[重组腺病毒AdE-SH2-Caspase 8对伊马替尼耐药K562/G01细胞系凋亡的影响]

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

作者信息

Wang Lin, Fei Chang, Huang Zheng-Lan, Li Hui, Liu Zhang-Lin, Feng Wen-Li

机构信息

College of Laboratorial Medicine, Hunan University of Medicine, Huaihua 418000, Hunan Province, China.

Key Laboratory of Clinical Examination and Medical Diagnostics, Ministry of Education, College of Laboratorial Medicine, Chongqing Medical University, Chongqing 400016, China.

出版信息

Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015 Aug;23(4):976-81. doi: 10.7534/j.issn.1009-2137.2015.04.013.

DOI:10.7534/j.issn.1009-2137.2015.04.013

PMID:26314429

Abstract

OBJECTIVE

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib.

METHODS

The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot.

RESULT

The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased.

CONCLUSION

The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

摘要

目的

研究重组腺病毒AdE-SH2-Caspase8-HA-GFP（SC）表达的SH2-Caspase 8融合蛋白对K562/G01细胞系凋亡的影响，K562/G01细胞系是一种BCR/ABL阳性的慢性髓性白血病细胞系，对伊马替尼耐药。

方法

用AdE-SH2-Caspase 8-HA-GFP腺病毒（SC）感染K562/G01细胞系，然后将细胞分为3组：AdE-SH2m-Caspase 8-HA-GFP（SmC）组、AdE-GFP（CMV）组和PBS组作为对照。在荧光显微镜下和通过流式细胞术观察感染效率。通过蛋白质免疫印迹法检测融合蛋白SH2-Caspase 8-HA的表达。用瑞氏染色检测细胞形态。通过流式细胞术和DNA梯状条带检测细胞凋亡。通过蛋白质免疫印迹法检测Caspase 3和PARP的表达。

结果

荧光显微镜和流式细胞术证实SC对K562/G01细胞的感染效率高。SH2-Caspase 8-HA融合蛋白在K562/G01细胞中正确表达。用SC处理后，通过显微镜可观察到K562/G01细胞的凋亡。流式细胞术结果显示，与对照组相比，K562/G01细胞的早期凋亡显著增加（P < 0.05）。DNA梯状条带显示，用SC处理后，K562/G01细胞中出现了典型的DNA梯状条带。蛋白质免疫印迹检测显示，凋亡相关蛋白Caspase 3和PARP的表达水平升高。