CD95抑制大肠癌细胞生长的实验研究

目的建立表达外源性CD95基因的大肠癌细胞株,观察CD95表达细胞株在CD95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CD95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的HT-29,用G418筛选克隆细胞。以Northern blot,Western blot检测转导细胞CD95基因的表达。MTT法和直接记数法以及软琼脂集落形成实验检测转导株在CD95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/CD95 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CD95基因表达株(HT-29 CD95 cells)。杂交结果表明、转导株CD95mRNA及其蛋白水平的表达均明显高于非转导株,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CD95抗体作用下效果更为显著,差异有非常显著性意义。结论 CD95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,CD95基因导入大肠癌细胞后,能有效地表达CD95mRNA及其蛋白。CD95表达细胞株在CD95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CD95诱导细胞凋亡有关。

Anti-tumor effects of CD95 gene in colon carcinoma cells

To establish CD95-expressing colon carcinoma cell line and to observe anti-CD95 antibody inducing growth inhibiton of CD95-expressing colon carcinoma cell line in vitro. Method: HT-29 cell lines were transfected by eucaryotic expression vector pBK-CMV coutaining CD95 gene using liposome transfection reagent, and selected by G418. CD95 expression o transfected cells were detected with Northern blot and Western blot. The level of cell proliferation, growth Curves and plating efficiency were assessed with MTI, direct count and colony formation on soft agar. Result: To construct eucaryotic expression vector pBK-CMV/CD95 cDNA and to transfect into HT-29 cells successfully. After transfecfecl cells were selected, 2 stably transfected cell lines were isolated, therefore, CD95-expressing colon carcinoma cell line (HT-29 CD95 cells) was established. Hybridization results demonstrate thatexpression levels of CD95 mRNA and protein ontransfected 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 are no significant differences between transected cells and non-transfectedcell. Treatedwith anti CD95 antibody, this effect was more obvius, the difference was extremely significant.concluslon: ecpression of CD95 in colon carcinoma cells is low; Colon carcinorma cells transfectedwith eucaryotic expression vector can efficiently expness CD95 mRNA and protein. Anti-CD95 antibody can obvious inhibit growth and proliferation of CD95-expressing colon carcinoma cells in vitro. A mechanistic explanation for this contributes to CD95 inducing cell apoptosis.