摘要
目的通过转基因细胞模型L02／HBx观察乙型肝炎病毒x基因（HBx）对DNA修复酶hMTH1转录表达的调控及其对人肝细胞系L02细胞生物学特性的影响。方法传代培养稳定表达HBx的转基因细胞模型L02／HBx及空白对照组L02细胞和空载体对照组L02／pcDNA3．1细胞，倒置显微镜下观察各组细胞的形态学特征，以四甲基偶氮唑盐（MTT）比色法、流式细胞术检测各组细胞增殖、细胞周期和凋亡状况，并进行软琼脂克隆形成实验观察细胞的恶性转化能力。同时应用实时定量PCR测定各组细胞DNA修复酶hMTH1的表达水平。结果镜下观察发现L02／HBx细胞与对照组L02细胞相比形态发生明显改变。MTT法显示L02／HBx细胞生长速度比两对照组显著加快。流式细胞术结果表明，HBx可加速细胞周期进程、抑制细胞凋亡。软琼脂克隆形成实验发现L02／HBx细胞的克隆形成率明显高于L02细胞和L02／pcDNA3．1细胞（P〈0．05）。实时定量PCR检测发现hMTHI在L02／HBx细胞中的表达显著高于L02细胞和L02／pcDNA3．1细胞（P〈0．05）。结论HBx可诱导L02细胞发生恶性转化，在此过程中DNA修复酶hMTH1可出现反应性的上调表达。
Objective To study the effects of the HBV x gene （HBx） on the biological characteristics and the expression of DNA repair enzyme hMTH1 mRNA of the LO2/HBx transgene cell model. Methods Light microscopy was used to observe the morphologic characteristics of gene-transfected cell strain L02/HBx that stably expressed the HBx protein and the control groups of L02 and L02/pcDNA3.1. The changes of L02/HBx on the proliferation, cell cycle and apoptosis were observed by MTT assays and flow cytometry analysis respectively. Moreover, the malignant transformation of L02/HBx was assayed by colony formation in soft agar and the expression of DNA repair enzyme hMTH1 mRNA was assayed in each group by real-time qPCR. Results Inversion phase contrast microscope showed that the morphologic characteristics of L02/HBx had changed obviously compared with control groups. The MTT showed that L02/HBx proliferated more quickly and flow cytometry analysis indicated that HBx could accelerate the progression of cell cycle and inhibit apoptosis. Colony formation in soft agar demonstrated that the rate of colony formation of L02/HBx was remarkably higher than the L02 and the L02/pcDNA3.1 cells （P 〈 0. 05）. The real-time qPCR detection showed that the expression of hMTH1 mRNA in L02/HBx was significantly higher than that in the control groups （ P 〈 0. 05 ）. Conclusion HBx could play an important role in the malignant transformation of L02/HBx and the over expression of hMTH1 mRNA.
出处
《中华内科杂志》
CAS
CSCD
北大核心
2008年第10期830-833,共4页
Chinese Journal of Internal Medicine
基金
国家自然科学基金资助项目（30570821）
