糖基化终末产物对体外培养牛眼小梁细胞氧化应激及凋亡的影响

Effects of advanced glycation end products on oxidative stress and apoptosis of bovine trabecular meshwork cells cultured in vitro

目的通过观察糖基化终末产物(advanced glycation end products,AGE)对体外培养牛眼小梁细胞凋亡的影响,研究AGE与原发性开角型青光眼(primary open angle glaucoma,POAG)之间的关系,进一步探讨其发病机制。方法体外培养牛眼小梁细胞,通过形态学观察和神经元特异性烯醇化酶染色对培养的细胞进行鉴定。将第3代小梁细胞接种于6孔培养板,在培养基中加入不同浓度(0μg·mL-1、50μg·mL-1、100μg·mL-1、200μg·mL-1)的AGE-BSA培养96 h。终浓度(200μg·mL-1)的AGE-BSA培养液处理细胞不同时间(48 h、72 h、96h)。应用流式细胞仪检测小梁细胞凋亡率;活性氧荧光探针2’,7’-二氯荧光黄双乙酸盐检测细胞内活性氧(ROS)水平。结果 50μg·mL-1、100μg·mL-1、200μg·mL-1AGE-BSA作用96h后细胞凋亡率分别为(5.60±0.25)%、(9.57±0.08)%、(17.68±0.21)%,与对照组(0μg·mL-1AGE-BSA)细胞凋亡率(4.45±0.12)%相比均明显增高,且差异均有统计学意义(均为P<0.05);200μg·mL-1AGE-BSA作用细胞48 h、72 h、96 h后凋亡率分别为(10.51±0.28)%、(13.47±0.42)%、(17.68±0.21)%,与对照组相比也均明显增高,差异均有统计学意义(均为P<0.05)。与对照组比较,AGE-BSA处理后细胞内ROS水平显著提高,差异有统计学意义(P<0.05),BSA组差异无统计学意义(P>0.05)。结论在体外培养的条件下,AGE可能通过刺激牛眼小梁细胞产生大量ROS介导小梁细胞凋亡。

Objective To investigate the effects of advanced glycation end products （AGE） on apoptosis of bovine trabecular meshwork cells （TM cells） cultured in vitro, and further explore the relationship between AGE and primary open angle glaucoma （POAG） ,probe its pathogenesis. Methods The bovine TM cells were cultured in vitro and identified by morphological evaluation and neuronspecific enolase staining. The third generation of cells were inoculated to 5-well plate, and different concentrations（0 μg·mL^-1 ,50 μg·mL^-1 ,100 μg·mL^-1 and 200 μg·mL^-1）AGE-BSA was added into the medium for 96 hours, or treated with AGE-BSA （200μg·mL^-1 ） for different time （48 hours, 72 hours and 96 hours）. Flow cytometry was performed to detect the cells apoptosis, and the level of reactive oxygen species （ROS） was evaluated by 2＇ ,7＇-dichlorofluorescein diacetate （DCFH-DA） method. Results The apoptotic rates after treating with different concentrations （50 μg·mL^-1, 100 μg·mL^-1 and 200μg·mL^-1） AGE-BSA for 96 hours were （5.60 ±0.25）% , （9. 57 ±0. 08）% and （17.68± 0. 21 ）% , respectively, which were obviously higher than the control group （4. 45 ±0.12）% （all P 〈0.05）. The apoptotic rates after treating with 200μg·mL^-1 AGEs-BSA for 48 hours,72 hours and 96 hours were （10. 51 ±0.28）% , （13.47 ± 0. 42）% and （17.68 ±0.21 ）% ,respectively,which were also obviously higher than the control group （ all P 〈 0.05 ）. Compared with control group, the ROS level in bovine TM cells after treating with AGE-BSA was increased （P 〈0.05） ,but there was no statistical difference after treating with BSA （P 〉 0.05）. Conclusion AGE can increase the ROS level in bovine TM cells cultured in vitro to mediate their apoptosis.