【摘要】：[研究目的] 通過研究自噬現(xiàn)象在乙肝病毒復(fù)制中所扮演的角色,尋找降低乙肝病毒復(fù)制的新途徑,并通過對(duì)其機(jī)制的研究,探討該現(xiàn)象對(duì)于宿主細(xì)胞生存的影響及其致癌性的可能機(jī)理。 [研究方法] 1.用HepG2.2.15細(xì)胞系和pUC1857-1HBV1.2質(zhì)粒轉(zhuǎn)染的HepG2細(xì)胞系構(gòu)建出有HBV復(fù)制的細(xì)胞系； 2.采用3-甲基腺嘌呤和無胎牛血清培養(yǎng)基作為自噬現(xiàn)象的干預(yù)手段,使用HepG2細(xì)胞系和pUC18質(zhì)粒轉(zhuǎn)染的HepG2細(xì)胞系作為對(duì)照,通過測(cè)量上清中HBV拷貝數(shù)評(píng)估HBV復(fù)制情況,并對(duì)細(xì)胞裂解液采用蛋白免疫印跡法(Western Blotting)對(duì)自噬水平進(jìn)行分析； 3.使用GFP-LC3質(zhì)粒轉(zhuǎn)染的細(xì)胞及HBcAg免疫熒光抗體,通過熒光顯微鏡對(duì)HBV復(fù)制和自噬的水平進(jìn)行評(píng)估： 4.采用WST-1試劑及酶標(biāo)儀測(cè)定,對(duì)細(xì)胞增殖狀態(tài)進(jìn)行評(píng)估。 [研究結(jié)果] 1. HepG2.2.15細(xì)胞系和pUC1857-1HBV1.2質(zhì)粒轉(zhuǎn)染的HepG2細(xì)胞均有HBV產(chǎn)生和分泌。 2.有HBV復(fù)制產(chǎn)生的細(xì)胞系——-HepG2.2.15細(xì)胞系或pUC1857-1HBV1.2質(zhì)粒轉(zhuǎn)染的HepG2細(xì)胞中,P62蛋白水平的下降,LC3-Ⅱ/LC3-Ⅰ比值升高,說明HBV復(fù)制有誘導(dǎo)自噬的作用。 3.在HepG2.2.15細(xì)胞系中,使用1mM濃度的自噬抑制劑3-MA,可以減少上清液中HBV的拷貝數(shù)。但是對(duì)于10mM濃度的3-MA,以及不含F(xiàn)BS的乏營(yíng)養(yǎng)培養(yǎng)基對(duì)上清中HBV拷貝數(shù)的影響尚缺乏具有顯著性差異的結(jié)論。 4.轉(zhuǎn)染pEGFP-C1-LC3質(zhì)粒HepG2細(xì)胞中,GFP-LC3融合蛋白彌散在胞漿中。而轉(zhuǎn)染pEGFP-C1-LC3質(zhì)粒的HepG2.2.15細(xì)胞可以看到多個(gè)明亮的綠色熒光斑點(diǎn)。并且LC3斑點(diǎn)數(shù)量與HBcAg熒光斑點(diǎn)數(shù)量有一定程度的相關(guān)性,但兩者未表現(xiàn)出共定位。 5.在無血清培養(yǎng)基的饑餓狀態(tài)下,有HBV復(fù)制的細(xì)胞系增殖更快,加入10mM的3-MA則會(huì)抑制這種增殖。提示HBV所上調(diào)的自噬有利于細(xì)胞在極端情況下的生存。 [結(jié)論] 乙肝病毒可以提高其宿主細(xì)胞中自噬的水平,但自噬對(duì)于乙肝病毒的復(fù)制、產(chǎn)生的意義以及具體的機(jī)制還有待于進(jìn)一步探索。同時(shí)乙肝病毒也許有利于宿主細(xì)胞在應(yīng)激情境下生存狀態(tài),但這種影響還需進(jìn)一步的試驗(yàn)驗(yàn)證。更深入的研究也許將為我們治療乙肝病毒感染,阻止或減緩慢性乙肝病毒感染向肝硬化、肝癌進(jìn)展提供新的思路。
[Abstract]:[objective] to study the role of autophagy in hepatitis B virus replication, to find a new way to reduce hepatitis B virus replication, and to study its mechanism. To explore the effect of this phenomenon on the survival of host cells and the possible mechanism of carcinogenicity. [research methods] 1. HepG2.2.15 cell line and HepG2 cell line transfected with pUC1857-1HBV1.2 plasmid were used to construct HBV replication cell line. 2. The culture medium of 3-methyladenine and fetal bovine serum was used as an intervention for autophagy. HepG2 cell line and HepG2 cell line transfected with pUC18 plasmid were used as control. The replication of HBV was evaluated by measuring the copy number of HBV in the supernatant. The level of autophagy was analyzed by Western blot (Western Blotting). The levels of HBV replication and autophagy were evaluated by fluorescence microscope using GFP-LC3 plasmid transfected cells and HBcAg immunofluorescence antibodies. Cell proliferation was evaluated by WST-1 reagent and enzyme labeling instrument. [research results] 1. Both HepG2.2.15 cell lines and HepG2 cells transfected with pUC1857-1HBV1.2 plasmid produced and secreted HBV. 2. 2. The decrease of P62 protein level in HepG2.2.15 cell line with HBV replication or HepG2 cells transfected with pUC1857-1HBV1.2 plasmid increased the ratio of LC3- 鈪，

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