乙肝病毒X基因变异对AR在乙肝致癌过程中作用的影响

发布时间：2018-03-19 17:28

本文选题：乙肝病毒　切入点：肝细胞癌　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的构建稳定表达人AR的Hep G2细胞系,分别转染空质粒、HBx野生型质粒和变异型质粒至Hep G2细胞,通过检测细胞的迁移力、增殖周期和细胞凋亡等,探究HCC相关HBx变异与AR的交互作用对肝癌恶性程度的影响。研究方法1.构建携带人AR外显子基因、绿色荧光蛋白基因(GFP)及嘌呤霉素基因(PM)的重组表达质粒(p CDH-CMV-MCS-EF1-GFP-Puro);2.重组表达质粒包装感染Hep G2细胞及嘌呤霉素加压筛选,构建能稳定表达人AR的Hep G2细胞系;3.分别以慢病毒空载体、携带HBx野生型全长基因的慢病毒载体以及携带A1727G+A1762T/G1764组合突变或3’端缺失突变的HBx基因的慢病毒载体,瞬时转染Hep G2-GFP-AR细胞,通过细胞迁移实验和流式细胞技术检测细胞周期和细胞凋亡,观察不同HBx与AR在乙肝相关性肝癌发生发展中的作用。结果1.成功构建了携带人AR基因的慢病毒表达载体p CDH-CMV-MCSEF1-GFP-Puro-AR。2.通过携带AR基因的慢病毒感染Hep G2细胞,筛选出稳定高表达人AR的Hep G2细胞株,经流式细胞术验证其GFP的表达量,传至第10代后,该细胞株GFP的表达量为97.7%;Western-blot实验鉴定单克隆细胞株传至第14代时AR的表达量,2号克隆AR的表达量较高。3.选取AR表达量较高的2号克隆用于后续功能实验,Transwell实验结果显示:HBx空转染组在加入DHT后,下室中细胞OD值(0.45±0.02)显著高于未加DHT组(0.33±0.03),p0.001;与未转染HBx基因的Hep G2-GFP-AR细胞(空-组)(0.33±0.03)比较,转染携带HBx A1727G+A1762T/G1764组合突变(167-组)(0.43±0.01,p=0.001)或3’端缺失突变(CT-组)(0.45±0.03,p0.001)的质粒后,下室中细胞OD值显著增高;Hep G2-GFP-AR细胞在分别转染HBx A1727G+A1762T/G1764组合突变(167-组)或3’端缺失突变(CT-组)的质粒后,加入DHT时,下室中细胞OD值显著降低(167-组vs 167+组,0.43±0.01 vs 0.36±0.02,p=0.009;CT-组vs CT+组,0.45±0.03 vs 0.35±0.04,p=0.001)。细胞周期实验结果显示:各组间G2+S期细胞总占比无显著统计学差异。细胞凋亡实验结果显示:各组间细胞总凋亡率无显著统计学差异。结论1.本实验成功构建了携带人AR外显子基因的慢病毒表达载体p CDH-CMVMCS-EF1-GFP-Puro-AR;并利用该载体,慢病毒感染Hep G2细胞,成功构建了稳定表达AR的Hep G2细胞系Hep G2-GFP-AR。2.通过AR高表达细胞系Hep G2-GFP-AR进一步功能实验,发现在高表达AR的Hep G2-GFP-AR细胞中,转染空载体组加入DHT后细胞迁移能力显著增强,而转染HBx组合突变或3’端缺失突变的HBx后亦能够增强细胞的迁移能力;但加入DHT后则对HBx A1727G+A1762T/G1764组合突变或3’端缺失突变提高肿瘤细胞迁移能力的效应可能具有抑制作用;本研究未发现HBx相关突变株以及AR对Hep G2细胞的增殖和凋亡有显著影响。
[Abstract]:Objective to construct a stable Hep G2 cell line expressing human AR and transfect empty plasmid HBX wild-type plasmid and variant plasmid into Hep G2 cell line. To explore the effect of HCC related HBx variation and AR interaction on the malignancy of hepatocellular carcinoma. 1. Construct the gene carrying human AR exon. The recombinant expression plasmids of green fluorescent protein (GFP) and purine mycin gene (PMM) were constructed by pCDH-CMV-MCS-EF1-GFP-Puromo-2.The recombinant expression plasmid was packaged to infect Hep G2 cells and purine mycin to construct Hep G2 cell lines which could stably express human AR. The lentivirus vector carrying HBx wild-type full-length gene and the lentivirus vector carrying A1727G A1762T / G1764 combination mutation or HBx gene with 3 'deletion mutation were transiently transfected into Hep G2-GFP-AR cells. Cell cycle and apoptosis were detected by cell migration assay and flow cytometry. To observe the role of different HBx and AR in the occurrence and development of Hepatitis B related liver cancer. Results 1. The lentivirus expression vector pCDH-CMV-MCSEF1-GFP-Puro-AR.2 was successfully constructed. Hep G2 cells were infected by the lentivirus carrying AR gene. Hep G2 cell lines with stable and high expression of human AR were screened. The expression of GFP was confirmed by flow cytometry and passed on to the 10th passage. The GFP expression of the cell line was 97.7 and the expression of AR was identified by Western-blot. The expression of AR in clone 2 was higher than that in clone 2 at the 14th passage. Clone 2 with high AR expression was selected for further functional experiment. After adding DHT to the empty transfection group, Compared with Hep G2-GFP-AR cells without transfection of HBx gene (0.33 卤0.03p0.001), the cells were transfected with HBx A1727G A1762T / G1762T- G177- group (0.43 卤0.01p0.001) or 3'terminal deletion mutation CT-group (0.45 卤0.03p0.001), compared with Hep G2-GFP-AR cells without HBx gene transfection (0.33 卤0.03p0.001). After transfection of HBx A1727G / A1762T / G1764 combination mutagenesis (167- group) or 3'deletion mutation (CT- group), Hep G2-GFP-AR cells were added to DHT. The OD value of the cells in the lower chamber decreased significantly in 167- group vs 167 group (0.43 卤0.01) vs 0.36 卤0.02p0.009 CT- group vs CT group (0.45 卤0.03 vs 0.35 卤0.04 p0. 001). The results of cell cycle test showed that there was no significant difference in the total proportion of cells in G 2 S phase between the three groups. Conclusion: 1. The lentivirus expression vector pCDH-CMVMCS-EF1-GFP-Puro-ARA was successfully constructed in this study. The stable Hep G2 cell line Hep G2-GFP-AR.2 was successfully constructed by lentivirus infection with AR. Through further functional experiments of AR overexpression cell line Hep G2-GFP-AR, Hep G2-GFP-AR cell line with high AR expression was found. After transfection of empty vector with DHT, the cell migration ability was significantly enhanced, and the migration ability was also enhanced after transfection of HBx combination mutation or HBx with 3 'deletion mutation. However, the addition of DHT may inhibit the effect of HBx A1727G A1762T / G1764 combination mutation or 3'deletion mutation on the ability of tumor cell migration, and no significant effect of HBx related mutant and AR on the proliferation and apoptosis of Hep G2 cells was found in this study.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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