Ⅰ型干扰素和HBV调控APOBEC3G的分子机制研究

发布时间：2018-02-05 02:24

本文关键词： APOBEC3G 乙型肝炎病毒干扰素-α STAT3 乙肝表面抗原　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:乙型肝炎病毒(HBV)属于嗜肝DNA病毒科,感染HBV不仅会引起急性肝炎,还可能导致慢性肝炎、肝硬化,甚至肝癌。HBV可通过多种途径感染人体,引起肝病的传播,严重的威胁到了人类的生命健康。干扰素(Interferon)是治疗慢性乙型肝炎的一线药物之一。干扰素可以通过激活JAK-STAT信号通路诱导机体细胞表达包括APOBEC3G在内的数百种干扰素刺激基因(IFN Stimulated Genes,ISGs),有研究报道,I型干扰素诱导APOBEC3G的表达并不依赖于转录因子STAT1,然而I型干扰素诱导APOBEC3G的表达机制还尚不明确。APOBEC3G(载脂蛋白B mRNA编辑酶催化多肽3G)是一种RNA/DNA编辑酶,具有胞苷脱氨酶活性,可使胞嘧啶(C)脱氨基转变为尿嘧啶(U),从而引起病毒基因组发生碱基突变,导致病毒整个复制周期终止。有研究表明,APOBEC3G能够抑制HBV在肝细胞中的复制,且在乙肝患者体内APOBEC3G的表达受到抑制,然而,HBV对APOBEC3G的调控机制还没有相关报道。因此,本课题预研究转录因子STAT1/3在I型干扰素(IFNα)诱导APOBEC3G基因表达过程中的作用及机制,同时探讨HBV对APOBEC3G的调控机制。方法:为了验证I型干扰素能够上调肝细胞内APOBEC3G基因表达,首先通过使用IFNα/β刺激HepG2细胞,进行Western Blot和q PCR实验,检测APOBEC3G基因在蛋白水平上和mRNA水平上的变化情况。鉴于I型干扰素诱导APOBEC3G的表达并不依赖于转录因子STAT1,而且I型干扰素诱导APOBEC3G的表达机制还尚不明确。为了研究I型干扰素诱导APOBEC3G基因表达的机制,利用CRISPR/Cas9技术,构建出了在HepG2细胞中将STAT3基因敲除的细胞系,然后使用IFNα刺激STAT1,STAT3基因敲除的细胞,通过Western Blot检测APOBEC3G蛋白表达水平;另一方面,在使用IFNα刺激HepG2细胞的同时,加入了STAT3抑制剂,通过Western Blot检测STAT3分子的磷酸化水平以及APOBEC3G蛋白表达水平,检测了STAT3在I型干扰素诱导APOBEC3G基因表达过程中的作用。本实验中又验证了APOBEC3G对HBV的抑制作用,通过分子克隆实验构建出了APOBEC3G真核表达载体,用脂质体转染方法将APOBEC3G表达载体和HBV表达质粒共转到HepG2细胞中,转染后,通过ELISA检测HBe Ag、HBs Ag水平,q PCR检测HBV DNA、pg RNA的水平。在研究HBV对APOBEC3G基因表达调控机制的过程中,首先,本实验采用了20例乙肝患者和20例正常人的全血样本,从全血中分离出血清和PBMC,又从PBMC中提取RNA,反转录为c DNA,通过q PCR检测了APOBEC3G的mRNA水平;然后为了进一步证明HBV中的哪种病毒因素能够抑制APOBEC3G表达,又分别使用HepG2.2.15细胞培养上清、乙肝患者血清,与IFNα共同刺激HepG2细胞,检测细胞内APOBEC3G蛋白水平和mRNA水平;最后用HBs Ag重组蛋白进一步验证,通过使用HBs Ag重组蛋白和IFNα共同刺激HepG2细胞,然后进行Western Blot实验,检测STAT3分子的磷酸化水平以及APOBEC3G蛋白水平,q PCR检测APOBEC3G的mRNA水平。结果:I型干扰素能够明显上调HepG2细胞中APOBEC3G基因的表达;在使用STAT3抑制剂处理后,细胞内APOBEC3G的诱导表达明显下降;在STAT1被敲除的293T细胞中,APOBEC3G的诱导表达在刺激8h之内是不受影响的,但是在STAT3被敲除的293T和HepG2细胞中,APOBEC3G的诱导表达明显被抑制,并且本底水平也降低。将APOBEC3G表达载体和HBV表达质粒共转到HepG2细胞后,发现HBe Ag、HBs Ag、HBV DNA、pg RNA水平都明显下降。HBV能够抑制APOBEC3G基因的表达,在乙肝患者体内APOBEC3G基因的表达低于正常人;经过HepG2.2.15细胞培养上清、乙肝患者血清刺激后,细胞内APOBEC3G基因的表达明显下降;经过HBs Ag刺激后,STAT3分子的磷酸化位点Ser727的磷酸化水平以及APOBEC3G的蛋白表达水平、mRNA水平都明显下降,但是STAT3分子的磷酸化位点Tyr705的磷酸化水平没有受到影响。结论:I型干扰素通过激活转录因子STAT3诱导APOBEC3G基因表达;HBs Ag通过抑制STAT3磷酸化位点Ser727的磷酸化,从而抑制I型干扰素对APOBEC3G的诱导作用,同时也抑制了I型干扰素的抗病毒效果。
[Abstract]:Objective: hepatitis B virus (HBV) belongs to the hepatotropic DNA virus, HBV infection not only causes acute hepatitis, may also lead to chronic hepatitis, liver cirrhosis, liver cancer and even.HBV can infect the human body through a variety of ways, cause liver disease spread, a serious threat to human life and health. Interferon (Interferon) is one of the first drug treatment of chronic hepatitis B with interferon. Through hundreds of interferon induced cellular expression including APOBEC3G activation of JAK-STAT signaling pathway stimulated gene (IFN Stimulated Genes, ISGs), studies have reported that the expression of type I interferon induced by APOBEC3G is not dependent on the transcription factor STAT1, but the expression mechanism of type I interferon induced APOBEC3G is still no clear.APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide 3G) is a kind of RNA/DNA editase, has cytidine deaminase activity, the cytosine deamination (C) Converted to uracil (U), which caused the virus genome mutation, resulting in the virus replication cycle termination. Studies have shown that APOBEC3G can inhibit HBV replication in liver cells, and the expression of APOBEC3G in vivo in patients with hepatitis B inhibition, however, the regulatory mechanism of HBV on APOBEC3G has not been reported. Therefore, this project pre study of transcription factor STAT1/3 in type I interferon (alpha IFN) effect and mechanism of the expression of APOBEC3G gene induced by HBV, and discuss the regulation mechanism of APOBEC3G. Methods: in order to verify the type I interferon can up-regulated APOBEC3G gene expression in liver cells, first through the use of IFN alpha / beta HepG2 cells