pAFP-P53-EGFP靶向诱导AFP阳性肝癌细胞凋亡

Targeting Pro-apoptotic effect of pAFP-P53-EGFP on AFP positive hepatoma Cells

目的研究pAFP-P53-EGFP对AFP阳性肝癌细胞的靶向促凋亡作用。方法将SD大鼠AFP启动子、沉默子和远端增强子Ⅲ组合为1.2kb的AFP基因调控序列,导入pEGFP-N1质粒的启动子处,构建pAFP-EGFP同时引入P53基因片段,构建pAFP-P53-EGFP重组质粒。转染HepG2、SMMC7721、HeLa细胞,流式细胞术分析各细胞凋亡情况。分别将质粒pAFP-EGFP和pAFP-P53-EGFP转染HepG2细胞进行DNA ladder分析及电镜观察细胞超微结构。结果 pAFP-P53-EGFP重组质粒转染HepG2、SMMC7721、HeLa细胞,各组细胞凋亡率分别为:HepG2组%gate=2.65±0.08;HeLa组%gate=0.42±0.025;SMMC7721组%gate=0.39±0.018。AFP阳性的HepG2细胞凋亡率明显高于SMMC7721、HeLa细胞(P<0.05)。且转染pAFP-P53-EGFP质粒的HepG2细胞出现明显的DNA ladder,细胞超微结构亦显示出现凋亡。结论 pAFP-P53-EGFP可以在AFP阳性细胞中相对专一表达,且pAFP-P53-EGFP可通过诱导细胞凋亡从而达到抑制肿瘤作用。

Objective To study the targeting pro-apoptotic effect of pAFP-P53-EGFP on AFP positive hepatoma cells.Method The AFP promoter,silencer and the most remote enhancer Ⅲ of rat were ligated to construct a gene expression regulation and acted as the promoter of pEGFP-N1 plasmid.Meanwhile,the tumor suppressive gene P53 was inserted in the downstream of the regulatory sequence,and constructed pAFP-P53-EGFP plasmid.The pAFP-P53-EGFP was transfected into HepG2,SMMC7721,HeLa cells,apoptosis of the cells were examined through flow cytometry（FCM）.The pAFP-EGFP and pAFP-P53-EGFP were transfected into HepG2 for DNA ladder analysis and electron microscope observation.Results The pAFP-P53-EGFP had transfected into HepG2,SMMC7721,HeLa cells,and the rate of apoptosis of HepG2,HeLa,and SMMC7721 were 2.65±0.08,0.42±0.025 and 0.39±0.018,the rate of apoptosis HepG2 cells was higher than SMMC7721,HeLa cells.DNA ladder and apoptosis of the cells can be found in HepG2 cells transfected with pAFP-P53-EGFP.Conclusions The plasmid pAFP-P53-EGFP can express in AFP positive cells specifically.Furthermore,pAFP-P53-EGFP can inhibit tumor through inducing the AFP positive cells to apoptosis.Result The expression of green fluorescent protein in HepG2 transfected pAFP-P53-EGFP was significant higher than in the other two kinds of cells.