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P〖STBX〗53〖STBZ〗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G1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 targeting proapoptotic effect of expression vector containing AFPregulation sequence on AFP positive hepatoma cells. Methods: AFP promoter, silencer and the most remote enhancer Ⅲ were ligated to construct a 12 kb AFP regulation sequence, which was then used to construct a pAFPEGFP plasmid. The pAFPEGFP was used to transfect human hepatoma HepG2 (AFP positive), human hepatoma SMMC7721 (AFP negative) and human cervical carcinoma HeLa (AFP negative) cells; the fluorescent protein expression intensities were observed under fluorescence microscope. A pAFPP53EGFP plasmid was further constructed by inserting P53 gene into pAFPEGFP, which was then transfected into HepG2, SMMC7721 and HeLa cells. P53 protein expressions were detected by Western blotting analysis in different groups; apoptosis rates and cell cycles were examined by flow cytometry. Results: pAFPEGFP and pAFPP53EGFP recombinant plasmids were successfully constructed. The expression of EGFP fluorescent protein in pAFPEGFPtransfected AFP positive HepG2 cells was significant higher than those in AFP negative SMMC7721 and HeLa cells; P53 protein expression in HepG2 cells transfected with pAFPP53EGFP was also significantly higher than those in SMMC7721 and HeLa cells. The G1 phase proportion and apoptosis rate of pAFPP53EGFPtransfected HepG2 cells were significantly higher than those of SMMC7721 and HeLa cells (\[66.7±0.25\]% 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), but S phase proportion of pAFPP53EGFPtransfected HepG2 cells was significantly lower than those of SMMC7721 and HeLa cells (\[20.1±022\]% vs \[29.8±0.18\]%, \[37.8±0.21\]%, P<0.05). Conclusion: The pAFPP53EGFP plasmid containing AFP regulation sequence can specifically target AFP positive hepatoma cells, inducing cell cycle arrest and apoptosis of hepatoma cells.

河北省科技支撑计划重点基金项目(No.09276426D)