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利用自行构建的稳定高表达Ｐ１５ＩＮＫ４Ｂ的人肝癌细胞，研究了抑癌基因Ｐ１５在细胞增殖中的作用．首先将抑癌基因 Ｐ１５的 ｃＤＮＡ构建到高效真核表达的质粒载体 ｐＸＪ４１－ｎｅｏ中的 ＥｃｏＲＩ／ＸｈｏＩ位点，构建成 Ｐ１５真核表达质粒 ｐＸＪＰ１５．通过脂质体法将 ｐＸＪＰ１５质粒转染人肝癌细胞ＳＭＭＣ－７７２１，进一步用 Ｇ－４１８筛选，获得了稳定高表达Ｐ１５的人肝癌细胞模型ＳＨＴ及相应的表达空载体的对照细胞模型ＳＶＸＪ．通过Ｎｏｒｔｈｅｒｎ，Ｗｅｓｔｅｒｎ分子杂交分析，表明ＳＨＴ细胞中Ｐ１５基因表达和蛋白水平都明显高于对照组细胞，证实成功地建立了Ｐ１５高表达的人肝癌细胞模型，与对照组相比，Ｐ１５高表达的ＳＨＴ１细胞的增殖受到抑制，流式细胞光度术和ＭＩ值测定表明Ｐ１５阻抑细胞由Ｇ１期向Ｓ期和由Ｇ２期向Ｍ期的转换．Ｗｅｓｔｅｒｎ免疫印迹分析结果表明，癌基因ｃ－ｍｙｃ，ｃ－ｆｏｓ的蛋白水平表达下降可能是Ｐ１５抑制细胞增殖的分子机理之一。
Using self-built human hepatoma cells stably expressing P15INK4B, the role of tumor suppressor gene P15 in cell proliferation was studied. First, the cDNA of tumor suppressor gene P15 was constructed into the EcoRI/XhoI site in highly efficient eukaryotic expression plasmid vector pXJ41-neo to construct the p15 eukaryotic expression plasmid pXJP15. The pXJP15 plasmid was transfected into human hepatocellular carcinoma cell line SMMC-7721 by liposome method, and further selected by G-418. The human hepatoma cell model SHT with stable expression of P15 and the corresponding control cell model SVXJ expressing empty vector were obtained. Northern and Western molecular hybridization analysis showed that the expression and protein level of P15 gene in SHT cells were significantly higher than those in the control group. It was confirmed that the P15-expressed human hepatocellular carcinoma cell model was successfully established. Compared with the control group, P15 was highly expressed. Proliferation of SHT1 cells was inhibited. Flow cytometry and MI measurements showed that P15 suppressor cells switched from G1 phase to S phase and from G2 phase to M phase. Western blot analysis showed that the decreased expression of c-myc and c-fos oncogene might be one of the molecular mechanisms of P15 inhibiting cell proliferation.