关键词：肺肿瘤；细胞系；p53基因；转染；基因治疗
【摘要 】 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，且明显抑制转染细胞的恶性生长。
Effects ofexogenous wild type p53 on malignant growth of human lung cancer cell line
Wang Hui , Lai Baitang, Li Jinzhao, et al. Cellular and Molecular Biology Lab, Beijing Thoracic TumorResearch Institute. Beijing 101149
【Abstract 】 Objective To study the effects of exogenous wild type p53 suppressor gene onmalignant growths of human lung cancer cell line with mutant type p53 gene. Method Four human lung cancer cell line were screened formutations in the exon 5 through exon 8 of the p53 tumor suppressor gene with immunohistochemistry, polymerase chain reaction (PCR)/single strand conformationpolymorphism (SSCP) and DNA sequence analysis. The recombinent plasmid PZIPneoSV-p53 wasconstructed, which express wild type p53 gene. A transfected cell line, 801-D-p53, wasobtained after transferred the plasmid into 801-D cell line by gene gun mediated andselected by G418. The exogenous p53 in the transfected cell line 801-D-p53 were inspectedwith PCR, and the alteration of growths of the transfected cell line in vitro and in vivowas observed. Result The point mutationwere CGG to CTT transversion at codon 248 in exon 7 was found in human lung cancer cellline 801-D by PCR-SSCP and DNA sequence analysis and nuclear accumulation of the p53protein was observed. The neo gene and exogenous wild type p53 gene were detected in thetransfected cell line 801-D-p53. The cell growth experiment in vitro showed that theparent cell line 801-D growth was very fast, from 1
× 105 /mlto 2.5 × 105 /ml within 6 days, the transfected PZIP-neo-SV cellline (plasmid without p53) growth as fast as 801-D, but the transfected cell line801-D-p53 growth was inhibited seriously. The clonogenic formation rate of the parent cellline 801-D, transfected cell line 801-D-PZIP and the transfected cell line 801-D-p53 were11.2%, 11.4% and 0.46% respectively. The clononogenic formation inhibition rate of thetransfected 801-D-p53 was 96% comparing with the parent 801-D cell line. In the experimentof xenogenic tumor transplantation, each cell line was injected subcutaneously into fourmice and the growth of xenogenic tumor transplant in nude mice was observed. xenograftgrowth in all 4 mice in both 801-D and 801-D-PZIP groups, but only 1 xenograft growthamong 4 mice of 801-D-p53 group during 2 months. The average volume of xenogenic tumortransplant of 801-D and 801-D-PZIP groups were 6.500cm3 and 2.231 cm3 respectively and the only xenograft volume of 801-D-p53 group was 0.940 cm3 .The tumorigenicity of 801-D-p53 in nude mice was significantly suppressed. Conclusion Exogenous wild type p53 gene may stably exist in the humanlung cancer cell line with mutant type p53 after plasmid transfection and suppressed themalignant growth of the transfected cell line 801-D-p53 in vitro and in vivo. Theseresults indicate that the recombinent plasmid expressing wile type p53 may be useful forgene therapy of human lung cancer.......
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