本文選題：戊型肝炎 + 戊型肝炎病毒��； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：[研究背景] 戊型肝炎(Hepatitis E, HE)是由戊型肝炎病毒(Hepatitis E virus, HEV)感染引起的病毒性肝炎,20世紀(jì)80年代首次確認(rèn)此種炳毒為傳染病的病原。隨著人們對(duì)HE研究的深入以及科學(xué)技術(shù)手段的提高,人們對(duì)HE的認(rèn)識(shí)更加深入：HE不僅流行于衛(wèi)生條件較差的地區(qū),而且發(fā)達(dá)地區(qū)被診斷為HE的病人也日益增多。近年來(lái),HE已成為威脅人類(lèi)健康的全球性問(wèn)題：患HE的孕婦病死率可高達(dá)30%,慢性肝病基礎(chǔ)的患者易感染HEV從而誘發(fā)重型肝炎,有關(guān)慢性HE的報(bào)道也逐漸增多。防控HE疾病已成為科學(xué)家日益關(guān)注熱點(diǎn)。 HEV為單股正鏈RNA病毒,分為4個(gè)基因型,其中基因1型、2型及基因3型基因組的結(jié)構(gòu)相似,與基因4型基因組相比,結(jié)構(gòu)差異比較大。我國(guó)主要流行基因1型和基因4型。HEV全長(zhǎng)約7.2kb,有3個(gè)開(kāi)放讀碼框(Open Reading Frame, ORF),其中ORF1編碼非結(jié)構(gòu)蛋白,ORF2編碼戊型肝炎病毒的衣殼蛋白,ORF3則編碼個(gè)小分子蛋白,且基因1型與基因4型病毒編碼的ORF3蛋白的結(jié)構(gòu)存在差異：基因4型ORF3蛋白N端較1型ORF3蛋白缺少9個(gè)氨基酸。由于缺乏合適的動(dòng)物模型和細(xì)胞模型,戊型肝炎的發(fā)病致病機(jī)理并未闡明,且HEV編碼的小分子蛋白ORF3蛋白的生物學(xué)功能也不明確,不同基因型的ORF3蛋白功能是否存在差異,尚無(wú)相關(guān)報(bào)道。也有文獻(xiàn)報(bào)道不同基因型致病力也是存在差異的,這種差異的原因值得探討。本研究擬以Huh7為細(xì)胞模型,探討不同型ORF3蛋白的功能及其存在的差異,并進(jìn)步研究相關(guān)的機(jī)制,初步探討不同基因型的HEV病毒對(duì)免疫刺激影響的差異,為戊型肝炎的研究提供一定的理論基礎(chǔ)。 [目的] 探討基因1型及基因4型ORF3蛋白對(duì)肝癌細(xì)胞系的影響及存在的差異和可能的機(jī)制,且初步探討基因1型及基因4型HEV病毒對(duì)免疫刺激的影響差異,為進(jìn)一步深入了解戊型肝炎的發(fā)病致病機(jī)理提供新的科學(xué)依據(jù)； [方法] 1.分別構(gòu)建1型及4型HEV完整ORF3蛋白(ORF3蛋白)的真核表達(dá)質(zhì)粒,同時(shí)利用實(shí)驗(yàn)室已有的綠色熒光蛋白標(biāo)記的ORF3真核表達(dá)質(zhì)粒。質(zhì)粒轉(zhuǎn)染Huh7細(xì)胞：CCK8檢測(cè)不同型ORF3蛋白對(duì)Huh7細(xì)胞增殖的影響；PI染色流式細(xì)胞儀檢測(cè)ORF3對(duì)細(xì)胞周期的影響；Annexin V/PI流式細(xì)胞術(shù)檢測(cè)不同型ORF3蛋白對(duì)細(xì)胞凋亡的影響。 2.用基因1型及基因4型ORF3質(zhì)粒分別(?)染Huh7細(xì)胞,Real-time PCR檢測(cè)細(xì)胞周期相關(guān)基因cyclinD1及cyclin E的表達(dá),western blot檢測(cè)cyclinD1、cyclinE、cyclin A、cyclin B等蛋白的表達(dá)；Real-time PCR檢測(cè)細(xì)胞凋亡相關(guān)Bax、Bcl-xl基因的表達(dá),Western blot檢測(cè)相關(guān)Bax、Bcl-x1、Caspase-9等蛋白的表達(dá)；免疫熒光觀(guān)察不同型ORF3蛋白對(duì)p65核轉(zhuǎn)移的影響,并用western blot證實(shí)。 3.采集健康人的血(檢測(cè)肝功能正常,戊肝抗體、乙肝抗體及丙肝抗體陰性),Ficoll法分離外周單個(gè)核細(xì)胞(peripheral blood mononuclear cell, PBMC),用不同型的戊型肝炎病毒刺激外周單個(gè)核細(xì)胞,倒置顯微鏡觀(guān)察PBMC的生長(zhǎng)情況,ELISA檢測(cè)INF-γ及IL-4細(xì)胞因子分泌情況。 [結(jié)果] 1.成功構(gòu)建了基因1型及基因4型ORF3的真核表達(dá)載體；基因1型ORF3可以抑制肝癌細(xì)胞系Huh7增殖活性(p0.05),基因4型無(wú)統(tǒng)計(jì)學(xué)差異；基因1型ORF3蛋白可阻滯細(xì)胞周期于G0/G1期,而基因4型ORF3蛋白則對(duì)細(xì)胞周期無(wú)明顯抑制作用；基因1型和基因4型ORF3蛋白均可抑制星形孢菌素所誘導(dǎo)的細(xì)胞凋亡。 2. Real-time PCR示ORF3蛋白的表達(dá)可以抑制cyclinD1及cyclin E的表達(dá),基因1型及基因4型ORF3蛋白均可。