三氧化二砷联合Ad-IκBαM诱导胃癌细胞凋亡的研究

A study on the apoptosis of gastric carcinoma cells induced by arsenic .trioxide combined with Ad-IκBαM

目的研究三氧化二砷(As_2O_3)对胃癌 SGC-7901细胞作用过程中细胞内核转录因子kB(NF-kB)的激活及重组腺病毒 Ad-IKBαM通过抑制 NF-kB 的活化增强 As_2O_3的诱导凋亡作用。方法培养胃癌 SGC-7901细胞,以非感染组及感染 Ad-IkBα组为对照,采用凝胶电泳迁移率实验(EMSA)及免疫组化法检测 As_2O_3处理后细胞核内 NF-kB 的激活情况,以及感染 Ad-IkBαM对 NF-kB活性的影响;四甲基偶氮唑盐(MTT)法、Hoechest 染色、原位末端标记(TUNEL)法分别检测感染Ad-IkBαM对 As_2O_3诱导细胞凋亡的影响。结果 EMSA 及免疫组化法显示 As_2O_3作用于胃癌细胞可使细胞内 NF-kB 激活,感染 Ad-IkBαM使 NF-kB 活性受到明显抑制;MTT 法证明,As_2O_3作用后,感染 Ad-IkBαM细胞的凋亡率(59.2±2.5)%较感染 Ad-IkBα组(47.5±2.3)%及未感染组(40.0±1.2)%明显升高,各组间比较 P<0.01;Hoechest 法显示,感染 Ad-IKBαM组的凋亡率为(27.7±2.6)%,明显高于感染 Ad-IkBα组(18.3±1.5)%及未感染组(11.0±1.7)%(P<0.05)。TUNEL 法结果与 Hoechest 法一致,感染 Ad-IkBαM组的凋亡率为(31.1±2.5)%,高于感染 Ad-IkBα组(20.7±2.1)%及未感染组(13.0±1.7)%(P<0.05)。可见,感染 Ad-IkBαM可明显提高 As_2O_3诱导的细胞凋亡。结论 As_2O_3作用于胃癌细胞可使细胞内 NF-kB 激活,从而表明 NF-kB 激活可能为胃癌细胞抗诱导凋亡作用的重要机制;感染 Ad-IKBαM可有效抑制 NF-kB 的活性,并增强 As_2O_3的诱导凋亡作用。

Objective To study the activation of nuclear facter -κB （NF-κB） in gastric carcinoma SGC-7901 cells after treating with As2O3 and the enhancement of the therapeutic effect of As2O3 with recombinant adenovirus IκBαM. Methods Culture of gastric carcinoma SGC-7901 cells was carried out. The cells both uninfected and infected with Ad-IκBα but not treated with As2O3 were used as control. Electrophoretic mobility shift assay （EMSA） and immunohistochemistry were used to detect the activation of NF-κB in the cells after treatment of As2O3 and the combination with Ad-IκBαM. MTT, Hoechest staining and TUNEL were used to assay the change of apoptosis induced by As2O3 after infection with Ad-IκBαM. Results The results of EMSA and immunohistochemical method showed that after the treatment of As2O3 the cells showed high activity of NF-κB. Simultaneous infection with Ad-IκBαM can inhibit the activation of NF-κB; MTT method indicated that after the treatment of As2 O3 infected with Ad-IκBαM apoptosis rate of the cells （59. 2± 2. 5） % was higher than that of the cells treated with As2 O3 and infected with Ad-IκBα but not treated with As2O3 （47.5 ±2. 3）% and these neither infected nor treated （40. 0 ±1.2% ）, P 〈0. 01. The result of Hoechest staining method indicated that, in the group of cells treated with As2O3 and infected with Ad-IκBαM, apoptosis rate is （27.7 ±2.6）%, which was higher than the that of the cells infected with Ad-IκBα（ 18.3 ± 1.5 ） % but not treated with As2 O3 and these neither infected nor treated （ 11.0 ± 1.7% ）, P 〈0. 05. Hoechest staining method was in accordance with TUNEL technique; it was shown that in the group of cells treated with As2O3 and infected with Ad-IκBαM, apoptosis rate was （31.1 ±2. 5）%, being still higher than that of cells infected with Ad-IκBα but not treated with A2O3 （ 20. 7 ± 2. 1 ） % and these neither infected nor treated （13.0 ± 1.7）%, P〈0.01. Therefore, infection with Ad-IκBαM can significantly increase the apoptosis induced by As2O3. Conclusions Gastric carcinoma cells treated with As2O3 show activity of NF-κB. It is indicated that the activity of NF-κB may be the mechanism of the antagonism of gastric carcinoma cells against the apoptosis induced by As2O3. Infection with Ad-IκBαM can effectively inhibit the activation of NF-KB in gastric carcinoma cells and increase the cell apoptosis induced by A2O3.