乙型肝炎病毒对核转录因子Nrf2表达影响的研究

发布时间：2018-06-12 00:41

  本文选题：乙型肝炎病毒 + Nrf2　； 参考：《中南大学》2014年硕士论文

【摘要】：背景和目的：乙型肝炎病毒(HBV)感染呈世界性分布,我国是HBV的高流行地区,一般人群中流行率为7.18%,大概有9300万慢性乙型肝炎患者。慢性HBV感染可以导致肝脏慢性炎症坏死和纤维化,甚至可发展为肝硬化及肝细胞癌,对患者造成极大危害。氧化应激反应是肝细胞炎症坏死、纤维化甚至肝硬化及肝癌发生发展过程中的重要机制之一。慢性HBV感染的肝细胞持续表达病毒蛋白可以引起肝细胞线粒体释放过量的活性电子和活性氧自由基,导致肝细胞脂质过氧化和DNA损害,从而造成肝细胞的损伤。核转录因子Nrf2作为抗氧化应激基因的调控者,在抗氧化应激反应中促进多种抗氧化蛋白的合成而发挥重要作用,它激活多种抗氧化蛋白如NQO1, HO-1等,能够减轻肝细胞所受的氧化损害。在对酒精性肝炎、非酒精脂肪肝、药物性肝炎等的临床研究发现.Nrf2的上调可以减轻肝细胞所受的氧化应激损害。但HBV对Nrf2表达影响的研究报道很少,并且作用机制也不清楚。 在本研究中,我们通过研究HBV质粒转染HepG2细胞及慢性乙型肝炎患者肝组织,观察HBV对Nrf2mRNA和蛋白质表达的影响,为慢性乙型肝炎病变机制提供实验基础。 方法：1.质粒转染：将HepG2细胞种植于6孔板,当细胞生长达培养皿70%时,分别用pcDNA和pHBV质粒转染HepG2细胞,转染后4小时更换培养基1次,继续培养48小时收集细胞进行后续实验。 2.实时定量聚合酶链式反应：首先分别提取已经转染质粒的HepG2细胞的总RNA,测定RNA的浓度后取2ugRNA,在逆转录酶作用下合成cDNA,然后行Realtime PCR。 3.蛋白免疫印迹法：以β-actin为内参照使用蛋白免疫印迹法检测Nrf2蛋白的表达。 4.免疫组织化学法：用免疫组织化学方法检测慢性乙型肝炎患者肝活检病理组织中Nrf2蛋白的表达,并且观察轻度、中度及重度炎症坏死肝组织中Nrf2蛋白的表达。 结果：1.用RT-qPCR法检测Nrf2mRNA的表达,以β-actin为内参照。结果表明HBV质粒转染组Nrf2mRNA的相对表达量与空载体组和未转染组表达相似,说明HBV对Nrf2mRNA的转录水平无上调作用。 2.用Western blot法检测Nrf2蛋白的表达,以P-actin为内参照。结果表明HBV质粒转染组能够表达HBx蛋白,空载体组和未转染组无表达。说明HBV转染组质粒转染成功。空载体组和未转染组中Nrf2表达量基本一致,而HBV质粒转染组Nrf2的表达量明显高于前两者。表明HBV可以上调Nrf2蛋白的表达。 3.用免疫组织化学法检测慢性乙肝患者肝活检病理组织中Nrf2蛋白的表达。结果显示慢性乙肝患者Nrf2蛋白的表达量明显高于正常组。轻度炎症坏死肝组织中Nrf2的表达量最高,中度炎症坏死肝组织中Nrf2的表达量次之,重度炎症坏死肝组织中Nrf2的表达量最低。说明HBV感染能够上调慢性乙型肝炎患者肝组织Nrf2蛋白的表达,但是持续HBV感染可以下调Nrf2蛋白的表达。 结论：1.HBV表达质粒成功转染至HepG2细胞中,并且可以上调Nrf2的蛋白表达,HBV对Nrf2mRNA的表达未见上调作用,表明HBV可能在转录后水平调节Nrf2蛋白的表达。 2.HBV感染可以上调慢性乙型肝炎患者肝组织中Nrf2蛋白的表达,但是持续HBV感染可以下调Nrf2蛋白的表达,这可能是肝组织炎症坏死程度加重的原因之一。
[Abstract]:Background and objective: hepatitis B virus (HBV) infection is a worldwide distribution. China is a high prevalence area of HBV. The prevalence rate of general population is 7.18%, and about 93 million patients with chronic hepatitis B. Chronic HBV infection can lead to chronic liver inflammation and necrosis and fibrosis, even cirrhosis and hepatocellular carcinoma, which can cause a great number of patients. The oxidative stress reaction is one of the important mechanisms in the process of hepatocyte inflammatory necrosis, fibrosis, even liver cirrhosis and liver cancer. Chronic HBV infected hepatocytes can cause excessive release of active electrons and reactive oxygen radicals in hepatocyte mitochondria, resulting in lipid peroxidation and DNA damage in liver cells. The nuclear factor Nrf2, as a regulator of the antioxidant stress gene, plays an important role in promoting the synthesis of a variety of antioxidant proteins in the antioxidant stress response. It activates a variety of antioxidant proteins such as NQO1, HO-1 and so on. It can reduce the oxidative damage to liver cells. In the case of alcoholic hepatitis and Nonalcoholic Fat The clinical studies of fatty liver and drug hepatitis have found that up regulation of.Nrf2 can reduce oxidative stress damage to liver cells. However, there are few reports on the effect of HBV on the expression of Nrf2, and the mechanism of action is not clear.
In this study, we studied the effect of HBV on the expression of Nrf2mRNA and protein by transfecting HBV plasmids into HepG2 cells and the liver tissues of patients with chronic hepatitis B and providing an experimental basis for the mechanism of chronic hepatitis B disease.
