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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。
Objective To establish a colorectal cancer cell line expressing exogenous CD95 gene and observe the inhibitory effect of CD95-expressing cell line on the colorectal cancer cells cultured in vitro. Methods The molecular cloning technique was used to insert the CD95 gene into the multiple cloning sites of eukaryotic expression vector pBK-CMV. The target gene was introduced into the HT-29 of the recipient cells by liposome-mediated method, and the cloned cells were selected using G418. Northern blot and Western blot were used to detect the expression of CD95 gene in transduced cells. MTT assay and direct counting method and soft agar colony formation assay were used to test the cell proliferation, growth curve and cell colony formation rate of transduced strains under the effect of CD95 antibody. Results The eukaryotic expression vector pBK-CMV/CD95 cDNA was successfully constructed. After transducing cells and screening, two stable resistant cells were obtained, and a CD95 gene-expressing strain (HT-29 CD95 cells) was established. The results of hybridization showed that the expression levels of CD95 mRNA and protein in transduced strains were significantly higher than those of nontransduced strains. The proliferation rate, doubling time, and logarithmic growth period of transduced cells were all slower and inhibited than nontransduced strains. The colony forming ability was low, but the difference was not significant. However, the effect was more significant under the effect of CD95 antibody, and the difference was very significant. Conclusion The CD95 gene is in a low expression state in colorectal cancer cells. Through the eukaryotic expression vector, CD95 gene can effectively express CD95 mRNA and its protein after being introduced into colorectal cancer cells. The CD95-expressing cell strain can significantly inhibit the growth and proliferation of colorectal cancer cells in vitro under the action of CD95 antibody, and its mechanism of action is related to the apoptosis induced by CD95.