western blot示表達(dá)基因1型ORF3的細(xì)胞其蛋白cyclinD1、cyclinE及相關(guān)的細(xì)胞周期依賴(lài)性蛋白激酶CDK4、CDK6、CDK2蛋白表達(dá)量與對(duì)照組相比均有下降,但是基因4型則無(wú)明顯差異。表達(dá)基因1型及基因4型ORF3蛋白的細(xì)胞其S期,和G2期相關(guān)的細(xì)胞周期蛋白cyclinA及cyclin B的蛋白表達(dá)量與對(duì)照組比較表達(dá)量無(wú)差異； 3.Real-time PCR示基因1型及基因4型ORF3蛋白均可抑制Bax及Bcl-2基因的表達(dá)。凋亡相關(guān)蛋白的檢測(cè)示：對(duì)照組及轉(zhuǎn)染空載質(zhì)粒組加入促凋亡劑后Bax、caspase9、細(xì)胞色素c的表達(dá)與轉(zhuǎn)染ORF3質(zhì)粒組相比,明顯增高。 4.激光共聚焦顯示,表達(dá)ORF3蛋白的細(xì)胞,加入TNF-α刺激后,p65并未出現(xiàn)核轉(zhuǎn)移,基因1型和基因4型無(wú)差異；western blot顯示：表達(dá)ORF3蛋白的細(xì)胞在受到TNF-α刺激后胞核p65的表達(dá)量較對(duì)照組相比下降,且基因1和4型間無(wú)顯著差異。 5.PBMC加入不同型HEV刺激后,隨著時(shí)間的推移可見(jiàn)PBMC從單個(gè)散在的狀態(tài)逐漸出現(xiàn)聚團(tuán),且聚團(tuán)的數(shù)量逐漸增多,體積變大,與基因4型相比,加入基因1型HEV的PBMC聚團(tuán)出現(xiàn)的時(shí)間更早,且聚團(tuán)的數(shù)量更多,體積更大；而沒(méi)有加入病毒的PBMC從2d-9d處于單個(gè)散在狀態(tài),未見(jiàn)明顯細(xì)胞聚集的現(xiàn)象。 6. ELISA檢測(cè)PBMC培養(yǎng)上清中細(xì)胞因子INF-γ及IL-4的分泌量,INF-γ的分泌量高于基因1型組及對(duì)照組,而各組IL-4分泌量則小于最低檢測(cè)濃度。 [結(jié)論] 1.基因1型ORF3蛋白和基因4型ORF3蛋白對(duì)Huh7細(xì)胞的影響是存在差異的,基因1型ORF3蛋白可以抑制細(xì)胞增殖活性且阻滯細(xì)胞周期于G0/G1期,而基因4型ORF3蛋白無(wú)統(tǒng)計(jì)學(xué)差異�；�1型和基因4型ORF3蛋白均可抑制新型孢菌素誘導(dǎo)的細(xì)胞凋亡。 2.基因1型ORF3可能通過(guò)抑制cyclinD1及cyclinE蛋白的表達(dá)使細(xì)胞停滯于G0/G1期；基因1型及基因4型ORF3蛋白通過(guò)線(xiàn)粒體途徑抑制星形孢菌素誘導(dǎo)的凋亡 3.基因1型及基因4型ORF3蛋白均可抑制TNF-α誘導(dǎo)的p65核轉(zhuǎn)運(yùn)。 4.與基因4型HEV相比,基因1型HEV可以誘發(fā)更早、更強(qiáng)烈的免疫反應(yīng)。
[Abstract]:[research background]
Hepatitis E (HE) is a viral hepatitis caused by hepatitis E virus (Hepatitis E virus, HEV) infection. In 1980s, it was first confirmed that this kind of Bin was the pathogen of infectious disease. With the deepening of research on HE and the improvement of scientific and technological means, people know more about HE: HE is not only popular in hygienic strips. In recent years, HE has become a global problem that threatens human health in poor areas. In recent years, HE has become a global problem that threatens human health: the mortality rate of pregnant women with HE can be as high as 30%, patients with chronic liver disease based on HEV are susceptible to severe hepatitis, and the report on slow HE is increasing. Prevention and control of HE disease has become an important problem. Scientists are increasingly focusing on hot spots.
