USP18在IFN-α抗乙肝病毒机制中的作用及调控研究

发布时间：2018-01-25 02:24

本文关键词： 乙型肝炎病毒(HBV) α干扰素(IFN-α) 泛素特异性蛋白酶18(USP18) 干扰素刺激基因(ISGs) RNA干扰　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(hepatitis B virus,HBV)感染是一个世界性的问题。尽管有效的乙肝疫苗已在临床上广泛运用。慢性HBV感染能够导致肝硬化和肝细胞肝癌等危害严重的终末期肝病。虽然近年来有多种抗HBV药物不断涌现,但α干扰素(IFN-α)是目前治疗慢性HBV感染的首选药物之一,其具有疗程固定、停药后复发率低、部分患者可出现HBs Ag阴转而获得“彻底治愈”等优点。长效聚乙二醇干扰素还具有用药间隔时间长、副作用相对较少等优点。但目前仍面临着价格昂贵和相当一部分患者的持续病毒学应答率低等问题。IFN-α应答率低的具体分子机制仍不十分明确,有待进一步研究。在前期的工作当中我们发现慢乙肝患者IFN-α治疗前应答组和无应答组肝细胞中存在大量差异表达的基因。其中干扰素刺激基因USP18和ISG15位于同一信号通路,USP18在治疗前在无应答组中高表达,提示其可能与IFN-α抗HBV的活性相关,HBV可能调控宿主细胞中USP18的表达,进而改变IFN-α的抗病毒活性。目的:1.HBV感染肝癌细胞对USP18表达的影响。2.构建sh RNA-USP18慢病毒载体,研究其对Hepg2.2.15细胞内HBV复制能力的影响。3.研究sh RNA-USP18慢病毒载体在不同浓度IFN-α作用下对HBV复制能力的影响。4.研究sh RNA-USP18慢病毒载体是否是通过调控JAK/STAT信号通路调节IFN-α的抗HBV作用。5.利用生物信息学初步分析Hepg2.2.15-sh RNA-USP18细胞中的差异表达基因及相关信号通路。方法:1.利用实时荧光定量PCR分析不同肝癌细胞系中USP18 m RNA的表达水平。转染HBV表达质粒,利用实时荧光定量PCR、Western blot及双荧光素酶实验检测HBV感染对USP18 m RNA、蛋白及启动子活性的影响。2.构建sh RNA-USP18慢病毒载体,利用实时荧光定量PCR筛选出干扰效率最佳的慢病毒载体,并筛选出稳定细胞株。利用实时荧光定量PCR、Western blot验证Hepg2.2.15-sh RNA-USP18细胞中对USP18的干扰效率。3.利用定量PCR检测干扰USP18表达对Hepg2.2.15细胞中HBV DNA、pg RNA和total RNA复制的影响。利用ELISA检测干扰USP18对Hepg2.2.15细胞中HBs Ag和HBe Ag分泌的影响。利用荧光定量PCR检测过表达USP18对Hepg2.2.15细胞中HBV DNA、pg RNA和total RNA复制的影响。利用ELISA检测过表达USP18对Hepg2.2.15细胞中HBs Ag和HBe Ag分泌的影响。4.利用荧光定量PCR检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBV复制的影响。利用ELISA检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBs Ag和HBe Ag分泌的影响。利用Western blot检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBc Ag生成的影响。5.利用荧光定量PCR检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中JAK/STAT下游的干扰素刺激基因ISG15、Mx A和IFIT1 m RNA的影响。利用Western blot检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中JAK/STAT下游的干扰素刺激基因ISG15、Mx A和IFIT1蛋白的影响。利用Western blot检测干扰USP18表达后IFN-α对Hepg2.2.15细胞中JAK/STAT信号通路中磷酸化STAT1及STAT1总蛋白表达的影响。利用Western blot检测干扰USP18表达后IFN-α处理不同时间点及不同加药方式对Hepg2.2.15细胞中JAK/STAT信号通路中磷酸化STAT1及STAT1总蛋白表达的影响。6.利用基因芯片检测干扰USP18表达后Hepg2.2.15细胞中差异表达的基因,并利用公共数据库及相关软件对差异表达的基因进行生物信息学分析。结果:1.Hepg2.2.15细胞中USP18的表达水平较其他肝癌细胞中的表达高,HBV明显促进宿主细胞中USP18的表达,选择Hep G2和Hepg2.2.15细胞用于后续实验。HBV感染能够促进Hep G2细胞中USP18 m RNA和蛋白的表达,并能增强其启动子活性。2.针对USP18筛选出干扰效率最佳的慢病毒载体并构建稳定细胞株Hepg2.2.15-sh RNA-USP18。Hepg2.2.15-sh RNA-USP18细胞中USP18m RNA和蛋白的表达受到明显抑制。3.干扰USP18表达后Hepg2.2.15细胞中HBV DNA、pg RNA和total RNA的复制受到明显抑制。Hepg2.2.15细胞中HBs Ag和HBe Ag的分泌受到明显抑制。过表达USP18后Hepg2.2.15细胞中HBV DNA、pg RNA和total RNA的复制明显增强。Hepg2.2.15细胞中HBs Ag和HBe Ag的分泌明显增强。4.干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBV DNA、pg RNA和total RNA复制的抑制作用明显增强。干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBs Ag和HBe Ag分泌的抑制作用明显增强。干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBc Ag生成的抑制作用明显增强,且在IFN-α不同浓度组中均有明显的抑制作用。5.干扰USP18表达后IFN-α对Hepg2.2.15细胞中JAK/STAT下游的干扰素刺激基因ISG15、Mx A和IFIT1 m RNA和蛋白的诱导作用明显增强。干扰USP18表达后IFN-α对Hepg2.2.15细胞中JAK/STAT信号通路中磷酸化STAT1表达的诱导作用明显增强。干扰USP18表达后IFN-α处理不同时间点,Hepg2.2.15细胞中JAK/STAT信号通路中磷酸化STAT1表达明显增强。不同加药方式下干扰USP18表达后Hepg2.2.15细胞中JAK/STAT信号通路中磷酸化STAT1表达明显增强。6.初步筛选出干扰USP18后Hepg2.2.15细胞中差异表达的基因及相关的生物学功能和信号通路。结论:1.肝癌细胞Hep G2中HBV感染可促进USP18的表达并增强USP18启动子的活性。2.干扰USP18表达后Hepg2.2.15细胞中HBV的复制和分泌受到明显抑制。3.干扰USP18表达后IFN-α对Hepg2.2.15细胞中HBV的抑制作用增强。4.USP18能够通过调节JAK/STAT信号通路的活性来调节IFN-α的抗病毒活性。
