CD40-CD40配体对胃癌细胞AGS增殖和侵袭作用研究

CD40-CD40 ligand on proliferation and invasion of human gastric cancer cell line AGS

目的探讨CD40-CD40配体(CD40-CD40L)信号通路在胃癌细胞AGS细胞增殖和侵袭过程中的作用。方法细胞培养分成对照组(仅加入含有10%FBS的F12培养基)、sCD40L组、LY294002组和sCD40L+LY294002组。通过酶联免疫吸附实验(enyme linked immunosorbent assay,ELISA)测定细胞上清中血管内皮生长因子(vascular endothelial growth factor,VEGF)的浓度、细胞计数和氚[3H]标记的胸腺嘧啶核苷渗入法测定观察细胞生长和增殖的情况,Transwell实验观察LY294002抗对sCD40L影响GAS胃癌细胞侵袭的作用。蛋白质印迹法检测细胞PI3K、p-Akt蛋白表达水平。结果胃癌细胞AGS表达较高水平CD40,sCD40L组的细胞计数为(3.8±0.3)×105,较对照组(5.3±0.4)×105明显减少,差异有统计学意义,P=0.007。sCD40L组VEGF浓度为(161.3±28.04)pg/mL,与对照组(48.67±6.03)pg/mL比较明显增高,差异有统计学意义,P=0.002;LY294002+sCD40L组VEGF的浓度为(60.67±8.02)pg/mL,明显低于对照组的(161.3±28.04)pg/mL,P=0.004。sCD40L组的刺激指数为(0.82±0.16),明显高于LY294002+sCD40L组的刺激指数(0.56±0.15),差异有统计学意义,P=0.046。对照组中每个视野平均细胞数为(72.67±5.76)个,明显高于sCD40L组的(46.67±2.53)个,差异有统计学意义,P=0.002;LY294002+sCD40L组平均每个视野细胞为(8.67±2.52)个,较sCD40L组的(46.67±2.53)个明显较少,差异有统计学意义,P<0.001;与对照组相比,sCD40L使PI3K、p-Akt蛋白表达升高,与LY294002联合作用后,PI3K、p-Akt蛋白表达明显降低,P<0.05。结论CD40-CD40L相互作用抑制胃癌细胞增殖及胃癌的侵袭,同时阻断PI3K-AKT信号通路,可以增强sCD40L对胃癌AGS增殖和侵袭的抑制作用。

OBJECTIVE To investigate the role of CD40-CD40 ligand interaction on proliferation and invasion of the gastric cancer cell line AGS. METHODS We treated AGS cells with soluble CD40 ligand（sCD40L） with or without in- hibitor of phosphatidylinositol 3-kinase（LY294002）,and 10% FBS/F12 medium was used as control. We determined the growth effects by cell counts or [3H]-thymidine incorporation assay. VEGF levels in cell-free supernatant were detected using enzymelinked immunosorbent assays and the ability of cellular invasion using Transwell invasion assay. The protein expression of PI3K,p-Akt was tested by western blotting. RESULTS There were high levels of CD40 in surface of gas- tric cell line AGS, the AGS cell counts of control group and sCD40L group （5.3±0.4） × 105 and （3.8±0.3） × 10^5 ,with significant difference （P= 0. 007）; VEGF secretions of control group, sCD40L group and LY294002-k sCD40L group were （48.67±6.03） pg/mL, （161.3±28.04） pg/mL and （60. 67±8.02） pg/mL, with significant difference （P〈 0.05）. Stimulation indexes of GAS cells in sCD40L group and LY294002＋ sCD40L group were （0. 82 ± 0. 16） and （0.56±0.15）, with significant difference （P〈0.05）. The average ceil numbers in each microscopic field of control group, sCD40L group and LY294002＋ sCD40L group were （72.67±5.76）, （46.67±2.53） and （8.67±2.52）, with significant difference （P〈0.05）. The protein expression of PI3K, p-Akt increased in cells treated with sCD40L, but re- duced drastically in cells treated with sCD40L plus LY294002,P〈0.05. CONCLUSION CD40-CD40L interaction could inhibit AGS cell line proliferation and invasion, inhibit PI3K-AKT signaling pathway and enhance the inhibitory effect of proliferation and invasion by sCD40L on AGS cells.