本文選題：乙型肝炎病毒 + Nrf2�。� 參考：《中南大學》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9300萬慢性乙型肝炎患者。慢性HBV感染可以導致肝臟慢性炎癥壞死和纖維化,甚至可發(fā)展為肝硬化及肝細胞癌,對患者造成極大危害。氧化應激反應是肝細胞炎癥壞死、纖維化甚至肝硬化及肝癌發(fā)生發(fā)展過程中的重要機制之一。慢性HBV感染的肝細胞持續(xù)表達病毒蛋白可以引起肝細胞線粒體釋放過量的活性電子和活性氧自由基,導致肝細胞脂質過氧化和DNA損害,從而造成肝細胞的損傷。核轉錄因子Nrf2作為抗氧化應激基因的調(diào)控者,在抗氧化應激反應中促進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,能夠減輕肝細胞所受的氧化損害。在對酒精性肝炎、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)可以減輕肝細胞所受的氧化應激損害。但HBV對Nrf2表達影響的研究報道很少,并且作用機制也不清楚。 在本研究中,我們通過研究HBV質粒轉染HepG2細胞及慢性乙型肝炎患者肝組織,觀察HBV對Nrf2mRNA和蛋白質表達的影響,為慢性乙型肝炎病變機制提供實驗基礎。 方法：1.質粒轉染：將HepG2細胞種植于6孔板,當細胞生長達培養(yǎng)皿70%時,分別用pcDNA和pHBV質粒轉染HepG2細胞,轉染后4小時更換培養(yǎng)基1次,繼續(xù)培養(yǎng)48小時收集細胞進行后續(xù)實驗。 2.實時定量聚合酶鏈式反應：首先分別提取已經(jīng)轉染質粒的HepG2細胞的總RNA,測定RNA的濃度后取2ugRNA,在逆轉錄酶作用下合成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Nrf2蛋白的表達。 4.免疫組織化學法：用免疫組織化學方法檢測慢性乙型肝炎患者肝活檢病理組織中Nrf2蛋白的表達,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。 結果：1.用RT-qPCR法檢測Nrf2mRNA的表達,以β-actin為內(nèi)參照。結果表明HBV質粒轉染組Nrf2mRNA的相對表達量與空載體組和未轉染組表達相似,說明HBV對Nrf2mRNA的轉錄水平無上調(diào)作用。 2.用Western blot法檢測Nrf2蛋白的表達,以P-actin為內(nèi)參照。結果表明HBV質粒轉染組能夠表達HBx蛋白,空載體組和未轉染組無表達。說明HBV轉染組質粒轉染成功。空載體組和未轉染組中Nrf2表達量基本一致,而HBV質粒轉染組Nrf2的表達量明顯高于前兩者。表明HBV可以上調(diào)Nrf2蛋白的表達。 3.用免疫組織化學法檢測慢性乙肝患者肝活檢病理組織中Nrf2蛋白的表達。結果顯示慢性乙肝患者Nrf2蛋白的表達量明顯高于正常組。輕度炎癥壞死肝組織中Nrf2的表達量最高,中度炎癥壞死肝組織中Nrf2的表達量次之,重度炎癥壞死肝組織中Nrf2的表達量最低。說明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,但是持續(xù)HBV感染可以下調(diào)Nrf2蛋白的表達。 結論：1.HBV表達質粒成功轉染至HepG2細胞中,并且可以上調(diào)Nrf2的蛋白表達,HBV對Nrf2mRNA的表達未見上調(diào)作用,表明HBV可能在轉錄后水平調(diào)節(jié)Nrf2蛋白的表達。 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,但是持續(xù)HBV感染可以下調(diào)Nrf2蛋白的表達,這可能是肝組織炎癥壞死程度加重的原因之一。
[Abstract]:BACKGROUND & OBJECTIVE : Hepatitis B virus ( HBV ) infection is a worldwide distribution . Our country is a highly epidemic area of HBV . The prevalence rate of chronic HBV infection is 7.18 % . There are about 93 million patients with chronic hepatitis B . Chronic HBV infection can lead to chronic inflammatory necrosis and fibrosis of liver cells . It can reduce the oxidative damage of liver cells .

In this study , we investigated the effects of HBV on Nrf2mRNA and protein expression by studying HBV plasmid transfection of HepG2 cells and chronic hepatitis B patients , and provided an experimental basis for the mechanism of chronic hepatitis B disease .

Methods : 1 . The HepG2 cells were transfected into 6 - well plates . When the cells grew up to 70 % , HepG2 cells were transfected with pcDNA and pHBV plasmid respectively . After transfection , the culture medium was replaced for 1 time , and the cells were cultured for 48 hours to carry out subsequent experiments .

2 . Real - time quantitative polymerase chain reaction : Firstly , the total RNA of HepG2 cells transfected with plasmids was extracted , the RNA concentration was measured , 2 ugRNA was obtained , cDNA was synthesized under reverse transcriptase , and then PCR was performed .

3 . Western blotting : Using 尾 - actin as internal reference , the expression of Nrf2 protein was detected by Western blotting .

4 . Immunohistochemical method : The expression of Nrf2 protein in liver biopsy specimens of patients with chronic hepatitis B was detected by immunohistochemical method , and the expression of Nrf2 protein in liver tissues with mild , moderate and severe inflammation was observed .

Results : 1 . The expression of Nrf2mRNA was detected by RT - qPCR . The results showed that the relative expression of Nrf2mRNA was similar to that of empty vector group and untransfected group , indicating that HBV could not regulate the transcription level of Nrf2mRNA .

2 . The expression of Nrf2 protein was detected by Western blot . The results showed that the expression of Nrf2 was similar in both empty vector group and untransfected group .

3 . The expression of Nrf2 protein in liver biopsy specimens of patients with chronic hepatitis B was detected by immunohistochemical method . The results showed that the expression of Nrf2 protein in patients with chronic hepatitis B was higher than that in the normal group .

Conclusion : 1 . HBV expression plasmid is successfully transfected into HepG2 cells , and the expression of Nrf2 can be regulated up . The expression of HBV to Nrf2mRNA is not up - regulated , suggesting that HBV may regulate the expression of Nrf2 at post - transcriptional level .

2 . HBV infection can up - regulate the expression of Nrf2 protein in liver tissues of patients with chronic hepatitis B , but the persistent HBV infection can downregulate the expression of Nrf2 protein , which may be one of the reasons for the degree of inflammation and necrosis of liver tissue .
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.62

【參考文獻】

相關期刊論文 前2條

1 Lisa Rossi;Richard W Lambrecht;Herbert L Bonkovsky;;Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells[J];World Journal of Gastroenterology;2009年36期

2 Daniela Gordillo-Bastidas;Edén Oceguera-Contreras;Adriana Salazar-Montes;Jaime González-Cuevas;Luis Daniel Hernández-Ortega;Juan Armendáriz-Borunda;;Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine[J];World Journal of Gastroenterology;2013年47期

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本文編號：2007431