稳定高效表达p21~(WAF1/CIP1)的HCT/8-WAF1细胞株的建立

目的 :建立高效、稳定表达 p2 1 WAF1/ CIP1大肠癌细胞株 HCT/8- WAF1。方法 :应用阳离子脂质体分别将带有目的基因 WAF1的 pc DNA3及空白 pc DNA3质粒转染大肠癌细胞株 HCT/8,经G41 8进行阳性筛选 ,将筛选的阳性细胞连续传代培养 ,应用免疫荧光技术监测 p2 1 WAF1/ CIP1的表达。结果 :经 G41 8筛选后 ,空白对照细胞全部死亡 ,转染目的基因及空质粒组的细胞存活 ,可连续传代培养 30代以上 ,并选择 HCT/8作对照 ;免疫荧光技术监测 p2 1 WAF1/ CIP1表达结果显示转染目的基因的 HCT/8细胞 p2 1 WAF1/ CIP1表达阳性细胞率 ( 89.96% )显著高于转染空质粒组 ( 8.0 2 % )和空白对照组 ( 3.39% ) ( F=1 6 60 8.99,P<0 .0 0 1 ) ;各代间差异无显著性 ( F=2 .1 71 ,P>0 .0 5 )。结论 :HCT/8- WAF1是一株高效、稳定表达 p2 1 WAF1/ CIP1的大肠癌细胞株

Establishment of Cell Line of HCT/8-WAF1 Expressing p21WAF1/CIP1 Efficently and Stably

Objective:To establish a cell line of HCT/8 WAF1 efficiently and stablely expressing p21 WAF1/CIP1 . Methods:The recombined vector, pcDNA3 WAF1, and vector pcDNA3 were transfected into HCT/8 cell line by lipofectin, respectively. These two cell line were selected by G418, and selected positive cells were subcultured. HCT/8 cell line was enclosed as control group. The expression of WAF1 was detected by immunofluorescence. Results: After selected by G418, control HCT/8 cell line was died, and transfected pcDNA3 WF1 and pcDNA3 cell lines were subcultured over 30 generations. The p21 WAF1/CIP1 expression in transfected pcDNA3 WAF1 was 88.96%, the results showed that recombined gene of WAF1 was more efficient expression than other two group ( F=16 608.99,P <0.001); There was not significantly difference in every generations( F=2.171,P >0.05). Conclusion:The cell line HCT/8 WAF1 expressing efficiently and stablely p21 WAF1/CIP1 is established.