外源性野生型p53基因对人肺癌细胞生长的抑制

Effects of exogenous wild type p53 on malignant growth of human lung cancer cell line

目的观察外源性野生型ｐ５３基因对有ｐ５３基因突变的人肺癌细胞系生长的影响。方法用多聚酶链反应单链构象多态性及ＤＮＡ测序，选择ｐ５３基因突变的人肺巨细胞癌系８０１Ｄ为受体细胞。构建野生型ｐ５３表达质粒ＰＺＩＰｎｅｏＳＶｐ５３。用基因枪介导外源基因。建立转染细胞系８０１Ｄｐ５３。用聚合酶链反应检测外源基因，观察转染细胞恶性生长的变化。结果转染细胞系８０１Ｄｐ５３体外长期传代有ｎｅｏ基因及外源ｐ５３基因存在，转染细胞生长明显受到抑制，克隆形成抑制率达９６％，裸鼠异种移植致瘤性降低，肿瘤生长明显缓慢。结论外源性野生型ｐ５３经基因枪导入有ｐ５３基因突变的人肺癌细胞后可长期存在于转染细胞中。

Objective To study the effects of exogenous wild type p53 suppressor gene on malignant growths of human lung cancer cell line with mutant type p53 gene. Method Four human lung cancer cell line were screened for mutations in the exon 5 through exon 8 of the p53 tumor suppressor gene with immunohistochemistry, polymerase chain reaction (PCR)/single strand conformation polymorphism (SSCP) and DNA sequence analysis. The recombinent plasmid PZIPneoSV p53 was constructed, which express wild type p53 gene. A transfected cell line, 801 D p53, was obtained after transferred the plasmid into 801 D cell line by gene gun mediated and selected by G418. The exogenous p53 in the transfected cell line 801 D p53 were inspected with PCR, and the alteration of growths of the transfected cell line in vitro and in vivo was observed. Result The point mutation were CGG to CTT transversion at codon 248 in exon 7 was found in human lung cancer cell line 801 D by PCR SSCP and DNA sequence analysis and nuclear accumulation of the p53 protein was observed. The neo gene and exogenous wild type p53 gene were detected in the transfected cell line 801 D p53. The cell growth experiment in vitro showed that the parent cell line 801 D growth was very fast, from 1×10 5/ml to 2 5×10 5/ml within 6 days, the transfected PZIP neo SV cell line (plasmid without p53) growth as fast as 801 D, but the transfected cell line 801 D p53 growth was inhibited seriously. The clonogenic formation rate of the parent cell line 801 D, transfected cell line 801 D PZIP and the transfected cell line 801 D p53 were 11.2%, 11.4% and 0.46% respectively. The clononogenic formation inhibition rate of the transfected 801 D p53 was 96% comparing with the parent 801 D cell line. In the experiment of xenogenic tumor transplantation, each cell line was injected subcutaneously into four mice and the growth of xenogenic tumor transplant in nude mice was observed. xenograft growth in all 4 mice in both 801 D and 801 D PZIP groups, but only 1 xenograft growth among 4 mice of 801 D p53 group during 2 months. The average volume of xenogenic tumor transplant of 801 D and 801 D PZIP groups were 6.500cm 3 and 2.231 cm 3 respectively and the only xenograft volume of 801 D p53 group was 0.940 cm 3. The tumorigenicity of 801 D p53 in nude mice was significantly suppressed. Conclusion Exogenous wild type p53 gene may stably exist in the human lung cancer cell line with mutant type p53 after plasmid transfection and suppressed the malignant growth of the transfected cell line 801 D p53 in vitro and in vivo. These results indicate that the recombinent plasmid expressing wile type p53 may be useful for gene therapy of human lung cancer.