摘要
目的探讨晚期糖基化终末产物(AEGs)对内皮细胞衰老以及内皮屏障功能的影响和作用机制。 方法体外分离和培养人脐静脉内皮细胞(HUVECs),随机分为3组,分别为空白对照组(无干预)、牛血清白蛋白(BSA)对照组(BSA干预72h)、AGEs组(AGEs干预72h),分别进行β行半乳糖苷酶(SA-β-Gal)染色;实时定量聚合酶链反应(RT-qPCR)、蛋白免疫印迹法(Westernblot)检测各组衰老相关基因p53、p21、p16的表达;2',7'-二氯荧光黄双乙酸盐法(DCFH-DA)测定细胞内活性氧;细胞电阻计测量各组的跨内皮细胞电阻(TER);蛋白免疫印迹法检测肌球蛋白轻链激酶(MLCK)、磷酸化肌球蛋白轻链(p-MLC)和肌球蛋白轻链(MLC)的表达。 结果与空白对照组(7.81±0.35)%、BSA对照组(7.64±0.91)%相比,AGEs干预HUVECs引起SA-ECs轻链染色阳性率明显增加(67.30±0.75)%(P<0.01),以及衰老相关基因p53、p21、p16的表达量增加(P<0.05)。空白对照组(48.0±6.3)Ω·cm^2与BSA对照组(42.0±7.8)Ω·cm^2TER比较差异无统计学意义(P>0.05),而AEGs组TER(27.0±4.2)Ω·cm^2降低,与空白对照组和BSA对照组相比,差异有统计学意义(P<0.01)。空白对照组(38.36±8.55)%与BSA对照组(41.67±6.93)%活性氧生成比较差异无统计学意义(P>0.05),而AEGs组(69.31±8.47)%活性氧生成增加(P<0.05),同时AEGs组的MLCK表达与p-MLC/MLC明显增加(P<0.05)。 结论AGEs通过促进活性氧生成和氧化应激,以及调节MLCK信号通路影响内皮细胞衰老和内皮屏障功能障碍。
Objective To investigate the effects and mechanism of advanced glycosylation end products(AGEs)on endothelial cell senescence and endothelial barrier dysfunction. Methods Human umbilical vein endothelial cells(HUVECs)were isolated and cultured.The cells were randomized into three groups: the control group(normal medium), the bovine serum albumin-treated group(BSA control group)and AEGs group(treated with AEGs-BSA). Senescence of HUVECs were detected by senescence-associated β-galactosidase(SA-beta-Gal)staining.The mRNA and protein expressions of senescence-related genes of p53, p21 and p16 in each group were determined by reverse transcription and real-time PCR(RT-qPCR)and Western blot.Reactive oxygen species(ROS)level was determined by dichlorodihdrofluorescence diacetate(DCFH-DA). The transendothelial electric resistance(TER)were measured by endothelial electric resistance meter.The protein levels of myosin light chain kinase(MLCK), phosphorylated myosin light chain(p-MLC), myosin light chain(MLC)were detected by Western blot. Results Compared with the control group and the BSA control group, the AGEs group showed the significantly increased positive rate of senescence-associated SA-beta-Gal staining(67.30±0.75% vs.7.81±0.35% and 7.64±0.91%, respectively, P <0.01)and the expressions of aging-related genes of p53, p21 and p16 were significantly increased(P<0.05). There was no significant difference in transendothelial electric resistance(TER)between the control group and the BSA group(48.0±6.3 Ω·cm2vs. 42.0±7.8 Ω·cm^2, P>0.05), while TER was lower in the AEGs group than in control group and the BSA group[(27.0±4.2)Ω·cm^2vs.(48.0±6.3)Ω·cm^2 and (42.0±7.8)Ω·cm^2, P<0.01]. ROS production had no significant difference between the control group and the BSA group[(38.36±8.55)% vs.(41.67±6.93)%, P>0.05], while was increased in the AEGs group versus control group and the BSA group[(69.31±8.47)% vs.(38.36±8.55)% and (41.67±6.93)%, P<0.05). The protein expression levels of MLK and p-MLC/MLC were hi
作者
程冕
严金华
阮磊
翟茂才
黄毅
张乐
张存泰
Cheng Mian;Yan Jinhua;Ruan Lei;Zhai Maocai;Huang Yi;Zhang Le;Zhang Cuntai(Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430060, China)
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2019年第4期362-366,共5页
Chinese Journal of Geriatrics
基金
国家自然科学基金(81700355,81571377).
关键词
糖基化终末产物
高级
内皮细胞
衰老
Glycosylation end products, advanced
Endothelial cell
Aging