Methods: 1. plasmid transfection: HepG2 cells were planted in 6 orifice plates. When cell growth reached 70%, HepG2 cells were transfected with pcDNA and pHBV plasmids respectively. After 4 hours of transfection, the culture medium was replaced for 1 times, and the cells were continued for 48 hours to collect the cells for subsequent experiments.
2. real time quantitative polymerase chain reaction: first extract the total RNA of the transfected plasmid HepG2 cells respectively, determine the concentration of RNA after 2ugRNA, synthesize cDNA under the reverse transcriptase, and then perform Realtime PCR.
3. protein immunoblotting: using beta -actin as internal reference, Western blotting was used to detect the expression of Nrf2 protein.
4. immunohistochemistry: immunohistochemical method was used to detect the expression of Nrf2 protein in the pathological tissue of liver biopsy in patients with chronic hepatitis B and to observe the expression of Nrf2 protein in mild, moderate and severe inflammatory necrotic liver tissues.
Results: 1. the expression of Nrf2mRNA was detected by RT-qPCR, and beta -actin was used as internal reference. The results showed that the relative expression of Nrf2mRNA in the HBV plasmid transfected group was similar to that of the unloaded and untransfected groups, indicating that HBV had no up regulation on the transcriptional level of Nrf2mRNA.
2. Western blot was used to detect the expression of Nrf2 protein, and P-actin was used as internal reference. The results showed that the HBV plasmid transfected group could express HBx protein, no expression in the unloaded and untransfected group. It indicated that the plasmid transfection of HBV transfected group was successful. The expression of Nrf2 in the unloaded and untransfected group was basically the same, but the expression of Nrf2 was significantly higher in the HBV plasmid transfection group. The former two indicated that HBV could upregulate the expression of Nrf2 protein.
3. the expression of Nrf2 protein in the pathological tissue of liver biopsy in patients with chronic hepatitis B was detected by immunohistochemistry. The results showed that the expression of Nrf2 protein in chronic hepatitis B patients was significantly higher than that in the normal group. The expression of Nrf2 in the mild inflammatory necrotic liver tissues was the highest, the expression of Nrf2 in the moderate inflammatory necrotic liver tissue was the second, and the severe inflammatory necrosis of the liver group The expression of Nrf2 is the lowest in the fabric. It shows that HBV infection can increase the expression of Nrf2 protein in liver tissue of patients with chronic hepatitis B, but continuous HBV infection can reduce the expression of Nrf2 protein.
Conclusion: the expression plasmid of 1.HBV can be transfected into HepG2 cells successfully, and the expression of Nrf2 protein can be up-regulated. The expression of Nrf2mRNA is not up regulated by HBV, which indicates that HBV may regulate the expression of Nrf2 protein at the post transcriptional level.
2.HBV infection can increase the expression of Nrf2 protein in the liver tissues of patients with chronic hepatitis B, but continuous HBV infection can reduce the expression of Nrf2 protein, which may be one of the reasons for the aggravation of inflammation and necrosis of liver tissue.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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