论文部分内容阅读
目的探讨乙肝病毒X蛋白(HBx)羧基端缺失了40个氨基酸的突变体(HBx3′-40)和野生型HBx对Huh7和SMMC-7721肝癌细胞的生物学行为的影响。方法脂质体和磷酸钙法介导pcDNA3HBx3′-40和pcDNA3HBx重组体转染HBV(-)的人肝癌细胞Huh7和SMMC-7721。Neo基因PCR及Western印迹方法检测质粒DNA片断插入和HBx蛋白质的表达。借助生长曲线、平板克隆形成、细胞周期、凋亡和裸鼠成瘤实验以及氯霉素乙酰转移酶(CAT)-ELISA法对转染细胞的生物学活性进行检测。结果pcDNA3HBx3′-40组细胞生长速度明显快于pcDNA3HBx和pcDNA3组;pcDNA3HBx3′-40组克隆形成率明显高于pcDNA3HBx和pcDNA3组(P<0·05);流式细胞仪检测结果显示pcDNA3HBx3′-40表达能加速Huh7细胞由G0/G1期→S期的进程;但细胞凋亡检测显示无血清诱导的SMMC-7721细胞HBx3′-40组能部分取消HBx的促凋亡效应,并且该组几乎丧失了HBx的反式激活作用;裸鼠成瘤实验显示,pcDNA3HBx3′-40组成瘤体积明显大于pcDNA3HBx组和pcDNA3组,瘤体重量间差异具有统计学意义(P<0·05)。表明HBx3′-40对肝癌细胞的生长具有明显促增殖作用。结论HBx碳端缺失了40个氨基酸的突变体转染细胞对比野生型HBx显示促增殖能力明显增强,推测HBx通过缺失突变修饰其生物学功能,取消了野生型HBx的反式激活功能和抗增殖效应。另一方面,HBx114~154个氨基酸位点的缺失可能导致p53无法发挥其抑癌作用。
Objective To investigate the effect of HBx3’-40 and wild-type HBx on the biological behavior of Huh7 and SMMC-7721 hepatocellular carcinoma cells. Methods Human hepatocellular carcinoma cells Huh7 and SMMC-7721 transfected with pcDNA3HBx3’-40 and pcDNA3HBx recombinant were transfected with liposome and calcium phosphate. Neo gene PCR and Western blotting were used to detect plasmid DNA insertions and HBx protein expression. The biological activity of transfected cells was detected by growth curve, plate clone formation, cell cycle, apoptosis and nude mice tumorigenesis experiment and chloramphenicol acetyltransferase (CAT) -ELISA. Results The growth rate of pcDNA3HBx3’-40 group was significantly faster than that of pcDNA3HBx and pcDNA3 group. The rate of pcDNA3HBx3’-40 group was significantly higher than that of pcDNA3HBx and pcDNA3 group (P <0.05). The results of flow cytometry showed that pcDNA3HBx3’- 40 could accelerate the process of Huh7 cells from the G0 / G1 phase to the S phase. However, the apoptotic test showed that HBx3’-40 group can partially cancel the pro-apoptotic effect of HBx in serum-free SMMC-7721 cells, And the transactivation of HBx was lost. Tumor formation in nude mice showed that the tumor volume of pcDNA3HBx3’-40 was significantly larger than that of pcDNA3HBx and pcDNA3 groups (P <0.05). The results showed that HBx3’-40 had a significant proliferative effect on the growth of hepatoma cells. Conclusions HBx transfected cells with a 40-amino acid deletion on the carbon end of the HBx gene show a significant increase in proliferative capacity compared with wild-type HBx. It is speculated that HBx modifies its biological function through deletion mutation and abolishes the transactivation function and anti-proliferation of wild-type HBx effect. On the other hand, the deletion of 114 to 154 amino acid residues in HBx may result in p53 being unable to exert its tumor suppressor effect.