本文選題：乙型肝炎病毒 + Nrf2��； 參考：《中南大學(xué)》2014年碩士論文

【摘要】：背景和目的：乙型肝炎病毒(HBV)感染呈世界性分布,我國(guó)是HBV的高流行地區(qū),一般人群中流行率為7.18%,大概有9300萬(wàn)慢性乙型肝炎患者。慢性HBV感染可以導(dǎo)致肝臟慢性炎癥壞死和纖維化,甚至可發(fā)展為肝硬化及肝細(xì)胞癌,對(duì)患者造成極大危害。氧化應(yīng)激反應(yīng)是肝細(xì)胞炎癥壞死、纖維化甚至肝硬化及肝癌發(fā)生發(fā)展過(guò)程中的重要機(jī)制之一。慢性HBV感染的肝細(xì)胞持續(xù)表達(dá)病毒蛋白可以引起肝細(xì)胞線粒體釋放過(guò)量的活性電子和活性氧自由基,導(dǎo)致肝細(xì)胞脂質(zhì)過(guò)氧化和DNA損害,從而造成肝細(xì)胞的損傷。核轉(zhuǎn)錄因子Nrf2作為抗氧化應(yīng)激基因的調(diào)控者,在抗氧化應(yīng)激反應(yīng)中促進(jìn)多種抗氧化蛋白的合成而發(fā)揮重要作用,它激活多種抗氧化蛋白如NQO1, HO-1等,能夠減輕肝細(xì)胞所受的氧化損害。在對(duì)酒精性肝炎、非酒精脂肪肝、藥物性肝炎等的臨床研究發(fā)現(xiàn).Nrf2的上調(diào)可以減輕肝細(xì)胞所受的氧化應(yīng)激損害。但HBV對(duì)Nrf2表達(dá)影響的研究報(bào)道很少,并且作用機(jī)制也不清楚。 在本研究中,我們通過(guò)研究HBV質(zhì)粒轉(zhuǎn)染HepG2細(xì)胞及慢性乙型肝炎患者肝組織,觀察HBV對(duì)Nrf2mRNA和蛋白質(zhì)表達(dá)的影響,為慢性乙型肝炎病變機(jī)制提供實(shí)驗(yàn)基礎(chǔ)。 方法：1.質(zhì)粒轉(zhuǎn)染：將HepG2細(xì)胞種植于6孔板,當(dāng)細(xì)胞生長(zhǎng)達(dá)培養(yǎng)皿70%時(shí),分別用pcDNA和pHBV質(zhì)粒轉(zhuǎn)染HepG2細(xì)胞,轉(zhuǎn)染后4小時(shí)更換培養(yǎng)基1次,繼續(xù)培養(yǎng)48小時(shí)收集細(xì)胞進(jìn)行后續(xù)實(shí)驗(yàn)。 2.實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)：首先分別提取已經(jīng)轉(zhuǎn)染質(zhì)粒的HepG2細(xì)胞的總RNA,測(cè)定RNA的濃度后取2ugRNA,在逆轉(zhuǎn)錄酶作用下合成cDNA,然后行Realtime PCR。 3.蛋白免疫印跡法：以β-actin為內(nèi)參照使用蛋白免疫印跡法檢測(cè)Nrf2蛋白的表達(dá)。 4.免疫組織化學(xué)法：用免疫組織化學(xué)方法檢測(cè)慢性乙型肝炎患者肝活檢病理組織中Nrf2蛋白的表達(dá),并且觀察輕度、中度及重度炎癥壞死肝組織中Nrf2蛋白的表達(dá)。 結(jié)果：1.用RT-qPCR法檢測(cè)Nrf2mRNA的表達(dá),以β-actin為內(nèi)參照。結(jié)果表明HBV質(zhì)粒轉(zhuǎn)染組Nrf2mRNA的相對(duì)表達(dá)量與空載體組和未轉(zhuǎn)染組表達(dá)相似,說(shuō)明HBV對(duì)Nrf2mRNA的轉(zhuǎn)錄水平無(wú)上調(diào)作用。 2.用Western blot法檢測(cè)Nrf2蛋白的表達(dá),以P-actin為內(nèi)參照。結(jié)果表明HBV質(zhì)粒轉(zhuǎn)染組能夠表達(dá)HBx蛋白,空載體組和未轉(zhuǎn)染組無(wú)表達(dá)。說(shuō)明HBV轉(zhuǎn)染組質(zhì)粒轉(zhuǎn)染成功�？蛰d體組和未轉(zhuǎn)染組中Nrf2表達(dá)量基本一致,而HBV質(zhì)粒轉(zhuǎn)染組Nrf2的表達(dá)量明顯高于前兩者。表明HBV可以上調(diào)Nrf2蛋白的表達(dá)。 3.用免疫組織化學(xué)法檢測(cè)慢性乙肝患者肝活檢病理組織中Nrf2蛋白的表達(dá)。結(jié)果顯示慢性乙肝患者Nrf2蛋白的表達(dá)量明顯高于正常組。輕度炎癥壞死肝組織中Nrf2的表達(dá)量最高,中度炎癥壞死肝組織中Nrf2的表達(dá)量次之,重度炎癥壞死肝組織中Nrf2的表達(dá)量最低。說(shuō)明HBV感染能夠上調(diào)慢性乙型肝炎患者肝組織Nrf2蛋白的表達(dá),但是持續(xù)HBV感染可以下調(diào)Nrf2蛋白的表達(dá)。 結(jié)論：1.HBV表達(dá)質(zhì)粒成功轉(zhuǎn)染至HepG2細(xì)胞中,并且可以上調(diào)Nrf2的蛋白表達(dá),HBV對(duì)Nrf2mRNA的表達(dá)未見上調(diào)作用,表明HBV可能在轉(zhuǎn)錄后水平調(diào)節(jié)Nrf2蛋白的表達(dá)。 2.HBV感染可以上調(diào)慢性乙型肝炎患者肝組織中Nrf2蛋白的表達(dá),但是持續(xù)HBV感染可以下調(diào)Nrf2蛋白的表達(dá),這可能是肝組織炎癥壞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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.62

【共引文獻(xiàn)】

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