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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。
This study was aimed to investigate the effects of tyrosine kinase inhibitor STI5 71 and P2 1WAF gene cloning on cell proliferation, cell cycle and apoptosis in K562 cells with acute myeloid leukemia. The P2 1WAF gene was amplified by RT-PCR and ligated to the T vector for sequencing after sequencing. The P2 1 pcDNA3. 1 vector was constructed and the P2 1 pcDNA3. 1 and empty vector were transfected into P2 1 protein by lipofectin The K562 cells lacking G418 were screened to obtain K462 cell lines which were resistant to G418. The expression of P2 1 protein was confirmed by Western blot. The cell viability was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The results showed that the growth rate of P2 1 pcDNA3. 1 K562 cells expressing exogenous P2 1 protein was significantly slower than that of the control K5 62 cell line. Flow cytometry showed that G0 / G1 phase cells were increased and P2 1 pcDNA3 was detected by MTT assay. When K562 cells were combined with STI571, the percentage of apoptotic cells decreased slightly and the cell viability decreased more slowly than that of K562 cells treated with STI571 alone. Conclusion: The expression of exogenous P2 1 protein can inhibit the proliferation of K562 cells and slightly inhibit the pro-apoptotic effect of STI571 and reduce the sensitivity of K562 cells to STI571.