[Abstract]:Hepatitis B virus (hepatitis B, virus, HBV) infection is a worldwide problem. Although effective hepatitis B vaccine has been widely used in clinical. Chronic HBV infection can lead to cirrhosis and hepatocellular carcinoma and other serious end-stage liver disease. In recent years there are many kinds of anti HBV drugs are emerging, but alpha interferon (IFN- alpha) is currently the primary drug for the treatment of chronic HBV infection and its treatment with fixed, low recurrence rate after drug withdrawal, some patients may appear HBs Ag to obtain advantages of "thoroughly cure Yin". Long-acting pegylated interferon medication has a long time interval, relatively few side effects and other advantages. But it is still facing the price is expensive and a considerable part of the molecular mechanism of sustained virologic response in patients with low rate of.IFN- a low response rate is still not very clear, needs further research. In previous work, when I They found that before the treatment response group and non response group of liver cells in the presence of a large number of differentially expressed genes of hepatitis B patients. The slow IFN- alpha interferon stimulated gene USP18 and ISG15 in the same signaling pathway, the expression of USP18 in non response group before treatment, suggesting that IFN- and alpha anti HBV activity, HBV the regulation of USP18 expression in the host cell, and then change the antiviral activity of IFN- alpha. Objective: 1.HBV infected liver cells on the expression of USP18.2. sh RNA-USP18 influence construct lentiviral vector, to study its effect on Hepg2.2.15 cells in HBV replication competent.3. RNA-USP18 lentiviral vector of SH in different concentrations of IFN- under the influence of alpha HBV replication the ability of the.4. research sh RNA-USP18 lentiviral vector is regulated by JAK/STAT signal pathway of IFN- alpha of the anti HBV effect of.5. by bioinformatics analysis of the Hepg2.2.15-sh RNA-USP18 Differential expression of genes and signaling pathways in cells. Methods: 1. using real-time fluorescence quantitative PCR analysis of the expression levels in different hepatoma cell lines USP18 m RNA. Transfection of HBV expression plasmid, using real-time fluorescence quantitative PCR, the USP18 m RNA Western blot infection and dual luciferase reporter assay HBV,.2. protein and promoter activity the construction of SH RNA-USP18 lentiviral vector, using real-time fluorescence quantitative PCR screened lentiviralvector efficiency, and selected stable cell lines. Using real-time fluorescence quantitative PCR,.3. interference efficiency of USP18 detected by USP18 quantitative PCR interference on the expression of Hepg2.2.15 cells in HBV DNA Western blot Hepg2.2.15-sh verification of RNA-USP18 cells, influence PG RNA and total RNA replication. The influence of using ELISA for detection of USP18 interference on the secretion of Hepg2.2.15 cells in HBs Ag and HBe Ag. Measured by fluorescent quantitative PCR. The expression of USP18 on Hepg2.2.15 cells in HBV DNA, PG RNA and total RNA replication effect. The detection of overexpression of USP18 on Hepg2.2.15 cells in HBs Ag and HBe Ag secretion of.4. by fluorescence quantitative PCR detection in USP18 knockdown IFN- alpha on the replication of HBV in Hepg2.2.15 cells affected by ELISA. By using ELISA interference detection USP18 IFN- HBs Ag alpha on Hepg2.2.15 cells and HBe Ag expression. After using Western blot to detect USP18 knockdown of IFN- alpha effect on Hepg2.2.15 cells in HBc Ag generated.5. using fluorescence quantitative PCR to detect the expression of USP18 after interferon alpha IFN- interference on the downstream of JAK/STAT in Hepg2.2.15 cells stimulated genes ISG15, A and IFIT1 Mx m RNA. Using Western blot detection USP18 gene ISG15 interference IFN- interferon alpha on the stimulation of downstream JAK/STAT expression in Hepg2.2.15 cells after A, effects of Mx and IFIT1 protein by Weste. RN blot detection in