慢病毒介导bcr/abl基因RNAi对白血病K562细胞生物学特性的影响  

Effects of lentivirus-mediated long term bcr/abl RNAi on biologic characteristics of human leukemia cell line K562

作　　者：吴枝娟[1,2] 许建华[1] 黄秀旺[1] 吴丽贤[1] 

机构地区：[1]福建医科大学药学院临床药理学研究所,福建福州350004 [2]福建医科大学基础医学院生理学与病理生理学系,福建福州350004

出　　处：《中国肿瘤生物治疗杂志》2009年第2期145-150,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.30873101)~~

摘　　要：目的:研究慢病毒介导RNAi致bcr/abl基因长期沉默对K562白血病细胞各种生物学特性的影响。方法:构建含bcr/ablRNAi序列的pNL-B/A-EGFP慢病毒重组质粒载体并包装病毒,感染K562细胞,挑取稳定转化的克隆(B/A-K562)。Real-time PCR及Western blotting验证干扰效应;锥虫蓝染色、集落形成实验检测细胞增殖能力变化;联苯胺染色观察细胞分化;ELISA法检测酪氨酸激酶活性;AO-EB染色观察细胞凋亡;比色法检测Caspase-3、Caspase-9活性;以上检测均以K562细胞和转染空质粒EGFP-K562作对照。结果:成功构建bcr/abl基因RNAi稳定转染的B/A-K562细胞,Real-time PCR及Westernblotting证实B/A-K562细胞中bcr/ablmRNA及P210bcr/abl蛋白含量明显下调。bcr/abl基因稳定下调后,K562细胞倍增时间明显延长(37.1vs20.4、23.3h)、集落形成能力减弱(P<0.01),K562细胞向红系分化,酪氨酸激酶活性下降(P<0.01),自发凋亡率显著提高(P<0.01),细胞中Caspase-3(P<0.05)及Caspase-9(P<0.01)活性明显提高。结论:慢病毒介导的RNAi能实现bcr/abl基因长期沉默,从而抑制K562细胞恶性增殖,诱导其分化及凋亡。Objective： To study the effects of lentivirus-mediated bcr/abl RNAi on the biological characteristics of human leukemia cell line K562. Methods： Bcr/abl RNAi lentivirus vector pNL-B/A-EGFP was constructed and was used to transfect K562 cells, the stable tansfectants（B/A-K562） were selected. RNAi efficiency was assessed by Real-time PCR and Western blotting. Cell proliferation was detected by trypan blue staining and colony formation assay, cell differentia- tion was investigated by benzidine staining, PTK activity was determined by ELISA, apoptosis was observed by AO-EB staining, and caspase-3 and caspase-9 activation were measured by chromometry. K562 ceils and mock transfected EGFP- K562 cells were used as controls. Results： pNL-B/A-EGFP stably transfeeted K562 cells （B/A-K562） was successfully constructed. Real-time PCR and Western blotting analysis confirmed that lentivirus-mediated bcr/abl RNAi down-regulated bcr/abl mRNA and P210bcr/abl protein expression in K562 ceils. The doubling time of B/A-K562 cells was obviously longer than those of K562 cells and EGFP-K562 cells （37.1 vs 20.4, 23.3 h）. Furthermore, B/A-K562 cells showed decreased colony formation ability, strengthened differentiation toward erythrocytes, decreased activation of PTK, increased apoptosis and enhanced caspase-3 and caspase-9 activation （ P 〈 0.05 or P 〈 0.01 ）. Conclusion： Lentivirus-mediated bcr/abl RNAi can result in long time silencing of bcr/abl gene, inhibit malignant proliferation and induce differentiation and apop- tosis in K562 cells.

关 键 词：RNA干扰 白血病细胞 BCR/ABL基因 慢病毒 稳定转染 

分 类 号：R733.72[医药卫生—肿瘤] R730.54[医药卫生—临床医学]