高糖条件下高表达ARNO对人肾小球内皮细胞通透性的影响及其作用机制  

Effects and mechanism of high ARNO expression on the permeability of human renal glomerular endothelial cells under high glucose condition

作　　者：王小龙 胡成芳 唐璐瑶 李艺 官涛 黄云剑 Wang Xiao-Long;Hu Cheng-Fang;Tang Lu-Yao;Li Yi;Guan Tao;Huang Yun-Jian(Department of Nephrology,Xinqiao Hospital,Army Medical University,Chongqing 400037,China)

机构地区：[1]陆军军医大学附属新桥医院肾内科

出　　处：《解放军医学杂志》2020年第1期55-61,共7页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81370821,81600583)~~

摘　　要：目的探讨高糖条件下高表达ARNO对人肾小球内皮细胞(HRGEC)通透性的影响及其作用机制。方法体外培养HRGEC,分别观察不同时间梯度(15、30、45和60 min)和浓度梯度(10、20、30和40 mmol/L)高糖作用对ARNO蛋白表达和内皮细胞通透性的影响。分别采用ARNOsiRNA和Arf6siRNA重组慢病毒载体感染HRGEC,获得沉默ARNO和Arf6的HRGEC细胞株,观察ARNO和Arf6基因沉默对内皮细胞通透性的影响。结果时间梯度实验中,与高糖对照组相比,高糖15、30、45、60 min组HRGEC ARNO表达明显升高(分别为0.670±0.051、0.960±0.106、0.716±0.026、0.531±0.030vs.0.242±0.029),差异有统计学意义(P<0.05),与高糖15 min组相比,高糖30 min组ARNO表达升高(P<0.05),与高糖30min组相比,高糖45 min组ARNO表达回落(P<0.05),与高糖45 min组相比,高糖60 min组ARNO表达降低(P<0.05)。与高糖对照组相比,高糖15、30、45、60 min组内皮细胞通透性明显增加(分别为1.196±0.004、1.399±0.012、1.301±0.052、1.184±0.030vs.1.000),差异有统计学意义(P<0.05),与高糖15 min组相比,高糖30 min组通透性增加(P<0.05),与高糖30 min组相比,高糖45 min组通透性下降(P<0.05),与高糖45 min组相比,高糖60 min组通透性下降(P<0.05)。浓度梯度实验中,与正常糖组相比,高糖20、30、40 mmol/L组HRGEC ARNO表达明显升高(分别为0.632±0.031、0.927±0.041、1.183±0.098 vs. 0.169±0.033),差异有统计学意义(P<0.05),且随高糖浓度增加,ARNO表达呈递增趋势,差异均有统计学意义(P<0.05)。与正常糖组相比,高糖10、20、30、40 mmol/L组内皮细胞通透性明显增加(分别为1.147±0.015、1.237±0.023、1.351±0.015、1.444±0.019 vs. 1.000),差异有统计学意义(P<0.05),且随高糖浓度增加,内皮细胞通透性呈递增趋势,差异均有统计学意义(P<0.05)。转染慢病毒后,与正常糖未转染组及正常糖空载体组相比,正常糖ARNO siRNA组ARNO mRNA转录明显降低(0.255±0.056 vs.1.000、1.183±0.297),ARNO蛋Objective To explore the effects and mechanism of high expression of ARNO(ARF nucleotide-binding-site opener) on the permeability of human renal glomerular endothelial cells(HRGECs) under high glucose conditions. Methods HRGECs were cultured in vitro, and ARNO expression and endothelial permeability were detected in high glucose time gradient(15, 30, 45 and 60 min) and concentration gradient(10, 20, 30 and 40 mmol/L) experiments, respectively. Cell lines of HRGECs with ARNO and Arf6 gene silencing were obtained by infecting HRGECs with ARNO si RNA and Arf6 si RNA recombinant lentivirus vectors, respectively. The effects of ARNO and Arf6 gene silencing on endothelial permeability were observed. Results In the time gradient experiments, compared with control group, ARNO expressions increased significantly in the groups with high glucose for 15, 30, 45 and 60 min(0.670±0.051, 0.960±0.106, 0.716±0.026, 0.531±0.030 vs. 0.242±0.029. P<0.05). Compared with high glucose 15 min group, ARNO expression increased in high glucose 30 min group(P<0.05);Compared with high glucose 30 min group, ARNO expression decreased in high glucose 45 min group(P<0.05);Compared with high glucose 45 min group, ARNO expression decreased in high glucose 60 min group(P<0.05). Compared with control group, endothelial permeability increased significantly in the groups with high glucose for 15, 30, 45 and 60 min(1.196±0.004, 1.399±0.012, 1.301±0.052, 1.184±0.030 vs. 1.000, P<0.05). Compared with high glucose 15 min group, endothelial permeability increased in high glucose 30 min group(P<0.05);Compared with high glucose 30 min group, endothelial permeability decreased in high glucose 45 min group(P<0.05);Compared with high glucose 45 min group, endothelial permeability decreased in high glucose 60 min group(P<0.05). In the concentration gradient experiments, compared with normal concentration glucose group, ARNO expressions increased significantly in the groups with high glucose of 20, 30 and 40 mmol/L(0.632±0.031, 0.927±0.041, 1.183±0.09

关 键 词：ARNO Arf6 高糖 人肾小球内皮细胞 通透性 

分 类 号：R692.6[医药卫生—泌尿科学]