摘要
目的探讨含有WW结构域的氧化还原酶基因（WWOX）表达对胆管癌QBC939细胞凋亡的影响及其作用机制。方法用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞；采用荧光定量逆转录-聚合酶链反应（RT—PCR）和Westernblot法鉴定WWOX在QBC939细胞中的表达；流式细胞仪（FCM）法检测转染前后各细胞凋亡率的变化；JC-1染色法检测细胞线粒体膜电位（△ψm）变化；荧光定量RT—PCR和Western blot法检测胆管癌细胞bcl-2表达的变化；将未转染和转染空质粒的细胞作为对照组接种到裸鼠皮下以检测荷瘤，TUNEL方法原位检测移植瘤的凋亡。结果建立了稳定表达WWOX基因的QBC939／WWOX细胞株，mRNA及蛋白表达明显增加。FCM显示QBC939／WWOX组的细胞凋亡率明显增高[（1．24±0．35）％比（1．73±0．48）％比（21．40±2．35）％，P〈0．01]，JC-1显示转染组的线粒体膜电位下降[（4．27±0．64）％比（4．96±0．52）％比（28．60±3．94）％，P〈0．01]，bcl-2 mRNA及蛋白的表达均显著降低（P〈0．05）。转染组的皮下肿瘤较对照组生长速度明显减慢（P〈0．05），TUNEL实验证实转染组的皮下肿瘤凋亡指数为（13．6±1．5）％，较对照组明显增高，差异有统计学意义（P〈0．01）。结论WWOX基因能促进胆管癌细胞的凋亡，其机制可能与下调bcl-2的表达，激活线粒体凋亡通路有关。
Objective To investigate the effect of transfection of WWOX gene on apoptosis of human cholangiocarcinoma QBC939 cells in vitro andin vivoand its possible mechanism. Methods The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection. The mRNA and protein expression levels in QBC939 cells stably transfected with WWOX were detected by using quantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting before and after transfection. Cell apoptosis was assessed by flow cytometry （FCM）. The alteration of mitochondria membrane potential （△ψm） was assayed by JC-1 staining method. The expression change of bcl-2 mRNA and protein was examined by using quantitative RT-PCR and Western blotting. All the experimental subjects were divided into three groups： QBC939 cells were cultured in natural status （nature control group）; QBC939 cells were transfected with blank plasmid （blank control group）, and QBC939 cells were transfected with WWOX/pmCherry-N1 （experimental group）. The three groups were subcutaneously inoculated into nude mice and the growth of xenografted tumor was observed. TUNEL was used to evaluate the apoptosis. Results QBC939 cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased. FCM analysis showed that the apoptosis rate after transfection was significantly promoted [ （ 1.24 ±0. 35 ） % vs （ 1.73 ± 0. 48） % vs （21.40 ±2. 35）% ,P〈 0. 01 ]. JC-1 staining method indicated that the experimental group was loss of △ψm [ （4. 27 ±0. 64） % vs （4. 96± 0. 52 ） % vs （ 28.60 ±3.94 ） %, P 〈 0. 01 ]. Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased （P 〈 0. 05）. The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down （P 〈 0. 05）. Number of apoptotic cells was significantly increased in the experimental group [ （4. 1± 0. 6）% vs （4.4±0.5）% vs （13.6 ±1.5）%,P〈0.01]. Conclusion WWOX gene can promote apoptosis of cholangiocarcinoma cells, which may be associated with downregulation of bcl-2 expression, and activation of the mitochondrial apoptosis pathway.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第11期1831-1834,共4页
Chinese Journal of Experimental Surgery
关键词
胆管癌
基因转染
脱噬作用
Cholangioearcinoma
Gene transfeetion
Apoptosis
