CAJ | 학술논문

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.
목적 탐토신경선알신호전도통로재위비소억제대장암세포조망중적작용급기궤제.방법 위비소、병곡알각설5개농도제도,분별위6.25、12.50、25.00、50.00、100.00 mg/L화8.00、16.00、32.00、64.00、128.00 mg/L.운용새서람(MTT)법관찰세포증식활력개변,학립5-태위비소화병곡알처리HT-29세포적최가농도；류식세포의역전록-취합매련반응(RT-PCR)、Western blot법분별검측각조세포조망솔、신경선알、p38린산화수평、B림파세포/백혈병-2(bcl-2)、bcl-2상관X단백(bax) mRNA급기단백표체변화.결과 위비소능촉진대장암HT-29세포적증식,병억제조망,기최가농도위25.00 mg/L(F =31.36,P＜0.05)；병곡알능명현길항위비소촉진HT-29세포적증식작용,기최가농도위32.00 mg/L(F =24.31,P＜0.05).위비소조세포조망솔명현저우대조조화위비소+병곡알조(q =4.23、4.06,P＜0.05).위비소조bax mRNA、단백상대표체솔이급신경선알、p38린산화수평명현저우대조조、병곡알조급위비소+병곡알조(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),이bcl-2 mRNA、단백상대표체솔여기상반(q =4.95、4.24、7.07;7.07、6.36、7.78,P＜0.05).결론 위비소능구통과신경선알신호전도통로억제대장암HT-29세포조망,기가능시통과억제p38활성、상조bcl-2급하조bax표체래실현적,단차신호전도통로능구피위비소수체길항제병곡알조단.
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.