γ-氨基丁酸及其A型受体在胃癌SGC-7901细胞的表达及对细胞增殖的影响

Expression of GABA and GABRP in gastric cancer SGC-7901 cells and its effect on cell migration ability

目的观察GABA、GAD65、GABRP在胃癌SGC-7901细胞中的表达及对细胞增殖、细胞周期分布的影响。方法 RT-PCR、IF及Western Blot检测胃癌细胞SGC-7901中GABA、GAD65及GABRP mRNA及蛋白表达;1、10、100μmol/L GABA,0.5、5、50μmol/L Muscimol,0.5、5、50μmol/L Bicucullin分别作用于胃癌细胞SGC-7901 48 h后,新型四唑氮盐(MTS)比色法检测细胞增殖情况,流式细胞仪检测细胞周期分布情况。结果 GAD65、GABRP mRNA及蛋白均表达于SGC-7901细胞中;GABA、GABRP及GAD65蛋白定位于SGC-7901细胞细胞膜和细胞质;与空白组比较,1μmol/LGABA,5、50μmol/L Muscimol作用SGC-7901细胞48 h后,细胞生长的增殖率上升,5、50μmol/L Bicucullin作用SGC-7901细胞48 h后,细胞生长抑制率上升;1μmol/L GABA可导致细胞G0/G1数量的减少,G2/M期数量的增加;3个浓度的Muscimol可导致G0/G1期数量的减少,5、50μmol/L Muscimol可导致细胞G2/M期数量增加;5、50μmol/L Bicucullin可导致G2/M期数量的减少(P<0.05)。结论 GABA及其A受体在SGC-7901细胞中的表达可能促进细胞增殖,扰乱细胞周期进程,促使细胞分裂。

Objective To investigate the expression of γ-aminobutyric acid（GABA）,it＇s a receptor（GABAAR） and L-glutamate decarboxylase 65（GAD65） in human gastric cancer SGC-7901 cells and their effects on cell proliferation and cell cycle proportion.Methods RT-PCR,Western blotting and immunofluorescent staining were used to detect the expression of GABA,GAD65 and GABAAR in SGC-7901 cells.SGC-7901 cells were also cultured for 48 h with 1,10 or 100 μmol/L GABA,0.5,5 or 50 μmol/L Muscimol（GABAAR agonist）,and 0.5,5 or 50 μmol/L Bicucullin（GABAAR antagonist） respectively.MTS assay was used to examine cell proliferation.Flow cytometry was used to analyze cell cycle distribution.Results The mRNA and protein of GABA,GAD65 and GABRP were all expressed in SGC-7901 cells.GABA,GAD65 and GABRP protein level were predominantly localized in the cell cytoplasm of SGC-7901.Compared with blank group,the cells proliferation were obviously stimulated after 1 μmol/L GABA and 5 and 50 μmol/L Muscimol treatment,but significantly inhibited after 5 and 50 μmol/L Bicucullin treatment.Flow cytometry analysis showed that the percentage of cells in G0/G1 phase were decreased while the cells in G2/M phase was increased after 1 μmol/L GABA treatment.Different concentrations of Muscimol reduced G0/G1 phase cell percentage,but both of 5 and 50 μmol/L concentrations of Muscimol elevated G2/M phase cell percentage.Bicucullin at 5 and 50 μmol/L resulted in reduction of the percentage of G2/M phase cells（P0.05）.Conclusion Higher expression of GABA and GABAAR in SGC-7901 cells may promote cell proliferation,interfere cell cycle proceeding,and accelerate cell division.