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  • 姚盼盼,盛敏杰,陈 丽,等.不同浓度葡萄糖对兔角膜上皮细胞凋亡的影响[J].同济大学学报(医学版),2020,41(4):431-436.    [点击复制]
  • YAO Pan-pan,SHENG Min-jie,CHEN Li,et al.Effect of glucose at different concentrations on apoptosis of rabbit corneal epithelial cells[J].同济大学学报(医学版),2020,41(4):431-436.   [点击复制]
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不同浓度葡萄糖对兔角膜上皮细胞凋亡的影响
姚盼盼,盛敏杰,陈丽,陆佳俊,李敏,李冰
0
(同济大学附属杨浦医院眼科,上海 200090;复旦大学附属中山医院眼科,上海 200032)
摘要:
目的 探讨葡萄糖不同浓度对兔角膜上皮细胞(rabbit corneal epithelial cells, RCECs)凋亡的影响。方法 将 离体培养的RCECs随机分为正常对照组(5.5mmol/L葡萄糖)、高糖组(33mmol/L葡萄糖)和高渗组(5.5mmol/L葡萄糖+27.5mmol/L甘露醇),釆用Western印迹法检测各组RCECs双链RNA依赖蛋白激酶样ER激酶(PKR like endoplasmic reticulum kinase, PERK)、磷酸化双链RNA依赖蛋白激酶样ER激酶(phosphorylated PKR like endoplasmic reticulum kinase, p-PERK)、真核翻译起始因子(eukaryotic initiation factor 2α, eIF2α)、磷酸化真核翻译起始因子(phosphorylated eukaryotic initiation factor 2α, eIF2α)和转录因子C/EBP同源蛋白(transcription factor C/EBP homologous protein, CHOP) 表达水平;显微镜下观察不同葡萄糖浓度(200~800mmol/L)RCECs生长情况的变化,甘露醇做渗透压对照。结果 Western印迹法结果显示,与正常对照组相比,高糖组和高渗组均可诱导RCECs p-PERK、p-eIF2α和CHOP蛋白表达水平升高(P<0.05),且这3种蛋白表达水平高糖组明显高于高渗组(P<0.05)。显微镜观察发现: 在一定的范围(200~800mmol/L)内,RCECs损伤面积随葡萄糖浓度的升高而增加;而葡萄糖与同浓度甘露醇相比,对RCECs损伤范围均较小。但当总浓度达到800mmol/L时,葡萄糖和甘露醇均导致RCECs短期内大片脱落死亡,高糖与高渗致细胞损伤程度相当。结论 高糖浓度依赖性诱导RCECs的凋亡,浓度越低(≤33mmol/L),高糖较高渗对RCECs的影响越大。因此,慢性低度高糖刺激对RCECs具有累积毒性效应(渗透压改变带来的影响小),对于糖尿病性干眼的临床诊治有一定的指导意义。
关键词:  角膜上皮细胞  葡萄糖  浓度  内质网应激  形态学  细胞凋亡
DOI:10.16118/j.1008-0392.2020.04.005
投稿时间:2019-10-23
基金项目:上海市自然科学基金(19ZR1450500)
Effect of glucose at different concentrations on apoptosis of rabbit corneal epithelial cells
YAO Pan-pan,SHENG Min-jie,CHEN Li,LU Jia-jun,LI Min,LI Bing
(Dept. of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine, Shanghai 200090, China;Dept. of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai 200032, China)
Abstract:
Objective To investigate the effect of glucose at different concentrations on apoptosis of rabbit corneal epithelial cells(RCECs). Methods RCECs cultured in vitro were treated by dierent concentrations of glucose and mannitol. The expressions of PKR like endoplasmic reticulum kinase(PERK), phosphorylated PKR like endoplasmic reticulum kinase(p-PERK), eukaryotic initiation factor 2α(eIF2α), phosphorylated eukaryotic initiation factor 2α(p-eIF2α) and transcription factor C/EBP homologous protein(CHOP) protein were determined by Western-bloting. The growth of RCECs cultivated in different glucose concentrations(200-800mmol/L) was observed with phase contrast microscope, and mannitol at corresponding concentrations was used as a osmotic pressure control. Results Western bloting demonstrated that the expressions of p-PERK, p-eIF2α and CHOP were increased significantly in RCECEs cultured with high concentrations of glucose and the control group, compared to the normal concentration group(P<0.05), but the expressions of PERK and eIF2α were not changed; and the expression level of the three protein in the high glucose concentration group was significantly higher than that of the osmotic pressure control(P<0.05). Phase contrast microscope showed that in a certain concentration range(200-800mmol/L), the damaged area of RCECs increased with the increase of glucose concentration, and the damage range of RCECs treated with glucose was lower than that of treated with the same concentration of mannitol. While both glucose and mannitol led the RCECs to shed and die in a short time when the glucose concentration was above 800mmol/L, the severity of RCECs injury caused by high glucose was comparable to that caused by high osmosis. Conclusion Glucose can induce the apoptosis of RCECs in a concentration-dependent manner. The results indicates that elevated glucose concentration has a cumulative toxic effect on RCECs in the long-term, and changes in osmotic pressure have little impact, which may have significance for clinical management of diabetic dry eye.
Key words:  corneal epithelial cells  glucose  concentration  endoplasmic reticulum stress  morphology  apoptosis

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