分选高纯度肾小球内皮细胞基础上应用小干扰RNA技术分析5-脂氧合酶激活蛋白的作用

Using siRNA technique to analyze the role of 5-lipoxygenase activating protein in highly purified glomerular endothelial cells

目的:采用多个内皮细胞特异性表达或吸收的蛋白分选高纯度的肾小球内皮细胞,观察5-脂氧合酶激活蛋白在细胞因子诱导的肾小球内皮细胞蛋白通透性中的可能作用。方法:实验于2005-12/2007-03在卫生部北京老年医学研究所完成。①实验材料:雌性Wistar大鼠2只,100～120g。②实验过程:用胶原酶消化大鼠肾小球细胞,先后用抗CD31和抗acetylated-LDL抗体标记细胞,通过流式细胞仪纯化肾小球内皮细胞,用抗CD54、CD106和CD62E鉴定分离的细胞。③评估:通过小干扰RNA技术抑制肾小球血管内皮细胞表达5-脂氧合酶;用干扰素-γ刺激细胞,通过免疫印迹杂交和免疫荧光组织化学检测5-脂氧合酶蛋白的表达;用FITC标记的BSA作为指示剂来测量体外培养的肾小球血管内皮细胞的蛋白通透性。结果:①分选高纯度肾小球内皮细胞:CD31磁珠分选得到83.3%CD31阳性细胞,其中12.5%为同时CD31+和高吸收acetylated-LDL。分离该双阳性细胞后得到纯度大于99.2%的肾小球内皮细胞。流式细胞仪检测显示这些细胞的CD54、CD106和CD6也均为阳性。在相差显微镜下观察,细胞形态呈鹅卵石状。②细胞膜上5-脂氧合酶定位:用干扰素-γ处理细胞后,5-脂氧合酶蛋白表达水平升高并呈时间和剂量依赖性。③细胞转染5-脂氧合酶小干扰RNA后结果:用干扰素-γ处理,干扰素-γ不能诱导5-脂氧合酶的高表达;用干扰素-γ处理细胞后转染5-脂氧合酶小干扰RNA,干扰素-γ诱导5-脂氧合酶蛋白表达水平被明显抑制。④在5-脂氧合酶小干扰RNA转染细胞中干扰素-γ处理和未处理细胞白蛋白的通透率:分别为(0.37±0.09)%和(0.31±0.06)%,差异没有显著性(P>0.05)。结论:干扰素-γ能够诱导肾小球内皮细胞表达5-脂氧合酶蛋白并导致细胞通透性增高。5-脂氧合酶蛋白介导了干扰素-γ诱导细胞通透性增高的过程。

AIM： Through the highly purified glomerular endothelial cells （GEnC） constructed by the protein absorbed or expressed by several endothelial cells, to investigate the possible role of 5-lipoxygenase activating protein （FLAP） in the protein permeability increasing in GEnC induced by interferon-γ. METHODS： The experiment was performed in Beijing Hospital/Beijing Institute of Geriatrics, Ministry of Health from December 2005 to March 2007. （1）Glomerular cells were isolated from 2 female Wistar rats of 100-120 g and digested with collagenase 11. After labeling with anti-CD31 and anti-acetylated-LDL antibody, the endothelial cells were purified by flow cytometry, and identified by CD54, CD106 and CD62E. （2）FLAP was knockdown by siRNA. Cells were treated with interferon-γ and the expression of FLAP was detected with Western blotting and immunofluorescence. FITC labeled albumin was added to the medium to measure the protein permeability ofmonolayer GEnC cultured in vitro. RESULTS： （1）CD31 beads selection attained 83.3% CD31 positive cells, in which 12.5% were both CD31 positive and acetylated-LDL high absorbed. Flow cytometry was used to select the double positive cells. The proportion of endothelial cells in selected cells was exceeded 99.2%. Flow cytometry assay showed these cells were also positive to CD54, CDI06 and CD62E. Under contrast phase microscopy, cells showed cobblestone shape. （2）The expression of FLAP was induced by the stimulation of interferon-γ in a time and dose dependent manner. （3）Interferon-γ could not induce the high expression of FLAP in cells transfected with FLAP siRNA. The expression of FLAP was also inhibited in cells treated with IFN-γ after the transfection of FLAP siRNA. （4）In cells tranfeeted with FLAP siRNA, the protein permeability ratio were （0.37±0.09）% in Weated cells and （0.31± 0.06）% in untreated cells, respectively. There was no significant difference （P 〉 0.05）. CONCLUSION： Interferon-γ can upregulate FLAP expression and increase the protein permeability of glomerular endothelial cells. FLAP mediates this process.