APO-1基因转染及其抑制大肠癌细胞生长的实验研究

Transfection and anti-tumor effects of APO-1 gene in colon carcinoma cells

目的建立表达外源性 APO-1基因的大肠癌细胞株,观察 APO-1表达细胞株在APO-1抗体作用下对体外培养的大肠癌细胞的抑制效应。方法采用分子克隆技术将 APO-1基因插入真核表达载体 pBK-CMV 的多克隆位点之间。以脂质体介导法将目的基因导入受体细胞 HT-29,用 G418筛选克隆细胞。以 Northern blot 检测转导细胞 APO-1 mRNA 的表达。MTI 法和直接记数法以及软琼脂集落形成实验检测转导株在 APO-1抗体作用下的细胞增殖水平、生长曲线及细胞克隆形成率。结果成功地构建了真核表达载体 pBK-CMV/APO-1 cDNA。转导细胞并经筛选后,获得了2株稳定的抗性细胞,从而建立了 APO-1基因表达株(HT-29 APO-1 cells)。杂交结果表明,转导株 APO-1mRNA 水平的表达显显高于非转导株。转导细胞增殖速度、倍增时间、对数生长期等均比非转导株更为缓慢和处于抑制状态,集落形成能力低下,但差异无显著性意义,而在 APO-1抗体作用下效果更为显著,差异有非常显著性意义。结论 APO-1基因在大肠癌细胞中处于低表达状态;通过真核表达载体的介导,APO-1基因导入大肠癌细胞后,能有效地表达 APO-1 mRNA。APO-1表达细胞株在 APO-1抗体的作用下可明显抑制体外培养的大肠癌细胞的生长增殖,其作用机制与APO-1诱导细胞凋亡有关。

objective To establish APO-l-expressing colon carcinoma cell line and to observe anti-APO-1 antibody inducing growth inhibition of APO-1-expressing colon carcinoma cell line in vitro. Method: HT-29 cell lines were transfected by eucaryotic expression vector pBK-CMV containing APO-1 gene using liposome transfection reagent, and selected by G418. APO-1 expression on transfected cells were detected with Northern blot. The level of cell proliferation, growth Curves and plating efficiency were assessed with MTT, direct count and colony formation on soft agar. Result: To construct eucaryotic expression vector pBK-CMV/APO-1 cDNA and to transfect into HT-29 cells successfully. After transfecfed cells were selected, 2 stably transfected cell lines were isolated. Therefore, APO-1-expressing colon carcinoma cell line(HT-29 APO-1 cells)was established. Hybridization results demonstrate that expression levels of APO-1 mRNA on transfected cells were much higher than those of non-transfected cells. The growth of transfected cells was inhibited. Proliferative rate, doubling time and logarithmic phase of growth were delayed and capability of colony formation was decreased, but there were no significant differences between transfected cells and non-transfected cell. Treated with anti-APO-1 antibody, this effect was more obvious, the difference was extremely significant. Conclusion: Expression of APO-1 in colon carcinoma [cells is low]Colon carcinoma cells transfected with eucaryotic expression vector can efficiently express APO-1 mRNA. Anti-APO-1 antibody can obvious inhibit growth and proliferation of APO-l-expressing colon carcinoma cells in vitro. A mechanistic explanation for this contributes to APO-1 inducing cell apoptosis.