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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 investigate the pro-apoptotic effect of pAFP-P53-EGFP on AFP positive hepatoma cells. Methods AFP gene regulatory sequence of 1.2 kb was combined with the AFP promoter, silencer and distal enhancer of SD rats, and then inserted into the promoter of pEGFP-N1 plasmid to construct pAFP-EGFP. At the same time, P53 gene fragment was constructed to construct pAFP- P53-EGFP recombinant plasmid. HepG2, SMMC7721 and HeLa cells were transfected, and the apoptosis of each cell was analyzed by flow cytometry. The plasmids pAFP-EGFP and pAFP-P53-EGFP were transfected into HepG2 cells for DNA ladder analysis and electron microscopy. Results The apoptosis rates of HepG2, SMMC7721 and HeLa cells transfected with pAFP-P53-EGFP recombinant plasmids were respectively:% gate = 2.65 ± 0.08 in HepG2 group;% gate = 0.42 ± 0.025 in HeLa group; ± 0.018. AFP-positive HepG2 cells apoptosis rate was significantly higher than SMMC7721, HeLa cells (P <0.05). The DNA ladder appeared in HepG2 cells transfected with plasmid pAFP-P53-EGFP, and the ultrastructure of cells showed apoptosis. Conclusion pAFP-P53-EGFP can express relatively specifically in AFP positive cells, and pAFP-P53-EGFP can inhibit tumor cells by inducing apoptosis.