论文部分内容阅读
目的研究外源性ｐ２１ＷＡＦ１基因超表达对ＨＣＣ－９２０４肝癌细胞系的作用和意义。方法通过脂质体介导方式将带有人全长ｐ２１ＷＡＦ１ｃＤＮＡ和潮霉素抗性基因的真核表达载体ＰＣＥＰ转入肝癌细胞中，用潮霉素筛选后，酶联免疫吸附测定（ＥＬＩＳＡ）法检测ｐ２１蛋白的表达情况，使用流式细胞仪检测细胞周期并判定有无细胞凋亡及程度。通过透射电镜进一步观察细胞的超微结构。结果ＥＬＩＳＡ法测定ｐ２１蛋白表达结果，对照组、空载体组和ＷＡＦ１组的吸光度（Ａ）值分别为０．１７５±０．０２、０．１８３±０．０３和０．３３±０．０４，提示转基因后ｐ２１蛋白量却明显增加（Ｐ＜０．０５）。流式细胞仪检测细胞周期表明Ｇ１期细胞显著增加，并在Ｇ１期前出现一凋亡峰，凋亡细胞占细胞总数的２２．５％。透射电镜可见ＷＡＦ１组部分细胞体积缩小，细胞完整，有出芽现象，细胞器结构完整，致密的染色体边集于核膜内侧，呈明显的凋亡细胞的形态。结论本实验成功地将ｐ２１ＷＡＦ１基因转入到培养的肝细胞中，获得了稳定表达ｐ２１蛋白的细胞株；外源性ｐ２１ＷＡＦ１基因的超表达可引起肝癌细胞自发凋亡。通过对该基因的进一步研究可为肝癌防治提供理论依据。
Objective To study the effect and significance of exogenous p21WAF1 gene overexpression on HCC-9204 liver cancer cell line. METHODS: The eukaryotic expression vector PCEP carrying human full-length p21WAF1 cDNA and hygromycin resistance gene was transferred into hepatoma cells by liposome-mediated method. After screening with hygromycin, enzyme-linked immunosorbent assay (ELISA) was used to detect The expression of p21 protein was measured by flow cytometry to determine the cell cycle and the degree of apoptosis. The ultrastructure of the cells was further observed by transmission electron microscopy. Results The expression of p21 protein was determined by ELISA. The absorbance (A) values of the control, empty vector and WAF1 groups were 0.175±0.02, 0.183±0.03 and 0.33±0.04, respectively. It was suggested that the amount of p21 protein was significantly increased after transgene (P<0.05). Flow cytometry showed that the cells in G1 phase increased significantly, and apoptotic peak appeared before G1 phase. Apoptotic cells accounted for 22.5% of the total number of cells. Transmission electron microscopy revealed that some of the cells in the WAF1 group had reduced cell size, cell integrity, budding, and complete organelle structure. The dense chromosomal edge was located inside the nuclear membrane and showed a distinct apoptotic cell morphology. Conclusion In this experiment, p21WAF1 gene was successfully transferred into cultured hepatocytes, and stable cell lines expressing p21 protein were obtained. Overexpression of exogenous p21WAF1 gene can cause spontaneous apoptosis of hepatoma cells. The further study of this gene can provide a theoretical basis for the prevention and treatment of liver cancer.