佛波酯处理人胃癌MGC80-3细胞过程中磷酯酶C-γ2的意义

THE IMPLICATION OF PLC-γ2 IN GASTRIC CANCER MGC80-3 CELLS TREATED BY TPA

目的探讨佛波酯(TPA)处理人胃癌MGC80-3细胞过程中磷酯酶Cγ-2(PLCγ-2)的意义。方法通过DAPI染色,荧光显微镜观察分析TPA对胃癌MGC80-3细胞的影响;借助核浆分离手段获得胃癌细胞核浆蛋白,并通过免疫印迹(Western blotting)检测TPA对PLCγ-2蛋白表达水平影响;通过免疫荧光技术处理,激光扫描共焦显微镜观察胃癌细胞内PLCγ-2蛋白的定位和转运;用PLCγ-2的抑制剂(U73122)预处理细胞,免疫印迹和激光扫描共焦显微镜分别检测其对TPA作用胃癌细胞内PLCγ-2的影响;以及荧光显微镜下观察分析U73122是否影响TPA对胃癌细胞的作用。结果TPA诱导胃癌MGC80-3细胞凋亡;同时TPA提高PLCγ-2蛋白表达水平,并诱导其发生核浆转运;其抑制剂(U73122)可以降低TPA对PLCγ-2蛋白表达水平的作用,但并不能影响TPA诱导胃癌细胞凋亡;而TPA诱导的PLCγ-2核浆转运却没有被PLCγ-2抑制剂(U73122)阻止。结论尽管TPA提高了胃癌MGC80-3细胞中PLCγ-2表达水平,但PLCγ-2表达水平和TPA诱导的胃癌MGC80-3凋亡没有直接关系,而其核浆转运可能与TPA诱导的胃癌细胞凋亡相关。

Objective To study the implication of phosphoinositide-specific phospholipase Cγ2（PLC-γ2） in gastric cancer MGC80-3 cells treated by TPA. Methods The effect of 12-O-tetradecanoylphorbol-1, 3-acetate（TPA） on gastric cancer MGC80-3 cells was detected and analyzed by DAPI staining under fluorescence microscope. The effect of TPA on the expression level of PLC-γ2 protein was detected by Western blotting, when nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation. Localization and translocation of PLC-γ2 were observed under laser scanning confocal microscope with immune- fluorescence technique. After MGC80-3 c ells were pretreated by U73122 （ PLC- 72 inhibitor ）, the effect of TPA on PLC- 72 was detected by Western blotting and laser scanning confocal microscope with immunefluorescence technique, and the effect of U73122 on MGC80-3 cells followed by TPA was observed and analyzed by DAPI staining under fluorescence microscope. Results TPA induced the apoptosis of gastric cancer MGC80-3 cells. Meanwhile, TPA increased the expression level of PLC-γ2 protein and induced its translocation from cytoplasm to nucleus. U73122 inhibited the effect of TPA on the expression level of PLC-γ2 protein, but it did not affect on the cell apoptosis induced by TPA. However, the translocation of PLC-γ2 protein induced by TPA was not inhibited by its inhibitor（U73122）.Conclusion Although TPA could increase the expression level of PLC-γ2 protein, it is not directly related to the cell apoptosis induced by TPA. However, its translocation might be related with the apoptosis of gastric cancer.