神经酰胺信号转导通路在胃泌素抑制大肠癌细胞凋亡中的作用及机制

Effects and mechanisms of ceramide signaling pathway in gastrin-inhibited cell apoptosis of large intestinal cancer

目的 探讨神经酰胺信号转导通路在胃泌素抑制大肠癌细胞凋亡中的作用及其机制.方法 胃泌素、丙谷胺各设5个浓度梯度,分别为6.25、12.50、25.00、50.00、100.00 mg/L和8.00、16.00、32.00、64.00、128.00 mg/L.运用噻唑蓝（MTT）法观察细胞增殖活力改变,确立5-肽胃泌素和丙谷胺处理HT-29细胞的最佳浓度；流式细胞仪逆转录-聚合酶链反应（RT-PCR）、Western blot法分别检测各组细胞凋亡率、神经酰胺、p38磷酸化水平、B淋巴细胞/白血病-2（bcl-2）、bcl-2相关X蛋白（bax） mRNA及其蛋白表达变化.结果 胃泌素能促进大肠癌HT-29细胞的增殖,并抑制凋亡,其最佳浓度为25.00 mg/L（F =31.36,P＜0.05）；丙谷胺能明显拮抗胃泌素促进HT-29细胞的增殖作用,其最佳浓度为32.00 mg/L（F =24.31,P＜0.05）.胃泌素组细胞凋亡率明显低于对照组和胃泌素＋丙谷胺组（q =4.23、4.06,P＜0.05）.胃泌素组bax mRNA、蛋白相对表达率以及神经酰胺、p38磷酸化水平明显低于对照组、丙谷胺组及胃泌素＋丙谷胺组（q=5.50、6.00、7.50;6.50、7.00、8.50;8.00、8.50、10.00;4.60、4.90、6.53,P＜0.05）,而bcl-2 mRNA、蛋白相对表达率与其相反（q =4.95、4.24、7.07;7.07、6.36、7.78,P＜0.05）.结论 胃泌素能够通过神经酰胺信号转导通路抑制大肠癌HT-29细胞凋亡,其可能是通过抑制p38活性、上调bcl-2及下调bax表达来实现的,但此信号传导通路能够被胃泌素受体拮抗剂丙谷胺阻断.

Objective To investigate the effects and mechanisms of ceramide signaling pathway in gastrin-inhibited cell apoptosis of large intestinal cancer.Methods Gastrin group and proglumide group were divided into five different densities with concentration gradient of gastrin and proglumide being 6.25,12.50,25.00,50.00,100.00 mg/L and 8.00,16.00,32.00,64.00,128.00 mg/L,respectively.The changes of proliferation of the HT-29 cells were detected by methyl thiazol tetrazolium （MTT） assay,and the optimal concentrations of gastrin and proglumide were determined.The changes of apoptosis rate of HT-29 cells were detected by using flow cytometry.The mRNA expression levels of gastrin receptor/cholecystokinin-B receptor （CCK-BR）,ceramide,p38,B lymphocytes/leukemia-2 （bcl-2） and bcl-2 associated X protein （bax） were detected by using reverse transcriptase-polymerase chain reaction （RT-PCR）.The protein expression levels of bcl-2,bax and ceramide,and p38 phosphorylation levels were detected by using Western blotting.Results Gastrin could promote the proliferation of HT-29 cells,and the optimal concentration was 25 mg/L （F =31.36,P ＜ 0.05）.Proglumide could significantly inhibit the proliferation of HT-29 cells stimulated by gastrin,and the optimal concentration was 32 mg/L （F =24.31,P ＜ 0.05）.The apoptosis rate of the gastrin group was significantly lower than in the control group and the gastrin ＋ proglumid group （q =4.23,4.06,P ＜0.05）.The mRNA and protein expression of bax and the levels of phosphorylated ceramide protein and phosphorylated p38 protein in the gastrin group were significantly lower than in the control group,the proglumid group,and the gastrin ＋ proglumid group （q =5.50,6.00,7.50; 6.50,7.00,8.50; 8.00,8.50,10.00; 4.60,4.90,6.53,P＜0.05）.On the contrary,the mRNA and protein expression of bcl-2 in the gastrin group was significantly higher than in the control group,the proglumid group,and the gastrin ＋ proglumid group （q =4.95,4.24,7.07; 7.07,6.36,7.78,P ＜ 0.05）.Conclusion Gastrin could inhibit the apoptosis of HT-29 cells by down-regulate the phosphorylation levels of ceramide and p38 protein,thus up-regulate bcl-2 and down-regulate bax through the ceramide signaling transduction pathway,while the effect can be restrained by gastrin receptor antagonist proglumide.