摘要
为探讨p2 1WAF1对K5 6 2细胞药物敏感性的调节作用及其对VP 16诱导的K5 6 2细胞凋亡的影响 ,构建了p2 1WAF1逆转录病毒表达载体 ,体外转染白血病细胞系K5 6 2。经RT PCR和Western印迹检测得到稳定表达p2 1WAF1的K5 6 2 p2 1WAF1细胞克隆后 ,用MTT法和存活细胞计数法检测转染 p2 1WAF1的K5 6 2细胞对VP 16在剂量和作用时间上的敏感性变化 ,用DNA凋亡片段分析和流式细胞术检测其对VP 16诱导的K5 6 2细胞凋亡的影响。实验结果显示 ,外源性 p2 1WAF1的表达明显降低了VP 16诱导的K5 6 2细胞凋亡 ,降低了K5 6 2细胞对VP 16的敏感性。以上结果表明 ,p2 1WAF1能通过抑制K5 6 2细胞的凋亡来降低其对VP 16的敏感性。
The objective of this study was to explore the effect of p21 WAF1 on the sensitivity to chemotherapeutic agent VP 16, etoposide, in leukemia cell line K562. A p21 WAF1 retroviral expression vector was constructed, and mediated by FuGENE TM 6, it was transfected into K562 cells, which without p21 WAF1 expression. The ecotropic expression of p21 WAF1 in K562 cells was identified by RT PCR and Western blot, and named K562 p21 WAF1 cell. The K562 p21 WAF1 cells were exposed to VP 16 at different concentrations and different times, then, the senitivity to VP 16 was examined by cell viability and MTT assay, the apoptosis induced by VP 16 was examined by DNA fragments electrophoresis and flow cytometric Annexin V PI dual labeling technique. The results showed that the ecotropic expression of p21 WAF1 decreased the sensitivity of K562 cells to VP 16. After treatment of VP 16, the DNA ladder was examined in control K562 neo cells at 20 μg/ml, but in K562 p21 WAF1 cells at 80 μg/ml. With FCM, the number of apoptotic K562 neo cells was 14.9% and 25.4% respectively after treated with 20 μg/ml VP 16 for 12 and 24 hours, and it was 6.94% and 10.96% in K562 p21 WAF1 cells. Our results suggest that the expression of p21 WAF1 could reduce the sensitivity of K562 cells to VP 16 and inhibit the apoptosis of K562 cells.
出处
《中国实验血液学杂志》
CAS
CSCD
2002年第1期31-34,共4页
Journal of Experimental Hematology
