反义端粒酶基因与顺铂诱导原代胃癌细胞凋亡作用的研究

The effect of apoptosis induced by telomerase antisense oligodeoxynucleotides and cisplatin in primary human gastric cancer cells

目的 探讨端粒酶反义寡核苷酸 (PS- ASODN)与顺铂 (DDP)联合应用对原代胃癌细胞凋亡作用的影响。方法 常规组织块培养法进行胃癌细胞原代纯化培养 ,取第 3代对数生长期细胞分六组进行实验。其中四组在培养 2 4小时及 4 8小时分别加入相同剂量的培养液 ,终浓度为 PS- ASODN3μM,N- ASODN3μM,DDP2 .0μg/ ml。作用 2 4小时后分别在 PS- ASODN组及 N- ASODN组加入终浓度为 2 .0 μg/ ml的 DDP;分别于培养后 2 4、4 8、72及 96小时收集各组细胞。以台盼蓝拒染法计算各组细胞生长抑制率 ,观察 PS- ASODN联合 DDP对原代胃癌细胞生长的影响 ;流式细胞学观察细胞凋亡率及细胞周期变化。结果 终浓度为 3μM的 PS- ASODN作用于原代胃癌细胞 2 4小时后加入 DDP 2 .0μg/ m l,能明显抑制胃癌细胞增殖 ,流式细胞学可检测到凋亡峰 ,细胞受阻于G0 / G1 期 ,作用 4 8及 72小时的凋亡细胞百分率 (35 .1%、4 5 .7% )明显高于 N- ASODN组、PS- ASODN组、DDP组及 N- ASODN+DDP组 ,差异有显著性 (P<0 .0 5 )。其作用呈时间依赖性及序列特异性。结论 以端粒酶 RNA模板区为靶点的 PS- ASODN可促进 DDP诱导的胃癌细胞凋亡 。

Objective To study the effect of apoptosis induced by telomerase antisense oligode- oxynucleotides(PS-ASODN) and cisplatin (DDP) in primary human gastric cancer cells.Methods Primary gastric cancer cells were purified by tissue culture method.The third generation cells were used in this experiment.After incubation for 24 hours and 48 hours,cells were treated with PS-ASODN of 3μM,N-ASODN of 3μM and DDP of 2.0μg/ml respectively.After incubation for 24 hours,DDP of 2.0μg/ml was added to group PS-ASODN and N-ASODN.Cells were continued to incubate for 48,72 and 96 hours.Inhibiting rate was determined by trypan blue.Flow cytometry was adopted to examine apoptotic rate and cell cycle.Results Treatment with DDP after 24 hours of exposure of cells to 3μM PS-ASODN inhibited significantly the growth of primary human gastric cancer cells.The apoptotic peak was detected with FCM and cells were blocked in G 0/G 1 stage.The percentage of apoptotic cells treated with DDP from 48 hours to 72 hours after 24 hours of exposure to PS-ASODN(35.1%,45.7%) was higher than that of cells treated with N-ASODN plus DDP or DDP alone (P<0.05).The effect was in time dependent and sequence specific manner.Conclusion PS-ASODN targeted hTR may increase the DDP-induced apoptosis of primary gastric cancer cells.Therefor it may be a new target to gastric carcinoma therapy.