酪氨酸激酶抑制剂STI571与P21WAF基因克隆治疗慢性粒细胞白血病的实验研究

本研究探讨酪氨酸激酶抑制剂ST1571和P21^WAF基因克隆对慢性粒细胞白血病急变K562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P21^WAF基因，胶纯化回收后连接到T载体测序，序列正确后构建P21—pcDNA3．1栽体，将P21—pcDNA3．1与空载体分别以脂质体转染入P21蛋白表达缺如的K562细胞，经筛选得到G418抗性的K562细胞株，Westernblot证实转染后有P21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P21蛋白的P21—pcDNA3．1-K562细胞株生长速度明显慢于对照K562细胞株，流式细胞仪显示G0／G1期细胞增多，MTT法显示P21—pcDNA3．1-K562细胞与ST1571联合应用后，与单用ST1571作用的K562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P21蛋白能抑制K562细胞增殖，同时对ST1571的促凋亡作用有轻度抑制，并降低K562细胞对ST1571的敏感性。

Experimental Research on Tyrosine-Kinase Inhibitor STI571 and P21WAF Gene Clone to Treat Chronic Myeloid Leukemia

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.