p53缺失和突变对人肺癌细胞系恶性表型影响的比较  

Study on the effects of p53 deletion and mutation on malignant phenotype of human lung cancer cell line

作　　者：汪惠[1] 赖百塘[1] 李金照[2] 杨学惠[1] 张春彦[1] 岳文涛[1] 张洪涛[1] 李曦[1] 湛秀萍[1] 王 

机构地区：[1]北京市结核病胸部肿瘤研究所细胞分子生物学实验室,101149 [2]中科院生物物理所

出　　处：《中国肺癌杂志》2002年第4期245-249,共5页Chinese Journal of Lung Cancer

摘　　要：目的　比较研究外源正义和反义 p5 3对所转染细胞系恶性表型的影响。 方法　构建正义和反义 p5 3cDNA真核细胞表达载体 pEGFP p5 3 (RS)和 pEGFP p5 3 (AS) ,用Lipofectin介导转染 80 1D细胞。PCR检测外源 p5 3和neo基因。荧光显微镜检查转染细胞绿荧光蛋白。免疫组化染色检测突变蛋白表达。比较pEGFP p5 3 (AS) 80 1D和 pEGFP p5 3 (RS) 80 1D的集落形成试验和裸鼠移植试验。用流式细胞术分析细胞周期。结果　PCR检测出外源 p5 3和neo基因存在于细胞 ,细胞可见绿色荧光。免疫组化检测示pEGFP p5 3(AS) 80 1D突变蛋白呈阴性 ,母系为阳性 ,表明反义 p5 3能封闭突变p5 3蛋白表达。pEGFP p5 3 (RS) 80 1D和pEGFP p5 3 (AS) 80 1D细胞集落形成率和裸鼠移植成瘤性均降低 ,pEGFP p5 3 (RS) 80 1D更为明显。pEGFP p5 3 (AS) 80 1D细胞周期阻滞于G1期。结论　在同一细胞背景下 ,p5 3缺失比p5 3突变对恶性增殖起更重要的作用。外源野生型p5 3在肿瘤细胞中可恢复重建其功能 ,外源反义 p5 3可封闭突变蛋白表达 ,阻止细胞停留于G1期。Objective To study the inhibition effects of both extraneous right sense and antisense p53 on malignant phenotype of human lung cancer cell line. Methods The named 801D cell line with p53 deletion and mutation at 248 code was selected as a model in vitro. The recombined plasmid pEGFP p53(RS) and pEGFP p53(AS) were constructed. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohistochemical stain of p53 antibody. The inhibition effect of extraneous p53 on tumor growth in vitro were determined by clonogenic survival assay. FCM analysis was carried out in cells. The inhibition effect on malignant growth of extraneous p53 in vivo was observed by heteroplastic transplant on nude mouse. Results The transfected cell lines, pEGFP p53(AS) 801D, pEGFP p53(RS) 801D and pEGFP 801D were established. Presence of extraneous p53 and neo genes in pEGFP p53(AS) 801D and pEGFP p53(RS) 801D was proved by PCR and green fluorescence was found out in those cells under the microscope. Mutant protein in pEGFP p53(AS) 801D was negative by immunohistochemical stain. The malignant growth of these transfected cell lines was inhibited comparing with parents in vivo and in vitro. Inhibition rate of colony formation was 62.0% for pEGFP p53(AS) 801D and 80.8% for pEGFP p53(RS) 801D. The tumorigenicity in nude mice was suppressed. Inhibition effects of extraneous right sense p53 on malignant growth of 801D was more distinct. FCM analysis showed that pEGFP p53(AS) 801D cells were arrested at G1 phase. Conclusion The transfected cell lines with extraneous right sense and antisense p53 appear that malignant growth can be inhibited in vivo and in vitro.

关 键 词：肺癌细胞系 恶性表型 P53基因 转染细胞 集落形成 基因缺失 基因突变 流式细胞术 

分 类 号：R734.2[医药卫生—肿瘤]