p53反义RNA对人肺癌细胞表型和顺铂敏感性的影响

Effects of p53 antisense RNA on malignant phenotype and sensitivity to cisplatin of human lung cancer cell line

目的 研究外源p53反义RNA对有p53基因 2 4 8密码点突变的人肺癌细胞系恶性表型和顺铂敏感性的影响。方法 构建p53反义RNA真核细胞表达载体PEGFP p53(AS) ,经酶切图证明反向联结质粒。Lipofectin介导转染有p53基因 2 4 8密码点突变的人肺癌细胞系 80 1D ,经G41 8筛选获耐受克隆。稀释法建立单细胞克隆系 ,应用PCR检测外源基因 ,荧光显微镜检测细胞绿色荧光蛋白表达 ,p53单抗免疫组化染色检测p53突变蛋白表达 ,体外集落形成实验检测细胞生长 ,流式细胞仪检测细胞周期 ,MTT法检测细胞对顺铂药物敏感性。结果 酶切图证明了p53 cDNA反向联结于质粒 ,构建了PEGFP p53(AS) ,并建立了转染单细胞克隆系PEGFP p53(AS) 80 1D及空载细胞系PEGFP 80 1D。PCR检测外源p53基因和neo基因存在于转染细胞系 ,而荧光显微镜下发现其胞浆有绿荧光蛋白表达。免疫组化染色p53突变蛋白在 80 1D细胞系为阳性 ,而PEGFP p53(AS) 80 1D为阴性 ,证明外源反义p53在转染细胞稳定表达并封闭内源突变蛋白表达。与母系相比 ,PEG FP p53(AS) 80 1D集落形成抑制率为 61 % (P <0 .0 1 ) ,流式细胞仪检测该细胞系G1期细胞数明显增加 ,出现G1期阻滞的表现。MTT检测PEGFP p53(AS) 80 1D对顺铂比母系更为敏感。结论 有p53基因 2 4 8密码点?

Objective To study the effects of extraneous p53 antisense RNA on malignant growth and sensitivity to cisplatin of human lung cancer cell line. Methods 801D cell line with p53 deletion and mutation at 248 codon was selected as a parent cell line. An 1.8 kb human p53 full length cDNA was inserted into a mammalian expression vector PEGFP to construct a p53 antisense RNA recombined plasmid PEGFP p53(AS) and GFP gene at plasmid was a report gene to monitor extraneous gene expression. The extraneous gene was detected by PCR. The p53 mutation protein was examined by immunohitochemical stain of p53 monoclonal antibody. The inhibition growth efficacy of extraneous p53 in vitro was determined by clonogenic survival assay. Sensitivity of cells to cisplatin was examined with MTT assay. FCM analysis was performed to measure the effect of p53 antisense RNA on cell cycle. Results Two cell lines, PEGFP p53(AS) 801D and PEGFP 801D, were established after transfection of 801 D cells by lipofection and selection. Presence of extraneous p53 gene in PEGFP p53(AS) 801D was proved by PCR and expression of extraneous p53 was estimated when green fluorescence in those cells was found out under the fluorescent microscopy. Mutated p53 protein in parent cell line 801D was positive and in PEGFP p53(AS) 801D was negative with immunochemical stain. The inhibition rate of colony formation was 61% for PEGFP p53(AS) 801D (P<0.001). The sensitivity of PEGFP p53(AS) 801D cells to cisplatin was increased. FCM analysis showed that the cell line was arrested at G1 phase. Conclusion p53 mutation at 248 code plays an important role on malignant growth and resistance to cisplatin of human lung cancer cell line 801D. Malignant growth of cells with p53 deletion and mutation at 248 codon can be inhibited by extraneous p53 antisense RNA, and simultaneously the sensitivity to cisplatin is also increased.