c-FLIP反义寡核苷酸对肺癌A549细胞抑制作用

Effects of c-FLIP antisense oligodeoxynucleotides on growth and apoptosis of lung cancer cell line A549

目的探讨细胞型自杀相关因子(Fas)相关死亡域样白介素-1β转换酶抑制蛋白(c-FLIP)反义寡核苷酸(ASODN)对肺癌细胞系A549生长、凋亡的作用。方法设计合成c-FLIP ASODN,按不同浓度转染肺癌A549细胞,另设置空白对照组、脂质体转染组、无义寡核苷酸(NSODN)转染组,采用台盼蓝拒染法计数活细胞绘制细胞生长曲线,免疫组化链霉素抗生素过氧化物酶(SP)法检测细胞c-FLIP蛋白和Ki-67抗原表达、噻唑蓝法测定细胞增殖抑制率、流式细胞仪分析细胞凋亡情况。结果c-FLIPASODN转染A549细胞后,各ASODN组A549细胞生长受到抑制,随着ASODN浓度的增加,细胞增殖抑制率逐渐升高、c-FLIP蛋白和Ki-67抗原表达逐渐下降,而细胞凋亡率逐渐升高,与其他各组比较,差异有统计学意义(P<0.05)。而其余各组之间上述指标比较,差异无统计学意义(P>0.05)。结论c-FLIP ASODN能有效地干扰肺癌A549细胞c-FLIP基因表达,并可抑制癌细胞生长增殖,促进其凋亡,并且存在量效关系,有望成为治疗肺癌的基因药物。

Objective To study the effects of cellular Fas - associated death domain - like IL - 1 beta converting enzyme inhibitory protein （c- FLIP） antisense oligodeoxynucleotides （ASODN） on the growth and apoptosis of lung cancer cell line A549. Methods Synthesized ASODN of target c - FLIP was transfected to lung cancer cell line A549 at different concentration. Control group, liposome group, non sense oligonucleotide （NSODN） group were set for comparison. Living cells were counted by trypanblue method to draw cellular growth curve. Immunohistochemistry（SP） technique was also used to observe the expression of c - FLIP and Ki - 67 antigen in the transfected tumor cells. MTT and flow cytometric were used to analyze the cell growth inhibition rate and apoptosis rate. Results After treated with c - FLIP ASODN, the growth and caryocinesis of A549 cell were inhibited significantly, especially in 4ug/ml ASODN group. With the increasing of ASODN concentration the expression of c - FLIP and Ki - 67 antigen was fall - off. The apoptosis rate of A549 cells was increased. Statistically significant difference was found compared with the other groups（P 〈 0.05）. No statistically significant difference was found among the control group, liposome group and NSODN group（P 〉0.05）. Conclusion The c- FLIP ASODN could effectively inhibit the expression of c - FLIP gene and the growth of lung cancer cell line A549 and induce the apoptosis with a dose- effect relationship. It may be one of the promising medicine for antisense gene therapy in lung cancer.