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(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)胞線粒體釋放過量的活性電子和活性氧自由基,導(dǎo)致肝細(xì)胞脂質(zhì)過氧化和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,說明HBV對(duì)Nrf2mRNA的轉(zhuǎn)錄水平無上調(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)染組無表達(dá)。說明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 & 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(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

【參考文獻(xiàn)】

相關(guān)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(hào)：2007431