摘要
乙型肝炎病毒(HBV)作为一种非细胞毒性的嗜肝病毒,感染机体后将导致急慢性肝炎、肝硬化、肝癌等一系列肝脏疾病的发生,成为危害人类健康的一项严重问题。HBV感染后,决定病程转归的一个主要因素是机体的免疫反应能力,尤其是特异性细胞毒性T淋巴细胞(CTL)的反应水平。表达抗原特异性受体细胞毒性T(CD8+T)淋巴细胞能够识别HBV感染肝细胞表面的MHC-Ⅰ抗原肽段复合物,对感染细胞的病毒进行清除,当特异性的CTL建立以后,感染的肝细胞表面如果不表达MHC-Ⅰ抗原肽段复合物,也没法对病毒进行清除,而且最近的研究指出氧化应激损伤与丙肝病毒感染协同作用减弱了肝细胞内负责处理加工MHC-Ⅰ抗原的蛋白酶体的活性,也有研究证据表明乙肝病毒蛋白与蛋白酶体亚基之间可以相互作用降低肝细胞本身的抗原提呈功能,有利于病毒病原体躲避机体的免疫反应。因此乙肝病毒感染肝细胞的处理加工病毒抗原的能力有待于进一步研究证实。
本研究利用肝癌细胞系HepG2以及在HepG2基础上转染了HBV的HepG2.2.15细胞为靶细胞模型,初步探讨比较HepG2和HepG2.2.15细胞对乙醇致氧化应激损伤的敏感性差异,通过Western bloting方法检测LMP2免疫蛋白酶体的亚基,研究氧化应激损伤对HepG2和HepG 2.2.15细胞处理加工病毒抗原能力的影响。实验过程中,分别用1.5%、3%、5%、8%浓度的乙醇作用HepG2和HepG2.2.15细胞16h以后收集上清检测谷草转氨酶,四甲基偶氮唑盐(MTT)计算细胞活性抑制率。两种细胞1.5%、5%乙醇处理16小时后用WesternBlot检测免疫蛋白酶体亚基LMP2的表达。通过上述研究方案获得如下结果：
HepG2.2.15细胞对3%、5%的乙醇诱导损伤更敏感。HepG2.2.15细胞的LMP2表达比HepG2细胞低,1.5%乙醇处理16小时以后,HepG2.2.15细胞的蛋白酶体亚基LMP2表达比HepG2细胞下调更明显。
我们的研究提示了HBV的复制和氧化应激损伤降低了蛋白酶体亚基LMP2的表达,推测HBV的复制可能会造成肝细胞的氧化应激损伤,会减弱肝细胞的对MHC-I类分子限制的病毒抗原的提呈。
目的：建立一种简便经济的HBV转基因小鼠的原代肝细胞的分离纯化方法,并进行培养,观察HBV转基因小鼠原代肝细胞的生物学功能,探索HBV转基因小鼠原代肝细胞作为抗乙肝病毒药物药效学研究的细胞模型的潜能。
方法：采用稍加改良的两步胶原酶灌流法分离获取HBV转基因小鼠的肝细胞,用percoll密度梯度离心法纯化原代肝细胞,并进行原代培养。以台盼蓝染色法测细胞活力,显微镜下观察肝细胞形态变化,检测培养体系不同时间培养上清液中白蛋白、乳酸脱氢酶(LDH)、尿素的含量以及不同时间培养上清液中乙肝表面抗原(HBS-Ag)、e抗原(HBe-Ag)、HBVDNA分泌水平,利用HBV转基因小鼠原代肝细胞模型,通过检测培养上清HBS-Ag、HBe-Ag、HBVDNA分泌水平对Bay-4109的抗乙肝病毒作用进行药效学评价。
结果：改良的两步胶原酶消化法结合percoll密度梯度离心法所获取的肝细胞活细胞达到90%,纯度95%以上,贴壁良好、活性高、功能强；LDH漏出量、白蛋白分泌及尿素合成等指标在1周内呈现规律性变化,第3和第4天时LDH漏出量最低,白蛋白分泌及尿素合成功能正常,HBS-Ag在一周内分泌水平稳定、HBe-Ag在1-2天分泌高峰、后逐渐下降,HBVDNA分泌水平表明所分离的肝细胞在培养第3-4天功能最佳。Bay-4109作用于HBV转基因小鼠原代肝细胞48小时以后能够影响培养上清中HBe-Ag的分泌。
结论：胶原酶原位灌注法可以用于分离HBV转基因小鼠的原代肝细胞,并且分离获得的肝细胞存活数目以及细胞活性均能够满足实验观察的需要。并且HBV转基因小鼠的原代肝细胞体外培养具有病毒复制以及肝细胞的各种生物学特性,具有作为抗乙肝病毒药物药效学研究的细胞模型的潜能。
Hepatitis B virus(HBV) as a hepatotropic,noncytopathic virus,primarily infects hepatocytes and causes a series of liver diseases such as acute and chronic hepatitis,liver cirrhosis and hepatocellular carcinoma, which are among the most important human health problems worldwide.Immune response to viral antigens plays a crucial role in the pathogenesis of hepatitis B viral infections. However, when clonal expansion of cytotoxic T-lymphocytes (CTLs) is established, the next important restriction for elimination of infected cells is the availability of peptide-major histocompatibility complex (MHC) classⅠcomplexes, which are recognized by CTLs on the surface of target cells (hepatocytes). With the new recognition that the synergistic action of ethanol with HCV results in the suppression of MHC classⅠ-restricted antigen presentation. Evidence for direct interaction with proteasome subunits has been obtained for the HBV protein to reduced MHC classⅠ-restricted antigen presentation.
