凝溶胶蛋白对胃癌细胞凋亡及细胞中活性氧水平的影响研究

Effect of GSN on apoptosis and ROS level in gastric cancer cells

摘要:

目的探讨凝溶胶蛋白（GSN）对胃癌细胞凋亡及细胞中活性氧（ROS）水平的影响。方法免疫印迹法（Western blot）检测胃癌细胞SGC7901、AGS、BGC823 及胃黏膜上皮细胞GES-1 中GSN的表达水平。细胞转染GSN siRNA（GSN siRNA 组）和siRNA 对照（siRNA control 组），同时以只加入转染试剂的细胞为对照组，培养48 h 后，噻唑蓝（MTT）检测细胞增殖，流式细胞术检测细胞凋亡，激光扫描共聚焦显微镜法检测ROS水平，Western blot检测细胞中GSN、Notch1 胞内段受体（NICD1）、Notch2 胞内段受体（NICD2）、DNA 结合蛋白1（HES1）多克隆抗体及活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）水平。结果GSN在 胃癌细胞SGC7901、AGS 及BGC823 中的表达水平高于胃黏膜上皮细胞GES-1（p <0.05），且在AGS 细胞中的表达水平最高，后期选用胃癌细胞AGS 为研究对象。siRNA control 组细胞增殖、凋亡及细胞中ROS 水平、GSN、NICD1、NICD2、HES1 及Cleaved Caspase-3 水平与对照组相比均差异无统计学意义（p >0.05）。GSN siRNA 组细胞增殖能力及细胞中GSN、NICD1、NICD2 及HES1 水平低于对照组（p <0.05），而细胞凋亡能力及细胞中ROS 水平、Cleaved Caspase-3 水平均高于对照组（p <0.05）。结论干扰GSN 表达后，胃癌细胞增殖能力减弱，凋亡增加，作用机制可能与细胞中ROS 水平及NOTCH信号通路有关。

Abstract:

Objective To investigate the effect of GSN on the apoptosis and the level of ROS in gastric cancer cells. Methods The expression of GSN in gastric cancer cell lines SGC7901, AGS, BGC823 and gastric epithelial cells GES-1 were detected by Western blot. Cells were transfected with GSN siRNA (the GSN siRNA group), siRNA control (the siRNA control group) and transfection vehicle (the vehicle control group), respectively. Cell proliferation was determined by MTT assay. Cell apoptosis was determined by flow cytometry. ROS level was measured by laser scanning confocal microscopy. The levels of GSN,NICD1,NICD2, HES1, and cleaved Caspase-3 were detected by Western blot. Results Expression level of GSN in gastric cancer cell lines including SGC7901, AGS and BGC823 was significantly higher than that in gastric epithelial cells GES -1 (p < 0.05). Given that AGS witnessed the highest expression of GSN, following experiments were performed on it. There was no significant difference in cell proliferation, apoptosis, ROS,GSN, NICD1, NICD2, HES1 or cleaved Caspase-3 level between the siRNA control group and the vehicle control group (p > 0.05). Cell proliferation and the levels of GSN, NICD1, NICD2, and HES1 in the GSN siRNA group were dramatically lower than those in the control group (p < 0.05), while cell apoptosis and the levels of ROS and cleaved Caspase-3 were notably higher than those in the vehicle control group (p < 0.05). Conclusions GSN decreases proliferation ability while increases apoptosis of gastric cancer cells through manipulation on ROS level and NOTCH signaling pathway.