【作者】 李尧；

【导师】 夏雪山；

【作者基本信息】 昆明理工大学 ， 生物化工， 2012， 硕士

【摘要】 丙型肝炎病毒(HCV)是丙型肝炎病的主要病原体。据1999年世界卫生组对于HCV感染情况的调查,全球感染HCV的患者约1.7-2.0亿,我国属于HCV的高度感染区,其感染者约占3.2%。高效病毒体外培养体系与合适实验动物模型的缺乏,是丙型肝炎药物与疫苗研究的主要障碍。2005年,高效的HCV病毒细胞培养体系的建立,可以经过体外培养获得高感染性的HCV病毒,使得动物模型的建立尤为紧迫。除丙型肝炎病毒的自然宿主——人和黑猩猩外,尚未发现其他自然动物对HCV易感。树鼩是一种主要分布于我国云南地区低等灵长类动物。研究发现,树鼩可被HCV患者的血清感染,但是由于HCV患者血清的来源不明、病毒滴度较低、感染不稳定、树鼩个体背景复杂等原因,丙型肝炎病毒对树鼩的感染特性未被最终阐明。本研究以人工驯化、繁育的树鼩为实验对象,采用二步灌流、胶原酶消化法,分离、获得树鼩原代肝细胞(PTH),优化灌流液、离心速度和培养基,用单因素比较和正交实验法进行比较各优化因素之间的差别,最终以台盼蓝染色法测定获得PTH细胞数量,用MTT法测定PTH的状态。实验确定的最佳灌流液为D-Hank’s、最佳离心速度为800rpm3min、600rpm3min、400rpm2min、最佳培养基配方为1×ITS、1%DMSO、200uM谷氨酰胺、0.15%牛血清白蛋白、100U/ml青霉素、100ug/ml链霉素、0.1uM地塞米松和WEM培养基。经过优化的PTH具有更长的生长周期和更好的生长状态,可用于受体分子转染和HCV感染实验。为进一步提高树鼩原代肝细胞对HCV的感染性,将已成功构建可表达人CD81受体的质粒,用脂质体介导转染进入PTH,使树鼩原代肝细胞表达人的CD81分子,用PCR定性和Q-PCR的方法测定出CD81转染后最高表达量约是未转染对照细胞的234倍。最后,采用J6/JFH1-Huh7.5.1培养体系获得的病毒载量为107IU/ml以上的HCV病毒,感染高表达人CD81的PTH,荧光定量PCR法检测培养上清中HCV病毒载量,发现转染和未转染的PTH感染产出的病毒载量基本在104-105IU/ml之间,转染的细胞感染的第7天产出最高病毒载量值为106IU/ml.综上所述,本研究以树鼩为实验动物,采用二步灌流技术得到树鼩原代肝细胞,优化了树鼩原代肝细胞的分离、培养技术,得到了生长时间更长、状态较佳的PTH。将人的CD81转染入PTH中使该细胞能高表达人的CD81,用J6/JFH-1HCV病毒感染转染的细胞,最终用Q-PCR和荧光定量PCR的技术检测转染和未转染细胞感染上清载量,未发现转染所造成HCV感染性增强或病毒增殖能力提高。本研结果为树鼩做为丙型肝炎病毒小型动物模型,奠定了前期研究基础,并有助于HCV感染与复制的机制研究。更多还原

【Abstract】 As the cause of Hepatitis C, hepatitis C virus is prevalent worldwide. According to the released data, it was estimated that about1.7-2.0hundred million population are infected with HCV. China is the mid-high HCV prevalence area with HCV infection rate of3.2%among general population. So far, the effective treatment and protective vaccine is still poor due to the lack of effective culture system and suit animal model. In2005, the robust HCV cell culture system had been established. It made the seeking and development of the suit small animal model become more urgent. However, there is no evidence showing any other animals was infected with HCV except for human being and chimpanzee. Tree shrews (Tupaia belangeri chinensis) are small animals, closely related to primates, mainly spreaded in Yunnan province, which adapt easily to a laboratory environment. Although it was reported that Tupaia could be infected by HCV derived from hepatitis C patient’s serum, the infection of HCV to Tupaia has not been confirmed yet because of uncertain origin of HCV, low HCV viral load, and unclear background of host animal.In this study, we further determine the suspection of tree shrew as hepatitis C animal model. The two-step perfusion, collagenase digestion technology was used to separate and get primary tupaia hepatocyte (PTH). The used perfusion fluid, centrifugal speed and the culture medium and other factors of PTH culture were optimized. Finally the goted number of PTH are determined with Trypan Blue method and the PTH vatality is evaluated with MTT method. It was showed that D-Hank’s is the best perfusion fluid, centrifugal speed of800rpm for3min,600rpm for3min,400rpm for2min are more suit to PTH operation. The best culture medium contains1x ITS formula,1%DMSO,200uM glutamine,0.15%cow serum albumin,100U/ml penicillin,100ug/ml Streptomycin,0.1uM dexamethasone and WEM medium. PTH could grow for longer period with better state under this optimizated circumstance. It is qualified for plsmid transfection and HCV infection.To further increase the PTH’s suspectivity for HCV, the constructed plasmid which expressing CD81receptor gene successfully, was introdueced into PTH with the lipidosome. The expressed RNA was qualitvely and quantitavitly determined with PCR and real time PCR method. The results showed transfected PTH could express the human CD81with234times than control cells which transfected with mimic plasmid. The transfected PTH was inoculated with HCV supernants with107copies/ml viral load, which from J6/JFH1-Huh7.5.1HCV culture system. It was found that the viral load of PTH infection is between104and105IU/ml both in transfected cells and non-transfected cells, and the highest the viral load in transfected cells is106IU/ml on the seventh culture day.In summary, Tupaia belangeri chinensis is conducted with the two step perfusion technique, to achieve the primary Tupaia hepatocytes. The culture factors was optimized to optimized to get qualified hepatocytes. Moreover, the human CD81was transfected into PTH and highly expressed. The tranfected PTH could support the infection and replication of HCV derived from J6/JFH1-Huh7.5.1HCV culture system. Our results further confirmed the possibility of Tupaia as a HCV small animal model, and may provide a useful technique for the research on HCV infection and replication.更多还原