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
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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,原代树齁肝实质细胞(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。
我们采用树齁的肝星状细胞(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,使用树齁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。
Approximately two billion people have been infected with human Hepatitis B virus (HBV) worldwide with over240million people are chronically infected. Among them about600,000die of HBV-related liver diseases each year. Available treatments are inadequate or non-effective to those who are chronically infected and at high risk of cirrhosis and hepatocellular carcinoma. More than50%of liver cancers worldwide are attributable to HBV and over90%in Asia.
For a long period of time, primary hepatocytes, including those from humans, chimpanzees or tree shrews are the only cell types that can support complete life cycle of HBV. However, these cells precipitously decline in viability and remain susceptible to infection for only a few days following their preparation from liver tissue. Moreover, the infection efficiency in these models usually is low, only5-10%of primary hepatocytes could be infected in vitro with regular infection dosage. In addition to the primary hepatocytes, a hepatoma cell line, HepaRG, is the only cell line that is susceptible to HBV infection. However, its susceptibility is subjected to a month long process with induction of corticoids and DMSO.
In this study, we used primary hepatocytes of treeshrew as a model and examined if co-culturing hepatocytes with other nonparenchymal cells can improve human HBV infection of PTHs. We also investigated molecular mechanisms that are involved in the enhanced infection, and whether the recently identified receptor NTCP plays a role.
We demonstrated the infection of both HBV and WMHBV on PTHs co-cultured with hepatic stellates cells (HSCs) from tupaia or primary tupaia fibroblasts (PTFs) was significantly enhanced. The enhancement of HBV infection in co-cultivation is direct intercellular contact dependent. Co-cultured hepatocytes exhibited enhanced hepatic functions and physiological characteristics as indicated by activity of cytochrome P450enzymes, expression patterns of multidrug resistance protein2(MRP2) and zonula occludens protein1(ZO-1). Moreover, the HBV susceptibility of PTH was found to be prolonged effectively through co-cultivation.
The enhanced infection of HBV was partially attributable to the up-regulated hepatic transcription factors in the co-cultured hepatocytes, whereas the expression level of the newly identified cellular receptor sodium taurocholate cotransporting polypeptide (NTCP) for HBV was not significantly changed. Nonetheless, NTCP remains the receptor mediating viral entry into the co-cultured hepatoctyes. Together, experimental HBV infection on primary hepatocytes co-cultured with supportive cells sheds new light on HBV entry and provides a useful model to study contributing factors for the efficiency of HBV infection in vitro.
在现有的HBV体外感染研究中,原代树齁肝实质细胞(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。
我们采用树齁的肝星状细胞(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,使用树齁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。
Approximately two billion people have been infected with human Hepatitis B virus (HBV) worldwide with over240million people are chronically infected. Among them about600,000die of HBV-related liver diseases each year. Available treatments are inadequate or non-effective to those who are chronically infected and at high risk of cirrhosis and hepatocellular carcinoma. More than50%of liver cancers worldwide are attributable to HBV and over90%in Asia.
For a long period of time, primary hepatocytes, including those from humans, chimpanzees or tree shrews are the only cell types that can support complete life cycle of HBV. However, these cells precipitously decline in viability and remain susceptible to infection for only a few days following their preparation from liver tissue. Moreover, the infection efficiency in these models usually is low, only5-10%of primary hepatocytes could be infected in vitro with regular infection dosage. In addition to the primary hepatocytes, a hepatoma cell line, HepaRG, is the only cell line that is susceptible to HBV infection. However, its susceptibility is subjected to a month long process with induction of corticoids and DMSO.
In this study, we used primary hepatocytes of treeshrew as a model and examined if co-culturing hepatocytes with other nonparenchymal cells can improve human HBV infection of PTHs. We also investigated molecular mechanisms that are involved in the enhanced infection, and whether the recently identified receptor NTCP plays a role.
We demonstrated the infection of both HBV and WMHBV on PTHs co-cultured with hepatic stellates cells (HSCs) from tupaia or primary tupaia fibroblasts (PTFs) was significantly enhanced. The enhancement of HBV infection in co-cultivation is direct intercellular contact dependent. Co-cultured hepatocytes exhibited enhanced hepatic functions and physiological characteristics as indicated by activity of cytochrome P450enzymes, expression patterns of multidrug resistance protein2(MRP2) and zonula occludens protein1(ZO-1). Moreover, the HBV susceptibility of PTH was found to be prolonged effectively through co-cultivation.
The enhanced infection of HBV was partially attributable to the up-regulated hepatic transcription factors in the co-cultured hepatocytes, whereas the expression level of the newly identified cellular receptor sodium taurocholate cotransporting polypeptide (NTCP) for HBV was not significantly changed. Nonetheless, NTCP remains the receptor mediating viral entry into the co-cultured hepatoctyes. Together, experimental HBV infection on primary hepatocytes co-cultured with supportive cells sheds new light on HBV entry and provides a useful model to study contributing factors for the efficiency of HBV infection in vitro.
引文
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