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目的:观察含AFP基因调控序列的载体对AFP阳性肝癌细胞的靶向致凋亡作用。方法:将AFP启动子、沉默子和远端增强子Ⅲ组合为1.2 kb的AFP基因调控序列,构建pAFP-EGFP载体,分别转染人肝癌HepG2(AFP阳性)、人肝癌SMMC7721(AFP阴性)和人宫颈癌HeLa(AFP阴性)细胞,荧光显微镜下观察EGFP荧光蛋白表达强度。引入P53基因片段,构建pAFP-P53-EGFP重组质粒,转染HepG2、SMMC7721和HeLa细胞,Western blotting检测各组细胞P53蛋白的表达,流式细胞术分析各组细胞凋亡率及细胞周期。结果:成功构建了pAFP-EGFP和pAFP-P53-EGFP重组质粒。pAFP-EGFP转染后,AFP阳性的HepG2细胞中EGFP荧光蛋白表达显著高于AFP阴性的SMMC7721和HeLa细胞。pAFP-P53-EGFP转染后,HepG2细胞中P53蛋白的表达量明显高于SMMC7721和HeLa细胞;HepG2细胞的G1期细胞及细胞凋亡率明显高于SMMC7721和HeLa细胞[(66.7±0.25)%vs(50.5±0.18)%,(51.0±0.20)%,P<0.05;(2.65±0.08)%vs(0.42±0.03)%,(0.39±0.02)%,P<0.05],但S期细胞明显低于转染后SMMC7721和HeLa细胞[(20.1±0.22)%vs(29.8±0.18)%,(37.8±0.21)%,P<0.05]。结论:含AFP基因调控序列的pAFP-P53-EGFP载体可专一性地作用于AFP阳性肝癌细胞,引起肝癌细胞周期阻滞和凋亡。
OBJECTIVE: To observe the target-induced apoptosis of AFP positive hepatocarcinoma cells with AFP gene regulatory vector. Methods: AFP promoter, silencer and distal enhancer Ⅲ were combined into 1.2 kb AFP gene regulatory sequence to construct pAFP-EGFP vector. The recombinant plasmids were transfected into human hepatocellular carcinoma HepG2 (AFP positive), human hepatoma SMMC7721 (AFP negative) and Human cervical carcinoma HeLa (AFP negative) cells were observed under a fluorescence microscope for the expression of EGFP fluorescent protein. P53 gene fragment was introduced to construct pAFP-P53-EGFP recombinant plasmids. HepG2, SMMC7721 and HeLa cells were transfected with pAFP-P53-EGFP. The expression of P53 protein was detected by Western blotting. The apoptosis rate and cell cycle were analyzed by flow cytometry. Results: The recombinant plasmids pAFP-EGFP and pAFP-P53-EGFP were successfully constructed. After transfection with pAFP-EGFP, EGFP fluorescent protein expression in AFP-positive HepG2 cells was significantly higher than that in AFP-negative SMMC7721 and HeLa cells. After transfection with pAFP-P53-EGFP, the expression of P53 protein in HepG2 cells was significantly higher than that in SMMC7721 and HeLa cells; the rate of G1 phase cells and apoptosis in HepG2 cells was significantly higher than that in SMMC7721 and HeLa cells [(66.7 ± 0.25)%) (50.5 ± 0.18)%, (51.0 ± 0.20)%, P <0.05; (2.65 ± 0.08)% vs (0.42 ± 0.03)%, (0.39 ± 0.02)%, P <0.05] (20.1 ± 0.22)% vs (29.8 ± 0.18)%, (37.8 ± 0.21)%, P <0.05] after transfected SMMC7721 and HeLa cells. CONCLUSION: The pAFP-P53-EGFP vector containing AFP gene regulatory sequence can specifically act on AFP positive hepatoma cells, causing cell cycle arrest and apoptosis of hepatoma cells.