p53C末端356～393氨基酸对人肺癌细胞恶性表型的影响

目的　研究去除C末端 3 56～ 3 93氨基酸p53对人肺癌细胞恶性增殖抑制的影响。方法利用DNA重组技术 ,构建去掉C末端 3 56～ 3 93区 3 7个氨基酸残基p53和全长p53真核细胞表达质粒[pEGFP p53 (del) ,pEGFP p53 ]。p53缺失和突变的人肺癌细胞系 80 1D为受体细胞 ,lipofectin介导质粒转染细胞。G418筛选 ,建立转染克隆细胞系。以PCR、荧光显微镜检查外源基因的表达 ;以集落形成和裸鼠移植瘤试验检测细胞体内外恶性增殖能力 ;以移植瘤或接种部位细胞团印片检查荧光蛋白表达。结果　建立了转染克隆细胞系pEGFP p53 80 1D、pEGFP p53 (del) 80 1D和pEGFP 80 1D ,证明有外源p53基因存在和外源绿色荧光蛋白基因表达。pEGFP p53 (del) 80 1D体外集落形成抑制率为 99.6% ,pEGFP p53 80 1D为 81.1% ,pEGFP p53 (del) 80 1D细胞恶性增殖能力明显低于pEGFP p53 80 1D (P <0 .0 1)。pEGFP p53 (del) 80 1D形成的少数集落也有外源p53基因和绿色荧光蛋白表达。裸鼠移植瘤试验显示 ,对照组 80 1D和pEGFP 80 1D移植瘤为阳性 (4/ 4,4/ 4) ,pEGFP p53 (del) 80 1D和pEGFP p53 80 1D移植瘤为阴性 ,接种细胞部位仍有残留细胞团存在 ,有少数表达荧光蛋白的活细胞。结论去除C末端 3 56～ 3 93氨基酸的p53 ,在体外可明显

Inhibitory effect of p53 with deletion of c-terminal 356 - 393 amino acids on malignant phenotype of human lung cancer cell line

Objective To study the effect of extraneous p53 gene with deletion of c-terminal 3 56-393 amino acids on inhibition of malignant phenotype of human lung cancer ce ll line. Methods Recombinant plasmid pEGFP-p53 (del) with codon deletion of c-ter minal 37 amino acids from 393 to 356 region and pEGFP-p53 (wild type) were cons t ructed. The human lung cancer cell line 801D served as a receipt cell had p53 d eletion and mutation at 248 codon. 801D cells, having been transfected by pEGFP-p53 (w ild type ), pEGFP-p53(del) or pEGFP, were selected by G418. Growing transfected cells wer e cloned respectively by method of dilution. Presence of extraneous gene was det ected by PCR, their expression in cells was examined by fluorescence microscopy . Cloning efficiency was in vitro tested to examine the cellular proliferating ab ility. The xenograft in nude mice was performed and xenograft tumors were weighe d one month later. Expression of GFP in tumor and transplanted cellular mass wer e detected by blot slices. Results pEGFP-p53(del)-801D, pEGFP-p53-801D and pEG FP-801D were established. Extraneous p53 gene and expression of GFP were found i n pEGFP-p53(del)-801D and pEGFP-p53-801D. Inhibitory rate of colony was 99. 6% f or pEGFP-p53(del)-801D and 81.0% for pEGFP-p53-801D. Inhibition of ma lignant pro liferation of extraneous p53(del) was higher than that of p53(wild type) (P <0.0 1 ). Even when inhibition of malignant proliferation extraneous pEGFP-p53(del) w as obvious, 0.2% colonies were formed, extraneous p53 and expression of GFP wer e observed. Animal test showed that tumor on the nude mice was positive (4/4, 4/ 4 ) in the control group (801D and pEGFP-801D), but negative (0/4, 0/4) in the exp eriment group [pEGFP-p53 (del) 801D and pEGFP-p53 (wild type) 801D]. Express ion of GFP in the cells of cellular mass transplanted by pEGFP-p53(del)801D or pEGFP-p53(wild type)801D was observed. Conclusion In vitro inhibitory effect of ex traneous p53 gene with deletion of C-terminal 356-393 amino acids on malignant g rowth of lung cancer cell with p53 mutation or deletion at 248 codon is marked. Inhibitory action of p53 on malignant proliferation of cancer cells is heterogen eous.