乙型肝炎病毒在原代肝细胞共培养模型上的增强感染研究

发布时间：2018-03-03 23:03

本文选题：乙肝　切入点：原代肝细胞　出处：《北京协和医学院》2013年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(hepatitis B virus, HBV)的感染是目前世界范围内最为严重的公共卫生问题之一。根据世界卫生组织统计,截至目前为止,每年由于急性或慢性的HBV感染造成的死亡人数达到60万左右。在现有的HBV体外感染研究中,原代树烝肝实质细胞(primary tupaia hepatocytes, PTH)是一种被成功使用的模型。尽管如此,该系统仍然存在着相当的局限性。体内外培养环境的差异使得从肝脏组织中分离出来的原代肝细胞仅能部分维持原有的HBV易感性,而且随着体外培养时间的增加往往会迅速丧失。此外,实验动物的个体差别和分离技术的复杂性等因素也会增加不同批次分离的原代细胞的差异。这些问题导致了PTH作为HBV研究模型显得不够高效和不够稳定,降低了检测结果的信噪比,加大了分析难度,使得一些真正影响感染过程的重要因素在实际研究过程中变得难以被发现。另一方面,短暂的易感性维持时间也限制了许多研究方法的应用。我们采用树烝的肝星状细胞(hepatic stellate cells,HSC)作为支持细胞,建立了一种新的PTH共培养模型。我们发现,HBV在这种共培养系统上,感染得到了显著增强。除了HSC之外,使用树烝皮肤来源的原代成纤维细胞(primary tupaia fibroblast, PTF)建立的共培养系统也能对HBV的感染起到类似的促进效果。与之相一致的是,共培养系统对肝细胞的部分生理功能,包括细胞色素酶P450(CYP450)超家族成员的活性等方面也起到了较为明显的增强效果。这样的共培养系统很好地解决了原有模型存在的低效和不稳定的问题,有利于利用该模型揭示更多感染过程中的关键因素。除了促进感染之外,我们还发现,共培养系统能够显著地延长体外培养的原代肝实质细胞对HBV易感性的维持时问。这一优势拓宽了PTH作为HBV感染模型在实际研究中的应用。我们进一步研究了共培养系统影响肝实质细胞的相关机制,发现支持细胞对肝细胞的影响需要通过细胞间的直接接触来实现,并且两种细胞接触的时间点也会对最终促进感染的程度产生影响。我们通过表达水平、分布以及功能等多方面的检测,发现新鉴定出的HBV受体,钠离子-牛磺胆酸共转运蛋白(NTCP)在共培养系统中的肝细胞上表达没有发生明显的变化。另一方面,我们发现细胞内与HBV复制相关的一些肝富集转录因子(liver enriched transcription factor, LETF)的表达量出现了上调。研究发现其中的两种,HNF4A和HLF,在通过RNA干扰下调其表达水平后,HBV的感染效率也出现了明显的下降。这些结果显示,共培养系统中HBV的感染很有可能是在病毒的复制与生物合成等环节受到了增强。我们对共培养系统中的肝细胞作了进一步研究,发现两种与细胞生理状态密切相关的标记蛋白,MRP2(multidrug resistance protein2)和ZO-1(zonula occludens protein1)在表达和分布上呈现出与体内肝脏组织中的肝细胞类似的特征,而这些特征是单培养的肝细胞所不具备的。这说明共培养系统中的肝细胞在生理特征上更加接近于体内微环境中的细胞。因此,我们建立的原代肝细胞的共培养系统,能够作为一种体外研究HBV感染更加高效的模型,并且能够模拟更为接近体内生理状态下发生的HBV感染过程。这对于我们进一步揭示HBV感染和致病的相关机制有着十分重要的意义。
[Abstract]:The infection of hepatitis B virus (HBV) is one of the most serious public health problems in the world. According to WHO statistics, so far, the number of deaths caused by acute or chronic HBV infection has reached 600 thousand so far.
In the study of HBV infection in vitro. The primary hepatocytes (primary tree, Tupaia hepatocytes, PTH) is a successful model used. However, this system still has considerable limitations. In vivo and in vitro culture environment makes the difference of primary hepatocytes isolated from the liver tissues can only maintain the original HBV susceptibility, and it increases with time in vitro will be lost. In addition, individual differences and separation technology of experimental animal and other factors will also increase the complexity of different batches of isolated primary cells. The difference in these problems led to PTH HBV as the research model is not efficient and stable enough the test results, reduce the signal-to-noise ratio, increase the difficulty of analysis, some important factors that really affect the infection process in the course of practical research has become difficult to be found. On the other hand, short The duration of susceptibility also restricts the application of many research methods.
We use the hepatic stellate cells (hepatic, the stellate cells tree, HSC) as support cells, a new PTH co culture model. We found that HBV co cultured in this system, the infection has been significantly enhanced. In addition to HSC, primary fibroblasts, skin source (using a tree primary Tupaia fibroblast, PTF) to establish a co culture system can promote a similar effect on HBV infection. In line with this, co culture system on the part of the physiological function of liver cells, including cytochrome P450 (CYP450) superfamily member activity also plays an enhancement effect more obvious. This co culture system is a good solution to the original model is inefficient and unstable, is conducive to the use of the model reveal more key factors in the process of infection.
In addition to promoting infection, we also found that co culture system can significantly prolong the maintenance of HBV susceptibility in primary cultured hepatocytes in vitro. This advantage widens the application of PTH as a HBV infection model in practical research.
We further studied the mechanism of co culture system affecting liver parenchymal cells, and found that the influence of supporting cells on hepatocytes needs to be achieved through direct contact between cells. And the time of two cell contacts will also affect the degree of infection ultimately.
We through the expression of multiple testing of distribution and function, found the newly identified HBV receptor, Na + - taurocholate co transporter (NTCP) in liver cells expression have no obvious changes in co culture. On the other hand, we found that cells with HBV replication related liver enrichment of transcription factors (liver enriched transcription factor, LETF) expression was upregulated. The study found that two of them, HNF4A and HLF, through RNA interference inhibit the expression, the infection efficiency of HBV also decreased significantly. These results suggest that the most likely in viral replication and biosynthesis link has been enhanced in co culture system of HBV infection.
We made further research on the system of the liver cells were co cultured, found two kinds of state and closely related to the physiological cell marker protein, MRP2 (multidrug resistance protein2) and ZO-1 (zonula occludens protein1) on the expression and distribution characteristics showed in vivo and in the liver of liver cells were similar, and these features are single cultured liver cells do not have. This means that the system of hepatocytes on the physiological characteristics of more close to the in vivo microenvironment in cell culture.
Therefore, we established the primary hepatocyte coculture system, can be used as a more efficient model of HBV infection in vitro, and can simulate the process is more close to the physiological state of the infection occurred under HBV. This is for us to further reveal the HBV infection and pathogenesis of the relevant mechanism has a very important significance.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

【共引文献】