【摘要】 人p53基因是一种抑癌基因,p53蛋白作为一种细胞增殖的负调节因子,调控细胞的生长和分化,维持基因组DNA的稳定,其突变、缺失或与细胞、病毒、肿瘤蛋白结合所致的p53失功能,在恶性肿瘤发生发展中起着非常重要的作用.为了进一步探讨野生型p53基因的抗肿瘤特性,我们采用分子克隆方法将人野生型p53基因(WT-p53)cDNA全长正向插入到哺乳动物表达载体(pREP9)的BamHl位点,构建了pREP9-p53重组体,用电穿孔方法将其导入有p53基因突变的人肠癌SW1116细胞,通过标记基因Neo~R筛选带外源p53基因的G418抗药性克隆,以观察抗药性克隆数量,同时对G418抗药性细胞的生长速度及细胞增殖周期进行观察.结果表明,导入WT-p53基因能使肠癌SW1116细胞的G418抗药性克隆形成减少,细胞生长速度较对照细胞明显减慢,细胞增殖周期GI期增加、S期下降.这些结果证明人野生型p53基因能抑制肠癌细胞的生长.本研究为大肠癌WT-p53实验性基因治疗提供了理论依据,说明基于恢复P53肿瘤抑制基因功能的基因治疗在治疗结、直肠癌方面有着广阔的应用前景.更多还原

【Abstract】 p53 gene is a 16-20 kb of cellular DNA located on the short arm of human chromosome 17 at position 17pl3.1. This gene encodes a 393-amino acid nuclear phosphoprotein which involves in the regulation of cell proliferation. Loss of normal p53 function is associated with the cell transformation in vitro and the development of neoplasms in vivo. More than one-half of human malignancies were shown to contain an altered p53 gene. Most p53 gene alterations are the missense mutations, giving rise to an altered protein. The inactivation of wild-type p53 is currently regarded as an important genetic pathway for haman carcinogenesis generated by endogenous factors and exogenous carcinogens, as well as several tumor viruses. To gain more insight into the functional role of wild-type p53 in human colo-rectal carcinoma, a 2. 1 kilobase human wild-type p53 cDNA with 5’ and 3’ untranslated sequences was cloned into the BamHI site of pREP9 (episomal mammalian expression vector) in sense orientation. We performed experiments to transfer wild-type p53 into human colon adenocarcinoma cell line (SW1116) harboring mutant p53 genes with electroporation method. We assessed G4I8-resistant clonal growth, cell growth properties and cell cycle pattern by flow cytometry. The results demonstrated that human wild type p53 gene can suppress the phenotype of SW1116 cell line. So gene therapy based on restoration of the defective or mutant p53 function plays an important role in colo-rectal cancer treatment.更多还原