重组腺病毒表达的融合蛋白SH2-caspase 8对K562细胞凋亡的影响  

Effect of SH2-caspase 8 fusion protein expressed by recombinant adenovirus on apoptosis of K562 cell line

作　　者：王林[1] 李会[1] 刘章玲[1] 祖白玲[1] 黄峥兰[1] 向华[1] 冯文莉[1] 

机构地区：[1]重庆医科大学检验医学院/临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《临床检验杂志》2013年第8期612-616,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(30871102)

摘　　要：目的研究表达融合蛋白SH2-caspase 8的重组腺病毒AdE-SH2-caspase 8-HA-GFP(SC)对BCR/ABL阳性的慢性粒细胞白血病K562细胞凋亡的影响。方法将重组的腺病毒SC转染K562细胞(SC组),分别设突变AdE-SH2m-caspase 8-HA-GFP(SmC)组、病毒空载体AdEGFP(CMV)组和PBS对照组。用荧光显微镜和流式细胞仪(FCM)检测病毒转染效率,western blot检测融合蛋白及凋亡相关蛋白caspase 3、多聚二磷酸腺苷核糖聚合酶(PARP)的表达情况,用光学显微镜观察经瑞氏染色后的细胞形态,FCM和DNA梯度电泳检测细胞凋亡情况。结果 FCM和荧光显微镜检查结果显示,重组腺病毒在K562细胞中的转染效率较高;融合蛋白SH2-caspase 8-HA和SH2m-caspase 8-HA可在K562细胞中表达;瑞氏染色后SC组出现明显的凋亡形态改变;FCM检测结果表明,SC组与SmC组、CMV组和PBS组相比较,其早期凋亡细胞明显增高(t分别为6.945、19.083和16.470,P均<0.05),DNA梯度电泳检测结果发现SC组和SmC组可出现细胞凋亡特异性的梯度条带,western blot结果表明,SC组和SmC组凋亡相关蛋白caspase 3、PARP活化片段的表达水平升高。结论重组腺病毒SC表达的SH2-caspase 8融合蛋白能明显诱导K562细胞的凋亡。Objective To study the effect of Src homology-2（SH2） domain-caspase 8 fusion protein expressed by recombinant adenovirus SH2-caspase 8-hemagglutinin（HA）-green fluorescent protein（AdE-SH2-caspase 8-HA-GFP,SC） on the apoptosis of K562 cells,the BCR / ABL-positive chronic myeloid leukemia cell line.Methods K562 cells were transfected with the recombinant SC adenovirus vector expressing SH2-caspase 8 fusion protein.The 4 experimental groups were as follows： SC group（AdE-SH2-caspase 8HA-GFP）,SmC group（SH2 mutant group in which SH2 contained mutant nucleotide sequence,AdE-SH2m-caspase 8-HA-GFP）,empty vector groups（AdE-GFP） and PBS control group.The transfection efficiency was measured by fluorescence microscopy and flow cytometry（FCM）.The expressions of fusion protein and apoptosis-related protein,i.e.,caspase 3 and poly ADP-ribose polymerase（PARP） were analyzed by western blotting.Cellular morphology with Wright＇s staining was observed under light microscope.The cell apoptosis was detected by FCM and DNA ladder electrophoresis.Results The high infection efficiency of SC adenovirus vector in K562 cells was verified by fluorescence microscopy and FCM.Both SH2-caspase 8-HA and SH2m-caspase 8-HA fusion protein were expressed correctly in K562 cells.After treatment with SC adenovirus vector the morphology of apoptotic K562 cell was visible.The results of FCM showed that early apoptosis of K562 cells in SC group increased significantly compared with that in SmC,empty vector and PBS control group（t = 6.945,19.083 and 16.470 respectively,P 0.05）.DNA ladder electrophoresis indicated that the classic DNA ladder pattern of apoptotic cell appeared in K562 cells after treatment with SC or SmC adenovirus vector.Western blotting revealed activated fragments of caspase 3 and PARP proteins significantly increased.Conclusion The recombinant SC adenovirus vector may express SH2-caspase 8 fusion protein which significantly induced the apoptosis of K562 cells.

关 键 词：细胞凋亡 SH2结构域 半胱天冬酶8（caspase 8） 融合蛋白 K562细胞 

分 类 号：R733.72[医药卫生—肿瘤]