HBx基因转染胆管癌QBC939细胞对其凋亡的影响及作用机制的研究

Effect of transfection of HBx gene on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism

目的:探讨乙型肝炎病毒x基因(hepatitis B virusx,HBx)转染胆管癌QBC939细胞引起凋亡及其发生的可能机制。方法:将构建有HBx基因的真核表达载体pcDNA3-x采用脂质体转染法瞬时转染QBC939细胞,RT-PCR及Western印迹法鉴定HBx在QBC939细胞中的表达;DAPI染色后荧光显微镜镜下观察细胞核的改变,结合FCM法检测细胞的凋亡;以未转染和转染空质粒pcDNA3的QBC939细胞为对照,Western印迹法检测Bax、Bak、Bid、Bcl-XL、CytC、p65的表达;JC-1染色法检测细胞线粒体膜电位(ΔΨm)的变化;RT-PCR法检测Fas和c-myc在mRNA水平的表达;双荧光素酶报告系统分析细胞核因子-κB(nuclear factor kappa B,NF-κB)的转录活性。结果:RT-PCR及Western印迹法检测均证实转染后的QBC939细胞中有HBx的表达;DAPI显示转染细胞核呈现固缩且边缘化等细胞凋亡特征性的改变;FCM检测结果显示,转染HBx组与对照组相比,凋亡细胞数量明显增多;Western印迹法检测结果显示,Bcl-2家族中Bax表达升高,Bcl-XL下降,p65在核内的表达水平升高,细胞质中细胞色素C(cytochrome C)表达升高;RT-PCR结果显示,Fas和c-myc的mRNA水平均高于对照组;JC-1染色法结果显示ΔΨm下降;双荧光素酶报告系统分析提示NF-κB的转录活性升高。结论:成功将HBx基因转入QBC939细胞中,HBx可能是通过调节Fas/FasL死亡受体途径,Bcl-2家族引起的线粒体路径及NF-κB的激活、c-myc等凋亡相关基因的表达而引起QBC939细胞的凋亡。

Objective：To investigate the effect of transfection of hepatitis B virus x （HBx） gene on apoptosis of cholangiocarcinoma QBC939 ceils and its possible mechanism. Methods：The eukaroytic vector pcDNA3-x containing HBx gene was transiently transfected into QBC939 cells by lipofectamine mediation. The expression of HBx was confirmed by RT-PCR and Western blotting. The morphological changes of nucleus were observed by DAPI staining and the apoptosis was detected by flow cytometry. Untransfected QBC939 cells and those trasfected with empty vector were used as control. Expression of Bax, Bak, Bid, Bcl-XL, CytC, and p65 were measured by Western blotting. The alteration of mitochondria membrane potential （△ψm） was detected by JC-1 staining method. The mRNA expression of Fas and c-myc was determined by RT-PCR. The transcriptional activity of NF-κB was analyzed by Dual-Luciferase Reporter Assay System. Resuits：RT-PCR and Western blotting results verified that HBx was expressed in QBC939 cells after transfection. DAPI showed apoptotic cellular features, including nuclear condensation and chromatin marginization. FCM analysis showed that the number of apoptotic cells was significantly increased in the HBx transfection group than that in the control group. Western blotting demonstrated that the expression of Bax was increased and Bcl-XL was decreased. The expression of p65 in nucleus was increased and the cytoplasmic level of cytochrome c was elevated. RT-PCR results showed that the mRNA expression levels of Fas and c-myc were significantly higher in the HBx transfection group than those in the control group. JC-1 staining method indicated loss of △ψm. Dual-Luciferase Reporter Assay System suggested that the transcriptional activity of NF-κB was increased after transfection of HBx into QBC939 cells. Conclusion： HBx gene is successfully transfected into QBC939 cells. Overexpression of HBx gene induces apoptosis by regulating Fas/FasL death receptor pathway, Bcl-2 family-induced mitochondrial pathway, activity of NF-κB, and expression levels of c-myc apoptosis-related genes.