In this study,we usede transfected cell line HepG2.2.15 and its parental cell Line HepG2 to research the effect of ethanol-induced oxidative stress and HBV replication on proteasome.Cells were stimulated by ethanol with gradient concentration(1.5%-8%) for 16 hours. The assay of AST release from the cultured cells was used to evaluate the cytotoxicity of ethanol. The inhibition rate of cells was determined by MTT method. Western Blot was utilized to detecte the expression of LMP2. Our major findings are shown as followe:
HepG2.2.15 was more sensitive to 3% and 5% ethanol induced cell injury. The expressions of LMP2 were much lower in HepG2.2.15 cells compared with HepG2 cells with or without 1.5% ethanol exposure.
Our study demonstrated ethanol and HBV down-regulated the expression of immunoproteasome subunit LMP2 in hepatoma cells.We deduce HBV replication and Ethanol-induced oxidative stress suppress the generation of peptides for MHC classⅠ-restricted antigen presentation.
AIM:The purpose of this study was to search a simplified, inexpensive method in isolating HBV-transgenic mice hepatocytes,and the primary hepatocytes of HBV-transgenic mice was observed. To explore the potency of HBV-transgenic mice hepatocytes as a vitro model in anti-HBV drugs.
METHOAD:Mice hepatocytes were isolated by modified two steps collagenase perfusion,and then purificated by 40% Percoll.The viability of cultured hepatocytes was assessed by trypan blue exclusion. The morphologic change of cultured hepatocytes was observed, and the concentrations of albumin, urea,lactate dehydrogenase (LDH),HBS-Ag,HBe-Ag and HBVDNA in the supernatant collected from different cultural period of better isolated system were examined. Primary hepatocytes were treated with Bay-4109 to evaluate their pharmacodynamics and mechanism of action.
RESULT:Hepatocytes obtained by modified two steps collagenase perfusion were intact and had a prosperous viability and an active function. The fluctuated changes of LDH leakage, albumin synthesis and urea levelwere displayed in one week, and the lesser LDH leakage and higher concentrations of albumin and urea were observed on the third day. The contents of HBS-Ag secretion mantained higher level from 1th to 7th day, HBe-Ag and HBVDNA decreased gradually over time in the period of culture. The HBS-Ag and HBe-Ag release from the cultrued cells decreased after Bay-4109 treatment.
CONCLUSION:It is concluded that the collagenase digestion method is feasible for isolation of HBV-transgenic mice hepatocytes. Primary hepatocytes have an potentiality as a vitro model in anti-HBV drugs.
引文
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