自噬现象与乙肝病毒复制的关系
发布时间：2018-09-05 14:08
【摘要】：[研究目的] 通过研究自噬现象在乙肝病毒复制中所扮演的角色,寻找降低乙肝病毒复制的新途径,并通过对其机制的研究,探讨该现象对于宿主细胞生存的影响及其致癌性的可能机理。 [研究方法] 1.用HepG2.2.15细胞系和pUC1857-1HBV1.2质粒转染的HepG2细胞系构建出有HBV复制的细胞系； 2.采用3-甲基腺嘌呤和无胎牛血清培养基作为自噬现象的干预手段,使用HepG2细胞系和pUC18质粒转染的HepG2细胞系作为对照,通过测量上清中HBV拷贝数评估HBV复制情况,并对细胞裂解液采用蛋白免疫印迹法(Western Blotting)对自噬水平进行分析； 3.使用GFP-LC3质粒转染的细胞及HBcAg免疫荧光抗体,通过荧光显微镜对HBV复制和自噬的水平进行评估： 4.采用WST-1试剂及酶标仪测定,对细胞增殖状态进行评估。 [研究结果] 1. HepG2.2.15细胞系和pUC1857-1HBV1.2质粒转染的HepG2细胞均有HBV产生和分泌。 2.有HBV复制产生的细胞系——-HepG2.2.15细胞系或pUC1857-1HBV1.2质粒转染的HepG2细胞中,P62蛋白水平的下降,LC3-Ⅱ/LC3-Ⅰ比值升高,说明HBV复制有诱导自噬的作用。 3.在HepG2.2.15细胞系中,使用1mM浓度的自噬抑制剂3-MA,可以减少上清液中HBV的拷贝数。但是对于10mM浓度的3-MA,以及不含FBS的乏营养培养基对上清中HBV拷贝数的影响尚缺乏具有显著性差异的结论。 4.转染pEGFP-C1-LC3质粒HepG2细胞中,GFP-LC3融合蛋白弥散在胞浆中。而转染pEGFP-C1-LC3质粒的HepG2.2.15细胞可以看到多个明亮的绿色荧光斑点。并且LC3斑点数量与HBcAg荧光斑点数量有一定程度的相关性,但两者未表现出共定位。 5.在无血清培养基的饥饿状态下,有HBV复制的细胞系增殖更快,加入10mM的3-MA则会抑制这种增殖。提示HBV所上调的自噬有利于细胞在极端情况下的生存。 [结论] 乙肝病毒可以提高其宿主细胞中自噬的水平,但自噬对于乙肝病毒的复制、产生的意义以及具体的机制还有待于进一步探索。同时乙肝病毒也许有利于宿主细胞在应激情境下生存状态,但这种影响还需进一步的试验验证。更深入的研究也许将为我们治疗乙肝病毒感染,阻止或减缓慢性乙肝病毒感染向肝硬化、肝癌进展提供新的思路。
[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，
本文编号：2224511
[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，
本文编号：2224511
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