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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
Objective: To establish an efficient and stable expression of p2 1 WAF1 / CIP1 colorectal cancer cell line HCT / 8-WAF1. Methods: pcDNA3 with the target gene WAF1 and blank pcDNA3 plasmid were transfected into HCT / 8 cell line by cationic liposome respectively. Positive clones were screened by G41 8, and the selected positive cells were subcultured continuously and immunized Fluorescence was used to monitor the expression of p2 1 WAF1 / CIP1. Results: After G41 8 screening, the blank control cells were all dead, and the cells transfected with the gene of interest and empty plasmid survived for more than 30 passages. HCT / 8 was selected as the control. Immunofluorescence staining was used to detect the expression of p2 1 WAF1 / The results of CIP1 showed that the positive rate of p21 WAF1 / CIP1 positive cells (89.96%) in HCT / 8 cells transfected with the target gene was significantly higher than that in the transfected empty plasmid group (8.02%) and blank control group (3.39%) (F = 1 6 60 8.99, P <0 01). There was no significant difference between passages (F = 2.71, P> 0.05). CONCLUSION: HCT / 8-WAF1 is a highly efficient and stably expressed p2 1 WAF1 / CIP1 cell line