为抗原,分别免疫新西兰大耳白兔,收集血清,制得六个针对HCV不同蛋白片段的多克隆抗体。2、HCV核心蛋白表达载体的构建。根据实验的需要,在真核表达载体pEGFP-N1多克隆位点(MCS)前引入原核启动子lac promoter序列,得到穿梭表达载体pEGFP-N1-lac,再把PCR扩增出的HCV核心蛋白序列(573bp)插入到构建的穿梭表达载体MCS区,得到重组质粒pEGFP-N1-lac-core,该质粒在原核细胞(E.coli)和真核细胞(HepG2)中均能表达HCV核心蛋白,可以作为实验中HCV蛋白检测的阳性对照。3、重组质粒pJFH1/GDD-2RB的构建。应用突变PCR的方法,在原始克隆pJFH1/GDD的HCV全长基因组cDNA的两端各加入一个核酶序列,并通过合适的酶切位点把两端含有核酶的HCV全长cDNA连接到真核表达载体pEGFP-N1的CMV启动子下游,得到目的质粒pJFH1/GDD-2RB。此外,将上述重组质粒pJFH1/GDD-2RB中HCV NS5B基因(编码产物为RNA依赖的RNA聚合酶)中GDD的碱基序列突变为GND,使表达产物失去RNA聚合酶活性,得到阴性对照质粒pJFH1/GND-2RB。4、质粒转染HepG2细胞及稳定细胞系的筛选。pJFH1/GDD-2RB及其对照质粒pJFH1/GND-2RB转染HepG2细胞后,加入G418溶液至终浓度为0.7mg/ml(为预实验确定的最佳筛选浓度),对转染细胞进行加压筛选。2个星期后,每孔均有大量细胞死亡,但是整合入转染质粒的细胞存活下来并分裂增殖。随后采用有限稀释法把存活下来的细胞倍比稀释铺于96孔板,最后挑取单克隆进行扩大培养,其中pJFH1/GDD-2RB稳定克隆命名为HepG2/GDD,pJFH1/GND-2RB稳定克隆命名为HepG2/GND。5、细胞培养上清中HCV RNA的检测。根据深圳匹基公司HCV PCR荧光定量检测试剂盒操作流程,提取细胞培养上清中的RNA,提取的RNA经RNase-freeDNase I处理,以去除基因组DNA的污染。然后经过酚抽得到纯净的RNA,按照试剂盒配制25μl反应体系,应用Roche Lightcycler 2.0完成反应。结果显示HepG2/GDD培养上清中含有HCV RNA,参照标准品,其滴度达到1x107,阴性对照(HepG2/GND培养上清)和空白对照(水)中均没有检测到HCV RNA。6、间接免疫荧光检测HCV核心蛋白的表达。消化细胞HepG2/GDD和原始HepG2细胞(空白对照),铺于抗原片上,待细胞贴壁后,用37℃预温的1×PBS洗3次,每次10min,然后用4%的多聚甲醛(PBS稀释)室温固定30min,洗涤(洗涤方式同上)。再用0.2% Triton X-100(PBS稀释)浸泡5min以增强细胞膜的通透性,洗涤。用山羊血清室温封闭固定好的细胞30min,PBS清洗后,加入1:400稀释的抗HCV核心蛋白单抗,37℃孵育1h,洗涤,加入1:1000稀释的FITC(异硫氰酸荧光素)标记的羊抗鼠IgG,37℃避光孵育1h,洗涤后加入含有DAPI(4’,6-联脒-2-苯基吲哚二盐酸盐)的封片剂,荧光倒置显微镜下观察到HepG2/GDD细胞胞浆区域有较强荧光出现,而空白对照则无此现象,由此表明,HepG2/GDD细胞中有HCV核心蛋白表达。7、蛋白免疫印迹检测HCV核心蛋白的表达。收集HepG2/GDD和原始HepG2细胞,裂解缓冲液裂解后离心取上清,按照4:1的体积比加入5倍上样缓冲液,沸水浴15分钟。上样进行SDS-PAGE电泳,之后转至PVDF(聚偏氟乙烯膜)膜上,5%脱脂牛奶37℃封闭2h,抗HCV核心蛋白单抗4℃孵育过夜,HRP(辣根过氧化物酶)标记的羊抗鼠IgG 37℃孵育1h,ECL显色,暗室内曝光于胶片上,观察到HepG2/GDD泳道有条带产生,大小与阳性对照相似,而空白对照(HepG2细胞)无条带呈现。8、HCV病毒颗粒的电镜观察。收集HepG2/GDD细胞培养上清(约15ml),慢慢搅动并加入NaCl至终浓度0.5mol/L,再加入PEG6000至终浓度为10%,4℃过夜。8000rpm离心30min,收集沉淀,溶于100μl PBS中。用细滴管吸取一滴样品悬液,滴于铜网上,进行负染操作。完成负染后,置于电子显微镜(Philips, TECNAI-10)下,观察到HCV颗粒,直径约为55nm。9、干扰素治疗。把HepG2/GDD细胞铺于6孔细胞培养板中,加入不同剂量的IFN-α至终浓度为10U/ml、100U/ml、500U/ml。3d后收集细胞上清,提取RNA,实时定量PCR检测病毒滴度,发现病毒滴度随干扰素浓度升高而降低,预示该细胞模型可以用来抗HCV药物的筛选。10、HCV转染小鼠模型探索研究。通过高压水动力法把质粒转染入C57小鼠肝脏细胞,3天后处死小鼠,收集血清并取肝。用实时定量PCR的方法没有检测到血清中含有HCV RNA,免疫组化检测小鼠肝脏中HCV核心蛋白发现,在血管周围细胞中有HCV核心蛋白表达。通过上述一系列的研究,我们成功构建了质粒pJFH1/GDD-2RB,通过把该质粒转染HepG2细胞,然后G418加压筛选,获得了整合有该质粒的细胞系HepG2/GDD,该细胞系能够有效表达HCV核心蛋白,实时定量PCR检测上清液中HCV滴度达到1×10~7,电镜观察HCV直径为55nm。应用经IFN-α治疗发现上清液中病毒滴度随干扰素-α浓度的升高而降低。在随后开展的HCV转染小鼠模型探索研究中发现,小鼠血清中没有HCV颗粒,但是免疫组化证明,小鼠肝细胞中有HCV核心蛋白的表达,这为我们深入开展小鼠模型研究打下良好的基础。更多还原

【Abstract】 The hepatitis C virus (HCV) is a major cause of liver disease and infects about 170 million people worldwide, about 3% of the population in the world. In China, more than 50 million people are infected by HCV, and the number is increasing sharply. According to the data reported from China CDC, the newly infected people rose by 20.96% in 2009, reaching 131849. The majority, about 85%, of HCV-infected patients fails to clear the virus, and many develop chronic diseases, including hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). At present, the optimal regimen is a combination of PEG-IFN and ribavirin. This combination therapy results in sustained virological remission (SVR) in only 50% of patients, so it is of necessity to find new antiviral targets, and develop drugs and efficient vaccines, which have been hampered by the lack of robust model systems. The development of the sub genomic and genomic replicon system is a major breakthrough to understand viral replication and viral-host interactions and provides a means to test therapeutic targets. However, as yet, most of these systems can not efficiently produce viral particles, nor do they produce infectious virions. So it is of significance to develop new robust model systems, including in vitro cell models and in vivo animal models.Up to date, all the efficient cells models come from JFH1 system developed by Wakita in 2005. However, its application was limited due to the difficulty to control and operate. The HCV animal models, such as Chimpanzee system and mouse system, progress