目的:研究野生型p53（wtp53）基因对人胆囊癌细胞生长及致瘤性的影响。方法: 应用免疫细胞化学染色、PCR产物直接测序方法分析细胞系遗传背景，在脂质体介导下将含有wtp53的真核表达质粒pCMV-p53，导入GBC-SD细胞中。用G418筛选，建立转染克隆细胞系。以PCR，RT-PCR和蛋白印迹证实外源p53基因的整合与表达；以细胞生长曲线和集落形成实验反映细胞增殖状况；以裸鼠移植瘤试验检测体内致瘤性的影响。结果: GBC-SD细胞P53蛋白过表达;直接测序发现第5外显子126位密码子存在TAC→AAC的碱基突变。外源p53基因已整合入转染后的GBC-SD细胞并获稳定表达。表达外源wtp53的GBC-SD-wtp53细胞生长速率减慢、集落形成能力下降及裸鼠致瘤性受到显著抑制。结论: 野生型p53基因可有效抑制人胆囊癌GBC-SD细胞的体内、外生长。

Objective:To investigate effect of exogenous wild-type p53 gene on the cell growth and tumorigenicity of human gallbladder cancer cell lines.Methods:After identification of the genetic status of p53 gene of GBC-SD cell lines with the immunocytochemistry staining and the direct sequencing technique of PCR products, eukaryotic expressing plasmid pCMV-p53 was introduced by lipofectamine-mediated into GBC-SD cell lines. Growing transfected cells were selected by G418. The presence and expression of exogenous p53 gene was detected by PCR, RT-PCR and Western blot. The cellular proliferating ability was assessed using the cell growth curve and cloning assay. The xenograft in nude mice was performed to examine the effect of tumorigenicity.Results: P53 protein overexpression was showed in GBC-SD cell lines. A transversion of TAC→AAC at codon 126 of exon 5 was confirmed. PCR, RT-PCR and Western blot showed exogenous p53 gene had successfully transfected into GBC-SD cells and obtained high expression. The growth and proliferation of the cells were greatly decreased, and the tumorigenicity was significantly inhibited after transfection wtp53.Conclusion:The expression of exogenous wild-type p53 gene could effectively inhibit the growth of gallbladder cancer GBC-SD cells in vitro and in vivo.

山东省科技厅发展计划项目（2001BBICJA10）