沉默DNA-PKcs对肝癌耐药细胞Bel7402/5-Fu凋亡的影响  

Effect of siDNA-PKcs promoting cell apoptosis of multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu

作　　者：李大玉[1] 余春波[1] 朱欣婷[1] 刘喜平[1] 范芳[1] 李长福[1] LI Dayu ,YU Chunbo , ZHU Xinting , LIU Xiping , FAN Fang, LI Changfu(Teaching and Researching Section of Biochemistry, Zunyi Medical College, Zunyi, Guizhou 563000 China)

机构地区：[1]遵义医学院生化教研室,贵州遵义563000

出　　处：《重庆医学》2018年第24期3134-3137,3141,共5页Chongqing medicine

基　　金：贵州省社发攻关项目[黔科合SY(2013)3004];2016年硕士科研启动资金[F-829]

摘　　要：目的研究DNA依赖蛋白激酶催化亚基(DNA-PKcs)基因沉默后对肝癌耐药细胞Bel7402/5-Fu细胞凋亡的影响。方法实验分为空白对照组、脂质体对照组、NC对照组、siDNA-PKcs实验组。采用噻唑蓝(MTT)法检测耐药细胞Bel7402/5-Fu的耐药性;采用阳离子脂质体法将siDNA-PKcs寡核苷酸片段转染Bel7402/5-Fu细胞;Real-time PCR和Western blot分别检测细胞DNA-PKcs mRNA及蛋白的表达情况;倒置荧光显微镜下观察细胞形态变化(Hoechst33342染色法);流式细胞术检测细胞凋亡情况(AnnexinV/PI双染法);Western blot检测B细胞淋巴瘤-2相关x蛋白(Bax)、B细胞淋巴瘤/白血病-x基因长片段(Bcl-xl)蛋白的表达情况。结果 MTT检测Bel7402/5-Fu细胞的半抑制浓度(IC50)明显增高,耐药细胞Bel7402/5-Fu的耐药指数为13.13;DNA-PKcs的mRNA与蛋白表达水平明显减少,设计的siDNA-PKcs特异序列能有效沉默细胞DNA-PKcs的表达;Hoechst-33342染色检测结果显示siDNA-PKcs实验组细胞体积缩小、细胞核边集、呈亮蓝色;流式细胞术检测结果显示,siDNA-PKcs实验组细胞凋亡率比其余组增加(P<0.01);Western blot检测结果显示siDNA-PKcs实验组Bax蛋白表达水平比其余组高,Bcl-xl蛋白表达水平比其余组低(P<0.01)。结论siDNA-PKcs能诱导肝癌耐药细胞Bel7402/5-Fu细胞凋亡,并能上调促凋亡蛋白Bax的蛋白表达水平,抑制抗凋亡蛋白Bcl-xl的蛋白表达水平。Objective To study the influence of DNA dependent protein kinase catalytic subunit（DNAPKcs）silence on cellular apoptosis of multidrug-resistant（MDR）hepatocellular carcinoma cell Bel7402/5-Fu.Methods The cases were divided into the blank control group,liposomes control group,negative control（NC）group and siDNA-PKcs experimental group.The drug resistance of Bel7402/5-Fu cell line was determined by using MTT.The cationic liposome method was adopted to transfect siDNA-PKcs oligonucleotide fragment to Bel7402/5-Fu.Real-time PCR and western blot were used to detect the expression levels of DNA-PKcs mRNA and protein.The cellular morphology change was observed by the inverted fluorescence microscope（Hoechst33342 staining method）.The cell apoptosis was detected by using flow-cytometry（Annexin V/P dual staining）.The expression levels of cell apoptosis related proteins Bax and Bcl-xl protein were determined by western blot.Results The hemi-inhibitory concentration（IC50）of Bel7402/5-Fu cells detected by MTT was significantly increased.The drug resistance index of drug resistance Bel7402/5-Fu was 13.13.The expression level of DNA-PKcs mRNA was significantly decreased.The designed siDNA-PKcs specific sequence effectively silenced the expression of DNA-PKcs.The Hoechst-33342 staining detection results showed that the cellular volume was shrunk,nuclear had margination with bright blue.The flow-cytometry detection results showed that the apoptosis rate in the siDNA-PKcs experimental group was increased compared with the other groups（P〈0.01）.The western blot detection results showed that the expression level of Bax protein in the siDNAPKcs experimental group was higher than those in the other groups（P〈0.01）and the Bcl-xl protein level in the experimental group was lower than those in the other groups（P〈0.01）.Conclusion siDNA-PKcs could induce the apoptosis of MDR hepatocellular carcinoma cells,up-regulate the expression level of apoptosis-promoting protein Bax and inhibit the expression le

关 键 词：肝肿瘤 药物耐受性 DNA依赖性蛋白激酶催化亚基 肝癌耐药细胞 细胞凋亡 机制 

分 类 号：R735.7[医药卫生—肿瘤]