ROBO4过表达抑制高糖条件下人肾小球内皮细胞高通透性的体外研究

发布时间：2018-01-30 02:26

本文关键词： ROBO ARF 通透性基因过表达糖尿病肾病　出处：《解放军医学杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的探讨ROBO4基因过表达对高糖诱导的人肾小球内皮细胞(HRGEC)高通透性的影响。方法体外培养HRGEC,以携带ROBO4基因的重组慢病毒载体感染HRGEC获得过表达ROBO4的HRGEC。将细胞分为低糖培养组和高糖培养组,采用Western blotting检测ROBO4和ARF6蛋白的表达,异硫氰酸荧光素右旋糖酐(FITC-Dextran)法检测HRGEC的通透性,CCK-8法测定细胞存活率。结果慢病毒在HRGEC中的72h转染率达80%,与空载体组相比ROBO4转染组HRGEC细胞的ROBO4蛋白明显过表达(P0.05),CCK8检测显示慢病毒转染后HRGEC细胞活性不受影响。与低糖组相比,高糖组ROBO4蛋白表达在12h时点明显增高(P0.05),24h回落,72h下降最为明显(P0.05),而ARF6蛋白的表达在12h开始明显升高,72h时升高最为明显(P0.05)。高糖组FITC-Dextran层HRGEC屏障的通透性在24h开始逐步升高,72h时升高最为明显(P0.05);与空载体组相比,ROBO4过表达可显著抑制高糖条件下ARF6的表达及降低体外单层HRGEC屏障的FITC-Dextran通透性。结论 ROBO4过表达可降低高糖条件下HRGEC的通透性,激活ROBO4有望成为治疗糖尿病肾病内皮功能障碍的新方向。
[Abstract]:Objective to investigate the effect of overexpression of ROBO4 gene on high permeability of human glomerular endothelial cells induced by high glucose. Methods HRGEC was cultured in vitro. The recombinant lentivirus vector carrying ROBO4 gene was used to infect HRGEC. The cells were divided into low glucose culture group and high glucose culture group. The expression of ROBO4 and ARF6 protein was detected by Western blotting. Fluorescein isothiocyanate (FITC-Dextranan) was used to detect the permeability of HRGEC. Results the transfection rate of lentivirus in HRGEC for 72 hours was 80%. Compared with the empty vector group, the ROBO4 protein of HRGEC cells transfected with ROBO4 was significantly overexpressed (P0.05). CCK8 analysis showed that the activity of HRGEC cells was not affected after lentivirus transfection. Compared with low glucose group, the expression of ROBO4 protein in high glucose group was significantly higher than that in low glucose group at 12h (P 0.05). The expression of ARF6 protein began to increase at 12 h. The permeability of HRGEC barrier in FITC-Dextran layer of high glucose group increased gradually at 24 h. At 72 h, the increase of P0.05 was the most obvious. Compared with empty carrier group. Overexpression of ROBO4 could significantly inhibit the expression of ARF6 and reduce the FITC-Dextran permeability of monolayer HRGEC barrier in vitro. Overexpression of ROBO4 can reduce the permeability of HRGEC under high glucose condition. Activation of ROBO4 is expected to be a new direction in the treatment of diabetic nephropathy endothelial dysfunction.
【作者单位】：第三军医大学新桥医院肾内科;
【基金】：国家自然科学基金(81370821) 重庆市自然科学基金重点项目(cstc2013jjB10023)~~
【分类号】：R692.9;R587.2
【正文快照】： 糖尿病发病率逐年上升,糖尿病肾病(diabeticnephropathy,DN)仍然是临床难以攻克的慢性疾病之一[1]。近年研究发现,肾小球高通透性(endothelial/glomerular hyperpermeability)可能是糖尿病视网膜病、糖尿病系统血管病变和DN微量白蛋白尿启动的重要原因。已知高糖、晚期糖基化

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