CAJ | 학술논문

目的通过转摹因细胞模型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ID2/HBx及空白对照组ID2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、流式细胞术枪测各组细胞增殖、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ID2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ID2/pcDNA3.1细胞(P<0.05).实时定量PCR检测发现hMTH1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.
목적 통과전모인세포모형L02/HBx관찰을형간염병독x기인(HBx)대DNA수복매hMTH1전록표체적조공급기대인간세포계L02세포생물학특성적영향.방법 전대배양은정표체HBx적전기인세포모형ID2/HBx급공백대조조ID2세포화공재체대조조L02/pcDNA3.1세포,도치현미경하관찰각조세포적형태학특정,이사갑기우담서염(MTT)비색법、류식세포술창측각조세포증식、세포주기화조망상황,병진행연경지극륭형성실험관찰세포적악성전화능력.동시응용실시정량PCR측정각조세포DNA수복매hMTH1적표체수평.결과 경하관찰발현L02/HBx세포여대조조ID2세포상비형태발생명현개변.MTT법현시L02/HBx세포생장속도비량대조조현저가쾌.류식세포술결과표명,HBx가가속세포주기진정、억제세포조망.연경지극륭형성실험발현L02/HBx세포적극륭형성솔명현고우L02세포화ID2/pcDNA3.1세포(P<0.05).실시정량PCR검측발현hMTH1재L02/HBx세포중적표체현저고우L02세포화L02/pcDNA3.1세포(P<0.05).결론 HBx가유도L02세포발생악성전화,재차과정중DNA수복매hMTH1가출현반응성적상조표체.
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 L02/HBx transgene cell model. Methods Light microscopy was used to observe the morphologic characteristics of gene-transfected cell strain Lff2/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/HBxwas 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/peDNA3. 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.