p27~(Kip1)诱导肝癌细胞凋亡的实验研究

目的:探讨过表达的p27Kip1对肝癌细胞的抑制作用。方法:利用脂质体介导法将真核表达载体pcDNA3.1/Myc-His(+)C-p27Kip1基因导入HHCC细胞中,用Westernblot检测p27Kip1是否导入到肝癌细胞中;用流式细胞仪检测过表达的p27Kip1对肝癌细胞HHCC的抑制作用;电子显微镜技术探讨过表达的p27Kip1抑制肝癌细胞生长的可能机制。结果:Westernblot检测表明,转染p27Kip1的HHCC细胞大部分有p27Kip1蛋白的表达;而转染空载体或未转染的HHCC细胞,均未见p27Kip1蛋白表达。经连续3次克隆化后,转染pcDNA3.1/Myc-His(+)C-p27Kip1的HHCC细胞100%表达p27Kip1蛋白。过表达的p27Kip1对HHCC细胞有明显的抑制作用,其抑制率为49.09%～56%;过表达的p27Kip1可以封闭G1期的进程和诱导肝癌细胞发生凋亡,其凋亡百分比为12.3%。结论:过表达的p27Kip1可以通过诱导肝癌细胞凋亡抑制肝癌细胞生长。

The apoptosis induced by overexpression of p27Kip1 on hepatocellar cells

Objective:To explore the inhibitory effects of overexpression p27Kip1 on hepatocellular cell.Methods:Plasmid mediation was used to transfer pcDNA3.1/Myc-His( )C-p27Kip1into HHCC cells;Western blot was used to detect whether p27Kip1 transferring into HHCC;The flowcytometry were used to determine inhibitory effects of overexpressed p27Kip1on HHCC.Possible mechanism of p27Kip1's inhibition of hepatocarcinoma cells was investigated by using flowcytometry and EM.Results:Western blot indicated that p27Kip1 protein was expressed in most of the cells transfected by pcDNA3.1/Myc-His( )C-p27Kip1,but there was no p27Kip1 protein signal in the pcDNA3.1/Myc-His( )C-transfected or non-transfected HHCC cells.The whole HHCC cells transfected by pcDNA3.1/Myc-His( )C-p27Kip1 expressed p27Kip1protein after cloning continually for 3 times.Overexpressed p27Kip1 had remarkable inhibitions on HHCC,inhibition rate is 49.09%~56%.Overexpressed p27Kip1could close the process of G1 and induce apoptosis of hepatocarcinoma cells,percentage of apoptosis is 12.3%.Conclusion:Overexpressed p27Kip1's inhibition on hepatocarcinoma is probably realized through inducing apoptosis of hepatocarcinoma cells.