树鼩原代肝细胞培养体系和乙型肝炎病毒体外感染模型的建立
文献类型：学位论文
| 作者 | 张顶 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 何有文 ; 张华堂 |
| 关键词 | 乙型肝炎病毒 树鼩 肝细胞原代培养 感染机制 药物筛选 |
| 其他题名 | The establishment of culture system of primary Tree Shrew hepatocytes and HBV in vitro infection model |
| 中文摘要 | 乙型肝炎病毒（Hepatitis Virus type B, HBV）感染是一种严重威胁人类健康的世界性问题。由于HBV感染具有宿主和组织特异性，长期以来一直缺乏有效的体外感染模型，严重制约了HBV感染机制和治疗药物的研究。树鼩（Tree Shrews, Tupaia Belangeri）与人和灵长类亲缘关系较为接近，作为小动物模型在乙型肝炎研究中备受关注。但由于各种原因的限制，HBV树鼩感染模型仍处于“国际公认但实际短缺，国内首创但实际空缺”的状况。因此，复建和优化HBV树鼩体内外感染模型成为我们长期研究方向所必备的前提基础和开端。而树鼩原代肝细胞的分离和培养则是建立HBV体外感染模型的关键的第一步。我们通过与机械分离法的直接比较，验证了两步灌注法在树鼩肝细胞分离中的优越性。进而发现，在分离后的体外培养过程中，二甲基亚砜不仅能够促进和维持原代肝细胞的分化，而且能够显著地抑制纤维状细胞群的出现。同时，肝细胞生长因子（HGF）和表皮生长因子（EGF）能够促进肝细胞在体外长期存活。在此优化的条件下，原代培养可持续4-5周，并且较多的细胞聚集形成类似肝窦结构的形态，从而成功地复制和改进了树鼩原代肝细胞培养体系，为建立HBV体外感染模型提供了基础。与此同时，我们采集了2种来源的HBV，即以乙肝病人血清和HepG2.2.15细胞培养上清为来源。我们采用蔗糖浓度梯度离心法以纯化病人血清病毒，能较好地将HBV颗粒和亚病毒颗粒区分开来。在收集HepG2.2.15细胞株来源的病毒时，我们详细研究了HepG2.2.15细胞的生长曲线和病毒产量曲线，找到病毒产量与细胞生长状态之间的联系，确定了最佳收集病毒时间，并通过放大培养条件，大量收集HepG2.2.15细胞的培养上清。我们采用浓缩和未浓缩的HepG2.2.15病毒、纯化的血清病毒等3种方式制备的病毒感染树鼩原代肝细胞，通过不同水平的指标来检测感染结果。首先，细胞内HBVx基因mRNA的检测结果表明，3种来源的病毒都可以感染树鼩原代肝细胞。同时，细胞上清中分泌的病毒颗粒及病毒蛋白的检测结果表明，HepG2.2.15病毒感染树鼩原代肝细胞的效果比血清病毒的感染效果好。免疫荧光的检测结果进一步证实了HepG2.2.15病毒能够感染树鼩原代肝细胞。因此，在我们的实验中，树鼩原代肝细胞对2种来源和不同制备方式的病毒均具有易感性，且以未浓缩的HepG2.2.15上清病毒感染力优于血清病毒。综上所述，我们现有的结果验证了树鼩原代肝细胞对HBV具有易感性。就感染效率而言，尽管感染效率依赖于病毒滴度及其感染力，我们在病毒细胞比例为0.02:1的条件下，仍能确认易感性，并获得了与国际同类研究相当的感染效率。就本研究的应用前景而言，我们所建立的HBV体外感染模型，能够作为实验室的通用平台，用以来研究HBV的感染机制和抗病毒药物的筛选与评价。 |
| 英文摘要 | Hepatitis Virus Type B (HBV) infection is a world-wise disease that imperils human health. Due to the host and tissue specificity, the HBV infection model is not readily available, which limits the study of HBV infection mechanism and drug-screening. Tree Shrews (Tupaia Belangeri) are phenogenetically close to humans and primates, which are prominent small animal model for studies of HBV. However, due to some practical limits, the usage of Tree Shrew hepatocytes as HBV infection model is still blank. So, establishing the Tree Shrew hepatocytes model for HBV infection in vitro and the optimization will be fundamental and significant for our long-term research on HBV. Isolating and culturing Tree Shrew hepatocytes is the first key step toward the cellular model of HBV infection in vitro. In this study, we validated the superiority of perfusion over mechanic dispersion for hepatocytes separation and isolation. Subsequent cultures showed that dimethyl sulphoxide (DMSO) could suppress the growth of fibroblast-like cells and maintain the hepatocytes in the differentiated status. Furthermore, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) could sustain growth and survival of the hepatocytes in the long-term culture. The combination of DMSO and HGF/EGF could maintain the hepatocytes in a longer and more stable differentiated status with clear trend to form liver sinus-like structures. Following the methods detailed here, we could obtain plentiful starting material for the establishment of HBV infection in vitro. We adopted two sources for collecting HBV: one is serum from hepatitis B patient and the other is HepG2.2.15 cell line. We used sucrose gradient centrifugation to purify HBV from serum, which resulted in separating HBV particles and the subviral particles distinctly. Before collecting HBV from HepG2.2.15 culturing, we performed the HepG2.2.15 cell growth curve and virus production curve, and the relationship between these two different curves were analyzed. Finally, we fixed on a best time point to collect virus and collected HBV from HepG2.2.15 culturing with magnified culturing scale. The Tree Shrew hepatocytes were infected by three sourced virus, which were condensed and uncondensed HBV from HepG2.2.15, HBV from serum. The infection result was detected in three different levels: virus nucleic acid, virus protein and virus particles. First, the detection of HBV x gene mRNA in the cells indicates that all three sourced virus are capable of infecting the Tree Shrew hepatocytes. Second, we found virus from HepG2.2.15 have higher infection ability than virus from serum by the detection result of HBV particles secreted into the supernate. Third, Immunofluorescence result further substantiated the infection of Tree Shrew hepatocytes by virus from HepG2.2.15. Therefore, our result showed that primary Tree Shrew hepatocytes are susceptible to three sourced HBV, and that HBV from HepG2.2.15 have higher infection ability than HBV from serum. Collectively, our result validated the susceptibility of primary Tree Shrew hepatocytes by HBV. Although the high infection efficiency depends on virus load and virus infection ability, we detected the infected cells in the virus to cell ratio of 0.02:1 and found the commensurate infection efficiency in our model with the international peers. The HBV in vitro infection model we established here, as a laboratory routine, would be a promising system for the studying of HBV infection mechanism and HBV drug-screening. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6307]  |
| 专题 | 昆明动物研究所_分子免疫生物学 |
| 推荐引用方式 GB/T 7714 | 张顶. 树鼩原代肝细胞培养体系和乙型肝炎病毒体外感染模型的建立[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
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