晚期糖基化终末产物对肠道L细胞凋亡及增殖活性的影响

Effect of AGEs on apoptosis and growth of intestinal L cells

目的探讨晚期糖基化终末产物(AGEs)对肠道L细胞株GLUTag细胞凋亡及细胞增殖活性的影响。方法肠道L细胞株GLUTag细胞由加拿大多伦多大学Druker实验室惠赠。将GLUTag细胞株分5组:空白对照组,牛血清白蛋白(BSA)对照组,AGEs 100μg/m L组,AGEs 200μg/m L组,AGEs 300μg/m L组。培养方法:空白对照组,将GLUTag细胞株培养于低糖DMEM中;BSA对照组,将GLUTag细胞株细胞培养于加入BSA的低糖DMEM中;AGEs 100μg/m L组,将GLUTag细胞株培养于终质量浓度100μg/m L AGEs的低糖DMEM中;AGEs 200μg/m L组,将GLUTag细胞株培养于终质量浓度200μg/m L AGEs的低糖DMEM中;AGEs 300μg/m L组,将GLUTag细胞株培养于终质量浓度300μg/m L AGEs的低糖DMEM中;每组细胞均培养24 h。将培养在终质量浓度200μg/m L AGEs中GLUTag细胞株分别培养0、12、24、48 h(即4组)。各组细胞干预结束后采用双染细胞凋亡检测方法检测细胞凋亡率;Hoechst33258荧光染色观察细胞形态;细胞计数试剂盒检测细胞增殖活性。结果各组细胞经药物干预24 h后,AGEs 100μg/m L组、AGEs 200μg/m L组、AGEs 300μg/m L组凋亡细胞百分率明显升高,均显著高于空白对照组和BSA对照组,且AGEs 300μg/m L组凋亡细胞百分率最高(P=0.000)。AGEs 200μg/m L作用12、24、48 h后凋亡细胞百分率显著高于阴性对照组(作用0 h),且作用48 h凋亡率最高(P=0.000)。100、200、300μg/m L AGEs作用GLUTag细胞24 h后,细胞活性光密度(OD)值显著低于空白对照组和BSA对照组,且AGEs 300μg/m L组OD值最低(P<0.05)。AGEs 200μg/m L作用12、24、48 h后细胞OD值显著低于阴性对照组(作用0 h),其中48 h组OD值最低(P<0.05)。结论 AGEs对肠道L细胞株GLUTag细胞的凋亡及增殖活性均产生影响,其细胞凋亡率在相同作用时间下随着AGEs的浓度增高而增多,且在相同剂量下随着AGEs的作用时间延长而增多。AGEs可诱导肠道L细胞凋亡,抑制细胞增殖活性,从而损伤肠道L细胞。

Objective To investigate the effect of advanced glycation end products （AGEs） on proliferation, apoptosis of intestinal L cells. Methods Intestinal L cell line GLUTag cells were incubated in different concentration of AGEs（5 groups）：blank control group, in which GLUTag was cultured without intervention factor;BSA control group, in which GLUTag was cultured with BSA as the positive control;100μg/mL AGEs group, in which GLUTag was cultured with 100μg/mL AGEs;200μg/mL AGEs group, in which GLUTag was cultured with 200μg/mL AGEs;300μg/mL AGEs group, in which GLUTag was cultured with 300μg/mL AGEs. GLUTag cells in the 200μg/mL AGEs were incubated at different time point：0-hour, 12-hour, 24-hour and 48-hour. The proliferation was evaluated by cell counting kit, while the cell apoptosis detection kit was used to monitor cell apoptosis. Results After cultured 24-hour, the results obtained from FCM showed that the apoptosis rate in 100, 200, 300 μg/mL AGEs group were significantly higher than that of control group and BSA group. Apoptosis rate in 300 μg/mL AGEs group was the highest（F=63.844, P=0.000）. The apoptosis rate in 12-hour, 24-hour and 48-hour subgroups were significantly higher than that of control group, and the apoptosis rate in 48-hour group was the highest（F=31.286, P=0.000）. The optical density（OD） value in 100, 200, 300μg/mL AGEs groups were significantly lower than that of control group and BSA group, OD value in 300μg/mL AGEs group was the lowest （F=144.42, P〈0.05）. The OD value in 12-hour, 24-hour, 48-hour subgroups were significantly lower than that of control group, and OD value in 48-hour group was the lowest（F=132.11, P〈0.05）. Conclusion The apoptosis of GLUTag cells become severer with both the increased concentration of AGEs at the same aging time and the longer time of exposure to AGEs atthe same dosage. The apoptosis of L cells are induced by AGEs in a dose, time-dependent manner.