戊型肝炎病毒ORF3蛋白的功能研究

发布时间：2018-05-17 05:23

本文选题：戊型肝炎 + 戊型肝炎病毒　；参考：《华中科技大学》2012年博士论文

【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世纪80年代首次确认此种炳毒为传染病的病原。随着人们对HE研究的深入以及科学技术手段的提高,人们对HE的认识更加深入：HE不仅流行于卫生条件较差的地区,而且发达地区被诊断为HE的病人也日益增多。近年来,HE已成为威胁人类健康的全球性问题：患HE的孕妇病死率可高达30%,慢性肝病基础的患者易感染HEV从而诱发重型肝炎,有关慢性HE的报道也逐渐增多。防控HE疾病已成为科学家日益关注热点。 HEV为单股正链RNA病毒,分为4个基因型,其中基因1型、2型及基因3型基因组的结构相似,与基因4型基因组相比,结构差异比较大。我国主要流行基因1型和基因4型。HEV全长约7.2kb,有3个开放读码框(Open Reading Frame, ORF),其中ORF1编码非结构蛋白,ORF2编码戊型肝炎病毒的衣壳蛋白,ORF3则编码个小分子蛋白,且基因1型与基因4型病毒编码的ORF3蛋白的结构存在差异：基因4型ORF3蛋白N端较1型ORF3蛋白缺少9个氨基酸。由于缺乏合适的动物模型和细胞模型,戊型肝炎的发病致病机理并未阐明,且HEV编码的小分子蛋白ORF3蛋白的生物学功能也不明确,不同基因型的ORF3蛋白功能是否存在差异,尚无相关报道。也有文献报道不同基因型致病力也是存在差异的,这种差异的原因值得探讨。本研究拟以Huh7为细胞模型,探讨不同型ORF3蛋白的功能及其存在的差异,并进步研究相关的机制,初步探讨不同基因型的HEV病毒对免疫刺激影响的差异,为戊型肝炎的研究提供一定的理论基础。 [目的] 探讨基因1型及基因4型ORF3蛋白对肝癌细胞系的影响及存在的差异和可能的机制,且初步探讨基因1型及基因4型HEV病毒对免疫刺激的影响差异,为进一步深入了解戊型肝炎的发病致病机理提供新的科学依据； [方法] 1.分别构建1型及4型HEV完整ORF3蛋白(ORF3蛋白)的真核表达质粒,同时利用实验室已有的绿色荧光蛋白标记的ORF3真核表达质粒。质粒转染Huh7细胞：CCK8检测不同型ORF3蛋白对Huh7细胞增殖的影响；PI染色流式细胞仪检测ORF3对细胞周期的影响；Annexin V/PI流式细胞术检测不同型ORF3蛋白对细胞凋亡的影响。 2.用基因1型及基因4型ORF3质粒分别(?)染Huh7细胞,Real-time PCR检测细胞周期相关基因cyclinD1及cyclin E的表达,western blot检测cyclinD1、cyclinE、cyclin A、cyclin B等蛋白的表达；Real-time PCR检测细胞凋亡相关Bax、Bcl-xl基因的表达,Western blot检测相关Bax、Bcl-x1、Caspase-9等蛋白的表达；免疫荧光观察不同型ORF3蛋白对p65核转移的影响,并用western blot证实。 3.采集健康人的血(检测肝功能正常,戊肝抗体、乙肝抗体及丙肝抗体阴性),Ficoll法分离外周单个核细胞(peripheral blood mononuclear cell, PBMC),用不同型的戊型肝炎病毒刺激外周单个核细胞,倒置显微镜观察PBMC的生长情况,ELISA检测INF-γ及IL-4细胞因子分泌情况。 [结果] 1.成功构建了基因1型及基因4型ORF3的真核表达载体；基因1型ORF3可以抑制肝癌细胞系Huh7增殖活性(p0.05),基因4型无统计学差异；基因1型ORF3蛋白可阻滞细胞周期于G0/G1期,而基因4型ORF3蛋白则对细胞周期无明显抑制作用；基因1型和基因4型ORF3蛋白均可抑制星形孢菌素所诱导的细胞凋亡。 2. Real-time PCR示ORF3蛋白的表达可以抑制cyclinD1及cyclin E的表达,基因1型及基因4型ORF3蛋白均可。western blot示表达基因1型ORF3的细胞其蛋白cyclinD1、cyclinE及相关的细胞周期依赖性蛋白激酶CDK4、CDK6、CDK2蛋白表达量与对照组相比均有下降,但是基因4型则无明显差异。表达基因1型及基因4型ORF3蛋白的细胞其S期,和G2期相关的细胞周期蛋白cyclinA及cyclin B的蛋白表达量与对照组比较表达量无差异； 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基因的表达。凋亡相关蛋白的检测示：对照组及转染空载质粒组加入促凋亡剂后Bax、caspase9、细胞色素c的表达与转染ORF3质粒组相比,明显增高。 4.激光共聚焦显示,表达ORF3蛋白的细胞,加入TNF-α刺激后,p65并未出现核转移,基因1型和基因4型无差异；western blot显示：表达ORF3蛋白的细胞在受到TNF-α刺激后胞核p65的表达量较对照组相比下降,且基因1和4型间无显著差异。 5.PBMC加入不同型HEV刺激后,随着时间的推移可见PBMC从单个散在的状态逐渐出现聚团,且聚团的数量逐渐增多,体积变大,与基因4型相比,加入基因1型HEV的PBMC聚团出现的时间更早,且聚团的数量更多,体积更大；而没有加入病毒的PBMC从2d-9d处于单个散在状态,未见明显细胞聚集的现象。 6. ELISA检测PBMC培养上清中细胞因子INF-γ及IL-4的分泌量,INF-γ的分泌量高于基因1型组及对照组,而各组IL-4分泌量则小于最低检测浓度。 [结论] 1.基因1型ORF3蛋白和基因4型ORF3蛋白对Huh7细胞的影响是存在差异的,基因1型ORF3蛋白可以抑制细胞增殖活性且阻滞细胞周期于G0/G1期,而基因4型ORF3蛋白无统计学差异。基因1型和基因4型ORF3蛋白均可抑制新型孢菌素诱导的细胞凋亡。 2.基因1型ORF3可能通过抑制cyclinD1及cyclinE蛋白的表达使细胞停滞于G0/G1期；基因1型及基因4型ORF3蛋白通过线粒体途径抑制星形孢菌素诱导的凋亡 3.基因1型及基因4型ORF3蛋白均可抑制TNF-α诱导的p65核转运。 4.与基因4型HEV相比,基因1型HEV可以诱发更早、更强烈的免疫反应。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

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