HEV is a single strand of positive chain RNA virus, which is divided into 4 genotypes, in which the structure of genotypes 1, 2 and gene 3 is similar. Compared with the gene 4 genomes, the structural differences are larger. The main epidemic gene 1 and gene 4.HEV in our country are about 7.2kb, and there are 3 open reading frame (Open Reading Frame, ORF), in which ORF1 encodes non structural protein, O, O. RF2 encodes the capsid protein of hepatitis E virus, ORF3 encodes a small molecular protein, and there is a difference in the structure of ORF3 protein encoded by gene 1 and gene 4 virus: the N terminal of the gene 4 ORF3 protein is less than 9 amino acids in the ORF3 protein, and the pathogenesis of hepatitis E is not due to the lack of appropriate animal model and cell model. The biological function of the small molecular protein ORF3 protein encoded by HEV is not clear. There is no related report on the difference of the function of ORF3 protein in different genotypes. It is also reported that the pathogenicity of different genotypes is also different. The reason for this difference is worth exploring. This study is to use Huh7 as the cell model to explore the different type of O. The function of RF3 protein and the difference in its existence, and progress in the research related mechanisms, preliminary study the difference of the influence of different genotypes of HEV virus on immune stimulation, and provide a theoretical basis for the study of hepatitis E.
[Objective]
To explore the effect of gene 1 and type 4 ORF3 protein on the hepatocellular carcinoma cell line, the difference and possible mechanism, and to explore the difference of the effect of gene 1 and type 4 type HEV virus on the immune stimulation, and provide a new scientific basis for further understanding the pathogenesis of hepatitis E.
[method]
1. the eukaryotic expression plasmids of type 1 and type 4 HEV complete ORF3 protein (ORF3 protein) were constructed respectively, and ORF3 eukaryotic expression plasmids labeled with green fluorescent protein in the laboratory were used. The plasmid transfected to Huh7 cells: CCK8 to detect the effect of different ORF3 proteins on the proliferation of Huh7 cells; PI stained flow cytometry was used to detect the effect of ORF3 on the cell cycle. Annexin V/PI flow cytometry was used to detect the effects of different ORF3 proteins on cell apoptosis.
