腺病毒介导的TRAIL基因诱导腺样囊性癌细胞凋亡研究

TRAIL gene induce apoptosis of adenoid cystic carcinoma cell line mediated by adenovirus vector

目的:探讨以凋亡基因TRAIL为靶基因,腺病毒(Ad)为载体的重组基因治疗载体AdCMV-TRAIL诱导腺样囊性癌细胞系ACC-2凋亡的功能。方法:用携带绿色荧光蛋白基因EGFP的AdCMV-EGFP检测腺病毒载体对ACC-2的转染效率;将携带TRAIL基因的AdCMV-TRAIL转染ACC-2;应用RT-PCR方法检测转染后TRAIL基因表达;应用倒置显微镜和荧光倒置显微镜观察细胞形态,MTT方法检测细胞存活率,应用流式细胞仪检测肿瘤细胞的凋亡率。结果:AdCMV-EGFP在1000p/cell时转染效率为100%;RT-PCR在实验组检测到594bp的TRAIL基因;AdCMV-TRAIL组和AdCMV-EGFP组的细胞存活率随着时间逐渐下降,且转染AdCMV-TRAIL的肿瘤细胞比转染AdCMV-EGFP的肿瘤细胞下降快,统计学分析有显著差异(P<0.001)。AdCMV-TRAIL和AdCMV-EGFP均能促进腺样囊性癌细胞凋亡,且转染AdCMV-TRAIL引发细胞凋亡的程度明显较AdCMV-EGFP组大,统计学分析有非常显著的差异(P=0.0005)。结论:Ad-CMV-TRAIL在体外能明显抑制ACC-2细胞的活性,有效促使其凋亡;AdCMV-TRAIL可以成为腺样囊性癌转基因治疗的新策略。

Objective：To study the effects of TRAIL（ TNF-related apoptosis inducing ligand） gene on the adenoid cystic carcinoma cell line ACC-2 mediated by adenovirus vector. Methods： ACC-2 cells were infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene EGFP. Then the transducing rate were investigated through fluorescence detection. After ACC-2 cells were infected with AdCMV-TRAIL in proper titre, TRAIL gene was detected by means of RT-PCR. The proliferation ability of ACC-2 cells was evaluated by MTT, and the apoptosis was detected with flow cytometer at day 1,3,5,7. Results： The ideal titre of infection was 1 000 p/cell, and ACC-2 cells were infected 100% in this titre. TRAIL gene could be detected by RT-PCR after ACC-2 cells were infected by AdCMV-TRAIL. The proliferation ability of ACC-2 cells decreased in both groups, but the proliferation ability of AdCMV-TRAIL group decreased more quickly than AdCMV-EGFP group, and there was statistic difference （P 〈 0.001 ）. Both of two recombinated adenovirus led to apoptosis of ACC-2 cells, especially AdCMV-TRAIL function stronger than AdCMV-EGFP, and there was statistically difference （ P = 0.005 ）. Conclusion ： AdCMV-TRAIL could decrease the proliferation ability of ACC-2 cells, and cause apoptosis of ACC- 2 cells. AdCMV-TRAIL can be a potential gene therapy agent for adenoid cystic carcinoma.