酪氨酸激酶抑制剂STI571与P21^(WAF)基因克隆治疗慢性粒细胞白血病的实验研究  

Experimental Research on Tyrosine Kinase Inhibitor STI571 and P21^(WAF) Gene Clone to Treat Chronic Myeloid Leukemia

作　　者：王玮[1] 孙秉中[2] 刘新平[3] 冯琦[2] 尚振川[2] 曹云新[4] 药立波[1] 

机构地区：[1]解放军第175医院,漳州363000 [2]第四军医大学西京医院血液科 [3]第四军医大学生物化学和分子生物学教研室,西安710032 [4]第四军医大学中心实验室,西安710032

出　　处：《中国实验血液学杂志》2004年第6期737-742,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金 ( 39770 336);陕西省科研基金 ( 98SM40 )资助

摘　　要：本研究探讨酪氨酸激酶抑制剂STI5 71和P2 1WAF基因克隆对慢性粒细胞白血病急变K5 62细胞株的细胞增殖、细胞周期及凋亡等的治疗作用。RT PCR扩增P2 1WAF基因 ,胶纯化回收后连接到T载体测序 ,序列正确后构建P2 1 pcDNA3 .1载体 ,将P2 1 pcDNA3 .1与空载体分别以脂质体转染入P2 1蛋白表达缺如的K5 62细胞 ,经筛选得到G4 18抗性的K5 62细胞株 ,Westernblot证实转染后有P2 1蛋白表达 ,MTT法检测细胞存活率 ,流式细胞仪检测细胞周期和凋亡。结果表明 :表达外源性P2 1蛋白的P2 1 pcDNA3 .1 K5 62细胞株生长速度明显慢于对照K5 62细胞株 ,流式细胞仪显示G0 /G1期细胞增多 ,MTT法显示P2 1 pcDNA3 .1 K5 62细胞与STI5 71联合应用后 ,与单用STI5 71作用的K5 62细胞相比 ,凋亡细胞比例轻度减少 ,细胞存活率下降较慢。结论 :表达外源P2 1蛋白能抑制K5 62细胞增殖 ,同时对STI5 71的促凋亡作用有轻度抑制 ,并降低K5 62细胞对STI5 71的敏感性。To explore the effect of a tyrosine kinase inhibitor STI571 and P21 WAF gene clone on the proliferation, cycle, apoptosis of leukemia cell line K562, P21 WAF gene was obtained by RT PCR, and its sequence was approved to be correct, then P21 pcDNA3.1 vector was constructed and transfected into K562 cell line. After selected with G418, P21 pcDNA3.1 K562 cell clone that stably expression P21 WAF was isolated. P21 WAF protein was identified by Western blot. The survival rate were tested by MTT. Cell cycle and apoptosis were tested by flow cytometry. The results showed that the expression of P21 WAF protein could be detected by Western blot in P21 pcDNa3.1 K562 cells. A strong inhibition of cell proliferation was observed in P21 pcDNA3.1 K562 cells as compared with that of the control. The cells cycle were arrested in G 0/G 1 phase. The percentage of apoptosis was declined slightly after P21 pcDNA3.1 K562 cells were combined with STI571, meanwhile its survival rate declined more slowly than that of K562 cell with STI571. In conclusion, P21 WAF inhibits the proliferation of K562 cell,meanwhile slightly inhibits its apoptosis induced by STI571and decrease its sensitivity to STI571.

关 键 词：酪氨酸激酶抑制刑 ST1571 P21^WAF基因 基因克隆 慢性粒细胞白血病 

分 类 号：R733.72[医药卫生—肿瘤] R979.1[医药卫生—临床医学]