晚期糖基化终产物对结肠癌干细胞增殖及凋亡的影响研究

Effects of advanced glycation end products on proliferation and apoptosis in colon cancer stem cells

目的探讨晚期糖基化终产物(AGEs)对结肠癌干细胞AGEs受体(RAGE)表达及增殖和凋亡的影响。方法以人SW480结肠癌干细胞为研究对象,不同浓度AGEs及50μmol/L PD98059、40mmol/L二甲双胍(MET)干预结肠癌干细胞后,分别应用RT-PCR和Western blot检测24h细胞RAGE mRNA和RAGE、细胞外信号调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)蛋白表达;CCK-8检测24h细胞增殖;流式细胞术检测细胞凋亡。结果应用不同浓度(100、200、300μg/ml)AGEs干预后,与Con组(未加AGEs)比较,200μg/ml AGEs组RAGE mRNA和蛋白表达均增加,(p-ERK1/2)/(ERK1/2)也升高(P<0.05)。PD98059干预后,RAGE蛋白表达及(p-ERK1/2)/(ERK1/2)均降低。与不同浓度(100、200、300、500μg/ml)BSA组比较,相应浓度(100、200、300、500μg/ml)AGEs组细胞吸光度(OD)升高(P<0.05),细胞活力分别增加11.6%、16.3%、13.8%、13.9%。200μg/ml AGEs组PD98059干预,细胞OD值下降(P<0.05)。24h后,200μg/ml AGEs组细胞凋亡率与Con组比较,差异无统计学意义(P>0.05),MET组细胞凋亡率升高(P<0.05);与MET组比较,MET+AGEs组凋亡率降低(P<0.05);与MET+AGEs组比较,MET+AGEs+PD组凋亡率升高(P<0.05)。结论 AGEs可促进结肠癌干细胞增殖,抑制其凋亡,作用机制可能是通过作用于RAGE,上调其表达量,激活ERK1/2通路来实现。

Objective To investigate the effects of advanced glycation end products（AGEs）on the expression of AGEs receptor（RAGE）,proliferation and apoptosis of the colon cancer stem cells.Methods Human SW480 cancer stem cells were selected in this study.After treatment of AGEs at different concentrations,50μmol/L PD98059,and 40 mmol/L Metformin（MET）,the expression of RAGE mRNA and RAGE,extracellular signal-regulated kinase（ERK）and phosphor-ERK（p-ERK）protein in24 hwere detected by RT-PCR and western blot respectively.Cell proliferation in 24 hwas detected by CCK-8 and cell apoptosis was tested by flow cytometry. Results After AGEs intervention with different concentrations（100,200,300μg/ml）,the expression of RAGE mRNA and protein was significantly increased in the 200μg/ml AGEs group than in Con group（without AGEs）.And（p-ERK1/2）/（ERK1/2）was also significantly increased in the 200μg/ml AGEs group than in control group（P〈0.05）.After the intervention of PD98059,the expression of RAGE protein and（p-ERK1/2）/（ERK1/2）were significantly reduced.The cell optical density（OD）value increased significantly,and the cell viability increased by 11.6%,16.3 %,13.8 % and 13.9 % respectively in AGEs groups with the corresponding concentrations（100,200,300,500μg/ml）than in BSA groups with different concentrations（100,200,300,500μg/ml）（P〈0.05）.The cell ODvalue decreased significantly when PD98059 intervention was applied in the 200 ug/ml AGEs group（P〈0.05）.The apoptosis rate after 24 hwas not significantly different between 200μg/ml AGEs group and Con group（P〉0.05）.The apoptosis rate increased significantly in MET group（P〈0.05）,while decreased significantly in MET＋AGEs group than in MET group（P〈0.05）.The apoptosis rate increased significantly in MET＋AGEs ＋PD group than in MET＋AGEs group（P〈0.05）. Conclusion AGEs could enhance the cell proliferation and inhibit cell apoptosis in the colon cancer stem cell.The possible implementation mechanism may be the activation of RAGE,increasing the expression of RAGE,and activating ERK1/2 pathway.