USP18 knockdown of IFN- alpha on the expression of phosphorylated JAK/STAT signal pathway of STAT1 and STAT1 protein in Hepg2.2.15 cells. Using Western blot to detect USP18 expression after IFN- interference treatment at different time points and different methods of adding to the differential expression of.6. gene expression by gene chip detection of USP18 interference in Hepg2.2.15 cells after effect the expression of phosphorylated JAK/STAT signal pathway of STAT1 and STAT1 protein in Hepg2.2.15 cells, bioinformatics analysis of differentially expressed using public databases and software related genes. Results: the expression level of USP18 in 1.Hepg2.2.15 cells compared with other cancer cells in high, HBV significantly promotes the expression of USP18 in the host cell, select Hep G2 and Hepg2.2.15 cells for subsequent experimental infection of.HBV can promote the expression of USP18 m and RNA Hep protein in G2 cells, and can increase The strong promoter activity of.2. in USP18 were screened out the best jamming efficiency of lentiviral vector and expression of RNA protein and construct a stable cell line USP18m Hepg2.2.15-sh RNA-USP18.Hepg2.2.15-sh in RNA-USP18 cells was significantly inhibited the expression of.3. USP18 interference in Hepg2.2.15 cells after HBV DNA, PG RNA and total RNA replication was significantly inhibited.Hepg2.2.15 cell secretion in HBs and Ag HBe Ag was significantly inhibited. Overexpression of USP18 in Hepg2.2.15 cells after HBV DNA, PG RNA and total RNA replication significantly enhanced secretion of.Hepg2.2.15 cells in HBs Ag and HBe Ag significantly increased the expression of.4. after USP18 interference of Hepg2.2.15 cells in HBV IFN- alpha DNA, PG RNA and total inhibited RNA replication significantly enhanced. Inhibitory effect of USP18 expression after IFN- interference of Hepg2.2.15 cells in HBs Ag alpha and HBe Ag secretion increased significantly. After the expression of USP18 interference on IFN- alpha Hepg2.2.1 Inhibition of HBc Ag generated 5 cells increased significantly, and IFN- in different concentrations of alpha group significantly inhibited.5. USP18 expression after IFN- interference of interferon alpha on the downstream of JAK/STAT in Hepg2.2.15 cells stimulated ISG15 gene induced by Mx A IFIT1 and m RNA and protein significantly enhanced. USP18 knockdown of IFN- alpha induction effect on the expression of phosphorylated JAK/STAT signal pathway in STAT1 Hepg2.2.15 cells increased obviously. After the expression of USP18 interference to IFN- treatment at different time points, phosphate JAK/STAT pathway in the expression of STAT1 was significantly enhanced in Hepg2.2.15 cells. The expression of USP18 signaling pathway in the phosphorylation of JAK/STAT increased the expression of STAT1.6. was screened differentially expressed in Hepg2.2.15 cell interference after the USP18 gene and the related biological function and signaling pathways significantly in Hepg2.2.15 cells after interference with dosing mode. Conclusion: 1. small hepatocellular carcinoma Can promote the expression of USP18 and enhance the activity of.2. USP18 interference USP18 promoter expression in Hepg2.2.15 cells after HBV replication and secretion by.4.USP18 through regulating the activity of the JAK/STAT signaling pathway to regulate the antiviral activity of IFN- alpha IFN- alpha inhibition on Hepg2.2.15 cells in HBV inhibited the expression of.3. USP18 after HBV infected cell interference Hep G2.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.62

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