摘要
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
Objective: To clone p14 ARF gene from cells of the human normal liver cell line L02 and explore its inhibitory effect on tumor cell proliferation. Methods: p14 ARF cDNA was cloned from L02 cells by using RT-PCR method and inserted into the BamHI and EcoRI sites of pcDNA3 (called pcDNA3ARF). Lipofectamine mediated pcDNA3ARF was transferred into tumor cell lines, such as Hep2, KB, KB-v200, HepG2, HLE, Huh7, MCF7 and MCF7/ADR. The number of colonies of these tumor cell lines were calculated after more than two weeks' screening in DMEM medium containing G418. Results:The colony-forming efficiency of the p14ARF -transfected KB-v200 and MCF7/ADR drug-resistant cells was 13±2.0 and 21±1.5 , respectively, compared with 86±3.1(15%)and 126±7.5(17%)in the cells transfected with empty vector pcDNA3, and in the p53 positive tumor cell lines, such as HepG2(47%), Hep2(57%), MCF7(58%) cells, the inhibition was also found,whereas in the p53 negative tumor cell lines, such as KB(74%), HLE(65%), Huh-7(80%) cells, the inhibitory effect was milder. Conclusions: p14 ARF can suppress the growth of the p53 positive tumor cell lines in a p53-relevant manner, while in the p53 negative, drug-resistant tumor cells, p14 ARF has strong inhibitory effect, indicating the inhibitory effect of p14 ARF on growth of drug-resistant tumor cells is independent on p53, at least in KB-v200 and MCF7/ADR drug-resistant cells.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2000年第2期104-107,共4页
Chinese Journal of Cancer
