可溶性CD40配体联合LY294002对胃癌细胞生长的抑制作用

Inhibitory effect of soluble CD40 ligand combined with LY294002 on growth of gastric cancer cells

目的 观察可溶性CD40配体（sCD40L）联合磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路特异性阻断剂LY294002对胃癌AGS细胞体外生长及凋亡的影响,并探讨其作用机制.方法 将细胞分成对照组（仅加入含10％胎牛血清的RPMI 1640培养液）、LY294002组（20 μmol/L）、sCD40L组（2 mg/L）和LY294002＋ sCD40L组.噻唑蓝（MTT）检测细胞增殖变化;流式细胞术观察细胞周期和凋亡变化;透射电镜检测细胞形态变化;Western blot检测细胞PI3K、磷酸化蛋白激酶B（p-Akt）蛋白表达的变化.结果 与单独使用sCD40L或LY294002比较,sCD40L与LY294002联合应用后细胞生存率显著降低,LY294002组、sCD40L组及联合用药组细胞生存率分别为（65.72±3.19）％、（70.14±2.98）％、（41.25±2.74）％（P＜0.05）.LY294002及sCD40L单独作用胃癌AGS细胞48 h后均可使细胞周期分布发生明显改变,细胞周期阻滞于G1期,两者联合作用比单独用药周期阻滞明显增强（P＜0.05）.对照组、sCD40L组、LY294002组、联合用药组细胞凋亡率分别为（4.10±0.86）％、（4.23±0.74）％、（8.97±1.23）％、（29.32±2.37）％,与对照组比较,sCD40L组细胞没有明显的凋亡,LY294002组细胞有一定的凋亡,两者联合作用后细胞凋亡明显增多（P＜0.05）.与对照组比较,sCD40L促进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 阻断PDK/Akt信号通路可显著增强sCD40L对人胃癌AGS细胞生长的抑制作用.

Objective To investigate the effects of soluble CD40 ligand combined with LY294002,a specific inhibitor of phosphatidylinositol 3 kinase （PI3K）,on the proliferation and apoptosis of gastric cancer cells （AGS）,and its potential mechanisms.Methods The effect of gradient concentration of soluble CD40 ligand （sCD40L） and its combination with LY294002 on cellular proliferation was determined by using methyl thiazol tetrazolium （MTT） assay.The cellular cycle and apoptosis were evaluated by flow cytometry （FCM）.The cellular morphologic change was detected by election microscope.The protein expression of PI3K and p-protein kinase B （p-Akt） was tested by using Western blotting.Results The inhibitory rate of cellular proliferation in LY294002,sCD40L and LY294002 ＋ sCD40L groups was （65.72 ±3.19）％,（70.14 ±2.98）％ and （41.25 ±2.74）％ respectively （P ＜0.05）.The cellular cycle arrest was increased in cells treated with sCD40L plus LY294002 when compared with those treated with sCD40L or LY294002 alone （P ＜ 0.05）.LY294002 could increase cellular apoptosis with the apoptosis rate being （8.97 ± 1.23） ％,and sCD40L did not obviously increase cellular apoptosis with the apoptosis rate bing （4.23 ± 0.74）％.As compared with LY294002 alone,the rate of cellular apoptosis was increased obviously after being treated by LY294002 in combination with sCD40L with the apoptosis rate being （29.32 ±2.37）％ （P ＜0.05）.The protein expression of PI3K and p-Akt was increased in cells treated with sCD40L,but reduced drastically in those treated with sCD40L plus LY294002 （P ＜ 0.05）.Conclusion Inhibiting PI3K/Akt signaling pathway can enhance the inhibitory effect of proliferation by sCD40L on AGS,which may provide a new way for biochemical therapy of gastric cancer.