slowly and work unsatisfactorily.Based on the above situation and urgent need for robust HCV model systems, we constructed an HCV-producing cell model and explored to develop a new mouse model. The research results are as following:1. Preparation of anti-HCV polyclonal antibodies. Six proteins was expressed in E.coli, and then purified. Subsequently, six rabbits were immunized with the recovered proteins respectively. Finally, the blood sera were collected and ELISA was performed to determine the antibody titer.2. Construction of HCV core protein expression vector. To construct the versatile expression vector pEGFP-N1-lac, the lac promoter, which came from pUC19, was inserted into pEGFP-N1 digested with EcoRI/BamHI. Then HCV core gene was amplified by PCR and inserted downstream of the CMV promoter of pEGFP-N1-lac. Western blot analysis showed that this recombinant plasmid can express HCV core protein both in E.coli and in HepG2.3. Construction of HCV-ribozyme plasmid pJFH1/GDD-2RB. The original pJFH1 was reconstructed by putting ribozymes on both 5’and 3’ends of full-length HCV cDNA. Then the fragment containing HCV cDNA and the ribozymes was cloned into pEGFP-N1 to construct target plasmid pJFH1/GDD-2RB. In addition, a mutation in the GDD motif of HCV NS5B, RNA-dependent RNA polymerase (RdRP), was introduced into this construct, and the mutated construct was named pJFH1/GND-2RB as negative control.4. Selection of stable cells lines HepG2/GDD and HepG2/GND. The above constructed plasmids (pJFH1/GDD-2RB and pJFH1/GND-2RB) were transfected into HepG2 cells respectively, and then G418 was added to the final concentration 0.7mg/ml for clone selection. 2 weeks later, the cells without plasmid were dead and the survival cell clones were selected for stably transfected cell line cultivation.5. Determination of HCV RNA in the cell line culture medium. HCV RNA level was quantitated by TaqMan real-time PCR method. HCV RNA was detected in HepG2/GDD culture medium, the level of which was at least 128-fold higher than the level of HepG2/GND culture medium.6. Detection of HCV core protein by immunofluorescence and Western blot analysis. HepG2/GDD cells or negative control cells HepG2 were analyzed by immunofluorescence and Western blot. As expected, the results showed the presence of core protein in HepG2/GDD cells but not in HepG2 cells.7. Detection of HCV particles by electron microscopy. HepG2/GDD cell culture supernatant was precipitated with PEG6000 and put on formavar/carbon-coated copper grids. The grips were negative stained with 2% pphosphotungstic acid (PTA, pH6.5) and observed under a transmission electron microscope. The result showed that these cell culture-derived HCV particles were approximate 55nm in diameter.8. Suppression of HCV replication by IFN-α. To test the sensitivity of HCV production to antiviral, HepG2/GDD cells were treated with IFN-α. 3 days later, HCV RNA was measured in the culture supernatants and the result showed that the HCV RNA level decreased with the increased concentration of IFN-α.9. Transfection of C57 mouse by hydrodynamic injection. A large volume of ribozyme-containing HCV expression plasmid pJFH1/GDD-2RB (10% of the mouse body weight) in a saline solution that is isotonic with blood was injected into mouse blood through the tail vein in less than 10sec. 3 days after injection, the blood serum and liver of C57 were obtained. Real-time RT-PCR showed that HCV RNA was not detectable in the blood, however, the core protein expression was detected in the liver by immunohistochemisty.According to the series of studies shown above, we successfully constructed a HCV-producing cell model based on self-cleaving ribozymes. IFN-treating experiment demonstrated that it can be utilized for anti-HCV drug screening and evaluation. The transfected HCV mouse model was also explored and the probability of using it as an in vivo drug evaluation tool need to be examined.更多还原