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目的 :从正常人肝细胞系L0 2细胞中扩增人的肿瘤抑制基因 p14 ARF,以研究其对人的多种肿瘤细胞生长的影响。方法 :应用RT -PCR技术 ,从培养的正常人肝细胞系L0 2细胞中扩增出 0 .4Kb的p14 ARFcDNA片段 ,测序正确后插入真核表达载体 pcDNA3上的BamHI和EcoRI位点 ,命名为 pcDNA3ARF。此质粒由脂质体Lipofec tamineTM介导转染肿瘤细胞Hep2、KB、KB -v2 0 0、HepG2、HLE、Huh7以及MCF7和MCF7/ADR ,经G4 18筛选 2周后统计形成的集落数。结果 :经抗生素G4 18筛选 2周后 ,发现p5 3阳性的肿瘤细胞HepG2、Hep2、MCF7形成的集落数较少 ,分别为 5 9± 2 .1,60± 2 .1和 66± 7.0 ,占各自对照组的 4 7% ,5 7% ,5 8% ;而 p5 3异常的肿瘤细胞KB ,HLE ,Huh - 7,则形成相对较多的集落 ,分别为 91± 2 .5 ,4 8± 2 .0 ,4 1± 2 .0 ,占各自对照组的 74 % ,65 % ,80 % ,但是二种p5 3异常的耐药肿瘤细胞株KB -v2 0 0和MCF7/ADR ,形成的集落数最少 ,分别为 13± 2 .0和 2 1± 1.5 ,占各自转染空载体 pcDNA3的对照组的 15 %和 17%。 结论 :p14 ARF能明显抑制 p5 3阳性肿瘤细胞的生长 ,具有 p5 3相关性 ;然而 p14 ARF对 p5 3阴性的耐药肿瘤细胞却具有强烈的抑制作用 ,由此提示 :p14 ARF抑制耐药肿瘤细胞的生长是 p5 3非依赖性的
Objective : To amplify human tumor suppressor gene p14 ARF from normal human liver cell line L02 cells in order to study its effect on human tumor cell growth. METHODS: RT-PCR technique was used to amplify a 0.4 Kb p14 ARF cDNA fragment from the cultured normal human hepatocyte cell line L02. After sequencing, it was inserted into the BamHI and EcoRI sites of eukaryotic expression vector pcDNA3. pcDNA3ARF. This plasmid was transfected with tumor cells Hep2, KB, KB-v200, HepG2, HLE, Huh7, MCF7 and MCF7/ADR by Lipofectin Lipofec tamineTM. The number of colonies formed was counted after 2 weeks of G418 screening. RESULTS: After two weeks of antibiotic G4 18 screening, the number of colonies formed by HepG2, Hep2, and MCF7 tumor cells that were positive for p53 was found to be 5 9 ± 2.1, 60 ± 2.1, and 66 ± 7.0, respectively. 47%, 57%, 58% of the control group; while the p53 abnormal tumor cells KB, HLE, Huh-7, formed a relatively large number of colonies, which were 91 ± 2 .5 and 48% respectively. 2 .0, 4 1±2. 0, accounting for 74%, 65%, 80% of the respective control group, but two p53-resistant tumor cell lines KB-v200 and MCF7/ADR, forming colonies The numbers were the lowest, 13 ± 2.0 and 21 ± 1.5, respectively, which accounted for 15% and 17% of the control group transfected with empty vector pcDNA3, respectively. Conclusion: p14 ARF can significantly inhibit the growth of p53-positive tumor cells and has a p53 correlation; however, p14 ARF has a strong inhibitory effect on p53-negative drug-resistant tumor cells, suggesting that p14 ARF inhibits drug-resistant tumors Cell growth is p53 independent