| 李 滢,何毅怀,杨方万,穆茂媛,林世德.慢性乙型肝炎病毒感染病毒复制与肝细胞内质网应激反应相关性的初步研究[J].,2019,19(4):690-695 |
| 慢性乙型肝炎病毒感染病毒复制与肝细胞内质网应激反应相关性的初步研究 |
| A Preliminary Study on the Correlation between the Replication of Hepatitis B Virus and Endoplasmic Reticulum Stress of Hepatocytes |
| 投稿时间：2018-06-06 修订日期：2018-06-30 |
| DOI：10.13241/j.cnki.pmb.2019.04.019 |
| 中文关键词: 乙型肝炎病毒 内质网应激 HepG2.2.15细胞 拉米夫定 |
| 英文关键词: Hepatitis B virus Endoplasmic reticulum stress HepG2.2.15 cells Lamivudine |
| 基金项目:国家自然科学基金项目（81160067/H0318） |
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| 中文摘要: |
| 摘要 目的：研究体内、体外乙型肝炎病毒（hepatitis B virus，HBV）复制水平与肝细胞内质网应激（endoplasmic reticulum stress，ER stress）反应基因表达的相关性。方法：选择7例慢性HBV感染者作体内研究，用液氮冷冻保存肝脏穿刺活检组织，检测肝功能并提取mRNA；体外研究分三组，转染组（HepG2.2.15）为HBV基因转染人肝癌细胞株，对照组（HepG2）为人肝癌细胞株，干预组（3TC）为50 ?滋g/mL拉米夫定作用于HBV基因转染人肝癌细胞。分别培养2天、4天、6天后收集细胞提取mRNA。HBV DNA载量的测定用实时定量聚合酶链反应（PCR）；活化转录因子4（ATF4）、转录因子C/EBP同源蛋白（CHOP）、活化转录因子6（ATF6）、X盒结合蛋白1（XBP1）及葡萄糖调节蛋白78（GRP78）的mRNA表达，用SYBR Green荧光定量逆转录聚合酶链反应（SYBR Green RT-PCR）检测，以?茁-actin为内参照；GRP78的表达用蛋白免疫印迹法（WB）检测。结果：体内研究结果表明：XBP1、GRP78、CHOP及ATF6基因表达水平与病毒载量、炎症程度及肝组织纤维化程度无显著相关性（P>0.05）；ATF6与GRP78基因表达水平和HBV DNA水平呈显著正相关（P<0.05）。体外研究结果表明：干预组细胞培养2天至6天HBV DNA水平下降，CHOP、GRP78、ATF6及XBP1 基因表达水平在三组之间差异无统计学意义（P>0.05）；WB结果表明，转染组细胞GRP78的表达培养4天与6天相比差异无统计学意义（P>0.05），干预组培养6天后与转染组比较差异无统计学意义（P>0.05）。结论：研究结果提示体内HBV复制水平与肝细胞ER stress反应显著相关，而肝脏炎症程度及肝脏纤维化程度等与ER stress反应无明显相关性；体外三组细胞之间ER stress基因的表达无显著差异（P>0.05）。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the possible correlation between hepatitis B virus (HBV) replication level and the related genes expression of hepatocyte endoplasmic reticulum stress (ER stress) in vivo and in vitro. Methods: 7 patients with chronic hepatitis B (CHB) were collected as vivo study. Liver biopsies were carried out and a fragment of liver tissue was immediately frozen in liquid, liver functions were tested and mRNA was extracted. Vitro study was divided as 3 groups, HBV genome transfected human hepatoma carcinoma cells were used as transfection group (HepG2.2.15), human hepatoma HepG2 cells were used as control group (HepG2), HBV genome transfected human hepatoma carcinoma cells were cultured with 50 μg/mL lamivudine were used as interference group (3TC). The cells were collected to extract the mRNA after cultured for 2 d, 4 d and 6 d HBVDNA levels were determined by the method of real-time quantitative polymerase chain reaction (PCR), the expressions of activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), activating transcription factor 6 (ATF6), X box-binding protein 1 (XBP1) and glucose regulated protein 78 (GRP78) were inves- tigated by the method of SYBR Green real-time quantitative reverse transcriptase-polymerase chain reaction, β-actin was choosed as inner reference. The expression of GRP78 was determined by the method of western-blotting (WB). Results: The results in vivo study suggest- ed that XBP1, GRP78, ATF6 and CHOP mRNA with HBV DAN load, intensity of liver inflammation and degree of liver fibrosis showed no significant correlations (P>0.05), while significantly positive correlations were found between GRP78 mRNA, ATF6 mRNA with HBV DNA levels (P<0.05). The result in vitro study suggested that HBV DNA levels in interference group decreased from 2 d to 6 d and no significant differences were found in the expressions of CHOP, GRP78, ATF6 and XBP1 mRNA among the there groups (P>0.05). The WB results suggested that GRP78 expression in transfection group increased from 4 d to 6 d but not significant and no significant differences were found in interference group and transfection group at 6 d(P>0.05). Conclusion: The results suggested that hepatocyte ER stress is correlated with HBVDNA levels in vivo study, while intensity of liver inflammation and degree of liver fibrosis showed no significant correlations. The gene expressions of ER stress are not changed among the there groups in vitro. |
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