幽门螺杆菌对人胃上皮细胞β-catenin蛋白信号途径影响意义的研究

Effect of Helicobacter pylori onβ-catenin signaling pathway and its biological significance in human gastric epithelial cells

目的:探讨幽门螺杆菌(Helicobacter pylori,H.pylori)裂解液对人胃上皮GES-1细胞β-catenin蛋白信号途径及其下游靶基因Cyclin D1和c-myc表达,以及对细胞增殖的影响。方法:用H.pylori超声裂解液处理GES-1,免疫荧光法和蛋白质印迹法检测β-catenin蛋白亚细胞定位以及核内蛋白表达量的变化;荧光定量聚合酶链反应(quantitative polymerase chain reaction,QRT-PCR)检测Wnt/β-catenin蛋白信号下游靶基因Cyclin D1和c-myc基因mRNA表达水平的变化;MTT法检测细胞的增殖。结果:H.pylori裂解液处理人胃上皮GES-1细胞后,H.pylori处理组引起了β-catenin蛋白从细胞膜至细胞质以及细胞核内的移位,呈时间依赖性。蛋白质印迹法检测结果证实,与对照组相比,H.pylori处理组在12、24h分别引起了细胞核内β-catenin蛋白表达量的增加,分别为0.31±0.05和0.55±0.04,P值均<0.01。QRT-PCR检测发现,与对照组相比,H.pylori处理组12hc-myc基因表达量没有变化,F=0.338,P>0.05;而24h的表达量明显增高,F=39.290,P<0.01;H.pylori处理组12hCyclin D1基因表达量明显增高,F=72.567,P<0.01;而24h的表达量虽然增高,但是差异无统计学意义,F=2.368,P=0.175;且明显低于12h的表达量,F=37.468,P<0.01。MTT法检测显示,5μL的H.pylori裂解液处理细胞12、24和36h的增殖抑制率分别为-13.3%、-28.5%和-62.5%;10μL的分别为-24.1%、-58.0%和-87.9%;20μL的分别为-39.8%、-131.3%和-165.2%。结论:H.pylori裂解液异常激活了β-catenin蛋白信号途径,促进β-catenin蛋白向细胞核内移位,通过上调其下游靶基因cyclin D1和c-myc的mRNA表达,促进细胞增殖,其效应呈时间以及浓度依赖。

OBJECTIVE：To investigate the effect of Helicobacter pylori （H. pylori） ultrasonic lysate on β-catenin signaling pathway, downstream target gene Cyelin D1, c-myc,and cell proliferation in human gastric epithelial cells GES-1. METHODS： The subcellular localization of β-catenin protein was detected by immunofluorescence method after human gastric epithelial cells GES-1 were treated with H. pylori ultrasonic lysate,and the expression of β-catenin protein in nu- cleus was detected by Western blotting. The mRNA expression of Cyclin D1 and c-myc was detected by fluorescent quanti- tative polymerase chain reaction （QRT-PCR）, proliferative potential of GES-1 cells were estimated by MTT method. RE- SULTS： The translocation of β-catenin protein from cell membrane into cytoplasm and nucleus was observed in H. pylori treatment group by aser scanning confocal microscope,which presented in a time-dependent manner. Compared to control group,the expression of β-catenin protein in nucleus in H. pylori treatment group was significantly increased after 12 hand 24 h, respectively （12 h, 0.31 ±0.05, P〈0.01〈24 h, 0.55 〈 0.04, P〈0.01 ）. The expression of c-myc gene showed no significant increase after 12 h treated with H. pylori ultrasonic lysate （F= 0. 338, P〉0. 05）, but significantly increased after 24 hs （F=39. 290,P〈0.01）. The expression of Cyclin D1 gene was significantly increased after treated with H. py- lori ultrasonic lysate for 12 hs （F=72. 567,P〈0.01）,however,compared to control group,the expression of Cyclin D1 gene showed no statistical difference after 24 h （F=2. 368,P〉0.05）,and which was significantly lower than that after 12 h （F=37. 468,P〈0.01）. After 12 h, the inhibition rate of cell proliferation in human gastric epithelial ceils GES-1 treated with H. pylori ultrasonic lysate in concentration of 5,10 and 20 μL respectively were -13.3%,-28.5% and -62. 5% ;after 24 h,they were -24. 1%,-58.0% and -87.9%;after 36hs, - 39. 8% , -131. 3% and -165. 2%. CONCLUSION： H. pylori ultrasonic Iysate could activat β-catenin signaling pathway abnormally, promot β-catenin protein translocation into the nucleus, and induc cell proliferation by up-regulation the mRNA levels of the downstream target genes Cyclin D1 and c-myc in a time-dependent and concentration-dependent manner.