本文關(guān)鍵詞： APOBEC3G 乙型肝炎病毒 干擾素-α STAT3 乙肝表面抗原　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:乙型肝炎病毒(HBV)屬于嗜肝DNA病毒科,感染HBV不僅會(huì)引起急性肝炎,還可能導(dǎo)致慢性肝炎、肝硬化,甚至肝癌。HBV可通過(guò)多種途徑感染人體,引起肝病的傳播,嚴(yán)重的威脅到了人類的生命健康。干擾素(Interferon)是治療慢性乙型肝炎的一線藥物之一。干擾素可以通過(guò)激活JAK-STAT信號(hào)通路誘導(dǎo)機(jī)體細(xì)胞表達(dá)包括APOBEC3G在內(nèi)的數(shù)百種干擾素刺激基因(IFN Stimulated Genes,ISGs),有研究報(bào)道,I型干擾素誘導(dǎo)APOBEC3G的表達(dá)并不依賴于轉(zhuǎn)錄因子STAT1,然而I型干擾素誘導(dǎo)APOBEC3G的表達(dá)機(jī)制還尚不明確。APOBEC3G(載脂蛋白B mRNA編輯酶催化多肽3G)是一種RNA/DNA編輯酶,具有胞苷脫氨酶活性,可使胞嘧啶(C)脫氨基轉(zhuǎn)變?yōu)槟蜞奏?U),從而引起病毒基因組發(fā)生堿基突變,導(dǎo)致病毒整個(gè)復(fù)制周期終止。有研究表明,APOBEC3G能夠抑制HBV在肝細(xì)胞中的復(fù)制,且在乙肝患者體內(nèi)APOBEC3G的表達(dá)受到抑制,然而,HBV對(duì)APOBEC3G的調(diào)控機(jī)制還沒(méi)有相關(guān)報(bào)道。因此,本課題預(yù)研究轉(zhuǎn)錄因子STAT1/3在I型干擾素(IFNα)誘導(dǎo)APOBEC3G基因表達(dá)過(guò)程中的作用及機(jī)制,同時(shí)探討HBV對(duì)APOBEC3G的調(diào)控機(jī)制。方法:為了驗(yàn)證I型干擾素能夠上調(diào)肝細(xì)胞內(nèi)APOBEC3G基因表達(dá),首先通過(guò)使用IFNα/β刺激HepG2細(xì)胞,進(jìn)行Western Blot和q PCR實(shí)驗(yàn),檢測(cè)APOBEC3G基因在蛋白水平上和mRNA水平上的變化情況。鑒于I型干擾素誘導(dǎo)APOBEC3G的表達(dá)并不依賴于轉(zhuǎn)錄因子STAT1,而且I型干擾素誘導(dǎo)APOBEC3G的表達(dá)機(jī)制還尚不明確。為了研究I型干擾素誘導(dǎo)APOBEC3G基因表達(dá)的機(jī)制,利用CRISPR/Cas9技術(shù),構(gòu)建出了在HepG2細(xì)胞中將STAT3基因敲除的細(xì)胞系,然后使用IFNα刺激STAT1,STAT3基因敲除的細(xì)胞,通過(guò)Western Blot檢測(cè)APOBEC3G蛋白表達(dá)水平;另一方面,在使用IFNα刺激HepG2細(xì)胞的同時(shí),加入了STAT3抑制劑,通過(guò)Western Blot檢測(cè)STAT3分子的磷酸化水平以及APOBEC3G蛋白表達(dá)水平,檢測(cè)了STAT3在I型干擾素誘導(dǎo)APOBEC3G基因表達(dá)過(guò)程中的作用。本實(shí)驗(yàn)中又驗(yàn)證了APOBEC3G對(duì)HBV的抑制作用,通過(guò)分子克隆實(shí)驗(yàn)構(gòu)建出了APOBEC3G真核表達(dá)載體,用脂質(zhì)體轉(zhuǎn)染方法將APOBEC3G表達(dá)載體和HBV表達(dá)質(zhì)粒共轉(zhuǎn)到HepG2細(xì)胞中,轉(zhuǎn)染后,通過(guò)ELISA檢測(cè)HBe Ag、HBs Ag水平,q PCR檢測(cè)HBV DNA、pg RNA的水平。在研究HBV對(duì)APOBEC3G基因表達(dá)調(diào)控機(jī)制的過(guò)程中,首先,本實(shí)驗(yàn)采用了20例乙肝患者和20例正常人的全血樣本,從全血中分離出血清和PBMC,又從PBMC中提取RNA,反轉(zhuǎn)錄為c DNA,通過(guò)q PCR檢測(cè)了APOBEC3G的mRNA水平;然后為了進(jìn)一步證明HBV中的哪種病毒因素能夠抑制APOBEC3G表達(dá),又分別使用HepG2.2.15細(xì)胞培養(yǎng)上清、乙肝患者血清,與IFNα共同刺激HepG2細(xì)胞,檢測(cè)細(xì)胞內(nèi)APOBEC3G蛋白水平和mRNA水平;最后用HBs Ag重組蛋白進(jìn)一步驗(yàn)證,通過(guò)使用HBs Ag重組蛋白和IFNα共同刺激HepG2細(xì)胞,然后進(jìn)行Western Blot實(shí)驗(yàn),檢測(cè)STAT3分子的磷酸化水平以及APOBEC3G蛋白水平,q PCR檢測(cè)APOBEC3G的mRNA水平。結(jié)果:I型干擾素能夠明顯上調(diào)HepG2細(xì)胞中APOBEC3G基因的表達(dá);在使用STAT3抑制劑處理后,細(xì)胞內(nèi)APOBEC3G的誘導(dǎo)表達(dá)明顯下降;在STAT1被敲除的293T細(xì)胞中,APOBEC3G的誘導(dǎo)表達(dá)在刺激8h之內(nèi)是不受影響的,但是在STAT3被敲除的293T和HepG2細(xì)胞中,APOBEC3G的誘導(dǎo)表達(dá)明顯被抑制,并且本底水平也降低。將APOBEC3G表達(dá)載體和HBV表達(dá)質(zhì)粒共轉(zhuǎn)到HepG2細(xì)胞后,發(fā)現(xiàn)HBe Ag、HBs Ag、HBV DNA、pg RNA水平都明顯下降。HBV能夠抑制APOBEC3G基因的表達(dá),在乙肝患者體內(nèi)APOBEC3G基因的表達(dá)低于正常人;經(jīng)過(guò)HepG2.2.15細(xì)胞培養(yǎng)上清、乙肝患者血清刺激后,細(xì)胞內(nèi)APOBEC3G基因的表達(dá)明顯下降;經(jīng)過(guò)HBs Ag刺激后,STAT3分子的磷酸化位點(diǎn)Ser727的磷酸化水平以及APOBEC3G的蛋白表達(dá)水平、mRNA水平都明顯下降,但是STAT3分子的磷酸化位點(diǎn)Tyr705的磷酸化水平?jīng)]有受到影響。結(jié)論:I型干擾素通過(guò)激活轉(zhuǎn)錄因子STAT3誘導(dǎo)APOBEC3G基因表達(dá);HBs Ag通過(guò)抑制STAT3磷酸化位點(diǎn)Ser727的磷酸化,從而抑制I型干擾素對(duì)APOBEC3G的誘導(dǎo)作用,同時(shí)也抑制了I型干擾素的抗病毒效果。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373.21

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本文編號(hào)：1491847