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目的 研究p2 7KIP1 基因对肝癌细胞的细胞周期及细胞增殖的影响。方法 构建一种含 0 .6kbp2 7KIP1 cDNA的可诱导性真核表达载体pMD KIP1 ,通过外加 1 0 0 μmol/LZn离子诱导目的基因的表达。脂质体转染法将 p2 7KIP1 cDNA转入肝癌细胞系HCC 92 0 4中 ,免疫印迹分析以及逆转录 聚合酶链反应 (RT PCR)检测 p2 7KIP1 基因在蛋白质和mRNA水平的表达 ;细胞活力实验显示转染p2 7KIP1 基因对细胞增殖的作用 ;流式细胞仪观察目的基因对该细胞的细胞周期的影响。结果 转染p2 7KIP1 的HCC 92 0 4细胞在mRNA和蛋白质水平均有高水平p2 7KIP1 的表达。细胞活力检测显示在外加Zn离子 48h后细胞生长被抑制 35 % ;p2 7KIP1 的过表达能够显著地增加G1 期的细胞数 ,由 35 .2 1 %增加到 68.2 8% (P <0 .0 1 )。结论 本研究中所制备的可诱导性真核表达载体pMD KIP1能有效介导 p2 7KIP1 基因在HCC 92 0 4细胞中的高表达 ,并使HCC 92 0 4细胞的G1 期延长 ,从而抑制细胞增殖
Objective To study the effect of p2 7KIP1 gene on the cell cycle and cell proliferation of hepatoma cells. Methods An inducible eukaryotic expression vector pMD KIP1 containing 0.6kbp7 7KIP1 cDNA was constructed and the expression of the target gene was induced by addition of 1000 μmol / L Zn2 +. The p2 7KIP1 cDNA was transfected into hepatocellular carcinoma cell line HCC 92 04 by lipofectamine. The expression of p2 7KIP1 gene at mRNA and protein level was detected by Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) The effect of transfected p2 7KIP1 gene on cell proliferation was observed. The effect of the target gene on the cell cycle was observed by flow cytometry. Results HCC 92 0 4 cells transfected with p2 7KIP1 expressed high levels of p2 7KIP1 at mRNA and protein levels. Cell viability assay showed that cell growth was inhibited by 35% after 48 hours with Zn 2+ addition; overexpression of p2 7KIP1 significantly increased the number of cells in G1 phase from 35.21% to 68.28% (P <0.01) ). Conclusion The inducible eukaryotic expression vector pMD KIP1 prepared in this study can effectively mediate the high expression of p2 7KIP1 gene in HCC 92 0 4 cells and prolong the G1 phase of HCC 92 0 4 cells and thus inhibit cell proliferation