2. gene 1 and gene 4 ORF3 plasmids were used to dye Huh7 cells, and Real-time PCR was used to detect the expression of cell cycle related genes cyclinD1 and cyclin E. Western blot was used to detect the expression of cyclinD1, cyclinE, cyclin, and other proteins. Expression of l-x1, Caspase-9 and other proteins. Immunofluorescence was used to observe the effect of different ORF3 proteins on p65 nuclear transfer, and confirmed by Western blot.
3. the blood of healthy people (normal liver function, hepatitis E antibody, hepatitis B antibody and hepatitis C antibody negative), peripheral blood mononuclear cell (PBMC) was isolated by Ficoll method, the peripheral mononuclear cells were stimulated with different type of hepatitis E virus, and the growth of PBMC was observed by inverted microscope. ELISA was used to detect INF- gamma and IL-4. The secretion of cytokine.
[results]
1. the eukaryotic expression vector of gene 1 and gene 4 ORF3 was successfully constructed; gene 1 type 1 could inhibit Huh7 proliferation activity (P0.05) of hepatoma cell line, and there was no statistical difference in gene 4; gene 1 ORF3 protein could block cell cycle in G0/G1 period, but gene 4 ORF3 protein had no obvious inhibitory effect on cell cycle; gene 1 and base Because type 4 ORF3 protein can inhibit the apoptosis induced by stellosin.
2. Real-time PCR showed that the expression of ORF3 protein could inhibit the expression of cyclinD1 and cyclin E. Gene 1 and gene 4 ORF3 protein could be the cell protein cyclinD1 of.Western blot expression gene 1 ORF3, and the expression of cyclinE and related cell cycle dependent protein kinase decreased, but the expression of protein was decreased compared with the control group. There was no significant difference in gene 4 type 4. The expression of cell cycle protein cyclinA and cyclin B related to the S phase of gene 1 and gene 4 type ORF3 protein, and the expression amount of the cell cycle protein and cyclin B, were not different from those in the control group.
3.Real-time PCR gene 1 and gene 4 ORF3 protein could inhibit the expression of Bax and Bcl-2 genes. The detection of apoptosis related proteins showed that the expression of Bax, caspase9, and cytochrome C in the control group and the transfected empty plasmid group were significantly higher than that of the transfected ORF3 plasmid group.
4. confocal laser confocal microscopy showed that the cells expressing ORF3 protein were stimulated by TNF- alpha, p65 had no nuclear transfer, and there was no difference between gene 1 and gene 4. Western blot showed that the expression of ORF3 protein expressed by TNF- alpha was lower than that of the control group, and there was no significant difference between the 1 and 4 genes.
After 5.PBMC was added to the different type of HEV stimulation, as time goes on, it can be seen that PBMC gradually appears from a single scattered state, and the number of the cluster increases gradually and the volume becomes larger. Compared with the gene 4 type, the PBMC cluster that joins the gene 1 type HEV appears earlier, and the number of clusters is more and the volume is larger; and no PBMC from the virus is from 2D. -9d was in a discrete state and no obvious cell aggregation was observed.
6. ELISA detected the secretion of cytokine INF- gamma and IL-4 in the supernatant of PBMC culture. The secretion of INF- gamma was higher than that of the gene 1 group and the control group, and the secretion of IL-4 was less than the lowest detection concentration.
[Conclusion]
The effect of 1. gene 1 ORF3 protein and gene type 4 ORF3 protein on Huh7 cells is different. Gene 1 type ORF3 protein can inhibit cell proliferation activity and block cell cycle in G0/G1 stage, but there is no statistical difference between gene 4 type ORF3 protein. Gene 1 and gene 4 ORF3 protein can inhibit the apoptosis induced by new type of sporosporin.
2. gene 1 ORF3 may stagnate the G0/G1 phase by inhibiting the expression of cyclinD1 and cyclinE protein; gene 1 and gene 4 ORF3 protein inhibit astrocystin induced apoptosis through mitochondrial pathway
3. genotype 1 and genotype 4 ORF3 protein can inhibit TNF- - induced p65 nuclear transport.
4. compared with gene 4 type HEV, gene 1 HEV can induce an earlier and stronger immune response.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R373

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