stimulated by Western. Blot and Q PCR experiments, change detection of APOBEC3G gene at the protein level and mRNA level. In view of the expression of type I interferon induced APOBEC3G is not dependent on the transcription factor STAT1, and The expression of type I interferon induced APOBEC3G mechanism is still not clear. In order to study the mechanism of type I interferon induced APOBEC3G gene expression, the use of CRISPR/Cas9 technology, constructed in STAT3 HepG2 cells in gene knockout cell lines, and then use the IFN stimulated STAT1, STAT3 gene knockout cells, the expression level of APOBEC3G protein was detected by Western Blot; on the other hand, in the use of IFN stimulated HepG2 cells at the same time, adding STAT3 inhibitor, detected by STAT3 molecular Western Blot phosphorylation and APOBEC3G protein expression were detected in STAT3 induced APOBEC3G gene expression of type I interferon process. And this experiment verified the inhibitory effect of APOBEC3G on HBV, by molecular cloning to construct APOBEC3G eukaryotic expression vector by liposome transfection method to vector and HBV expression of APOBEC3G plasmid were transferred to HepG2 Cells, after transfection was detected by ELISA HBe Ag, HBs Ag HBV DNA Q PCR level detection, PG RNA level. The process control mechanism on the expression of APOBEC3G gene in HBV, first of all, this experiment used the whole blood samples of 20 patients and 20 normal persons, separated serum and PBMC from whole blood, and the extraction of RNA from PBMC, C DNA by reverse transcription, Q PCR detected the APOBEC3G level of mRNA; then in order to further prove which viral factors in HBV can inhibit the expression of APOBEC3G and HepG2.2.15 respectively using cell culture supernatant, serum of patients with chronic hepatitis B, and IFN alpha co stimulation HepG2 cell detection the intracellular APOBEC3G protein level and mRNA level; finally HBs Ag recombinant protein further verification, common stimulation of HepG2 cells by using HBs recombinant protein Ag and IFN alpha, Western and Blot experiments, the detection of STAT3 molecules and the phosphorylation level of AP The protein level of OBEC3G, Q PCR detection of APOBEC3G mRNA level. Results: type I interferon can markedly increase the high expression of APOBEC3G gene in HepG2 cells; in the use of STAT3 inhibitor, induced expression was significantly decreased in APOBEC3G cells; knock on STAT1 expression in 293T cells, 8h stimulation within is not affected induced by APOBEC3G, but was knocked out in STAT3 293T and HepG2 cells, the expression was significantly inhibited the induction of APOBEC3G, and the bottom level is lower. The APOBEC3G expression vector and HBV expression plasmid were transferred to HepG2 cells, HBe Ag, HBs Ag, HBV DNA, PG RNA levels were significantly decreased to.HBV inhibition of the expression of APOBEC3G gene, is lower than that of the normal expression of APOBEC3G gene in patients with hepatitis B in vivo; after HepG2.2.15 cell culture supernatant and serum of patients with chronic hepatitis B after stimulation of intracellular APOBEC3G gene expression decreased significantly after H; Bs after Ag stimulation, the phosphorylation level and protein expression level of APOBEC3G phosphorylation site of Ser727 molecule STAT3, mRNA levels were significantly decreased, but the phosphorylation site of Tyr705 molecule STAT3 phosphorylation was not affected. Conclusion: the type I interferon through activation of transcription factor STAT3 gene expression induced by APOBEC3G; HBs inhibited by Ag the STAT3 phosphorylation sites of Ser727 phosphorylation, thereby inhibiting the induction of type I interferon for APOBEC3G, but also inhibit type I interferon antiviral effect.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.21

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