作者：李敏，甄瑾，朱润秀，梁子红，崔蓝心，姚远

单位：

内蒙古自治区人民医院神经内科

Units：

Department of Neurology, Nner Mongolia People's Hospital, Hohhot 010017, China

关键词：

血脑屏障; 氧糖剥夺; 人脐静脉内皮细胞; 紧密连接相关蛋白;

Keywords：

　Blood-brain barrier; Oxygen-glucose deprivation; Human umbilical vein endothelial cells; Tight junction-related proteins

CLC：

R 322.81

DOI：

10.12114/j.issn.1008-5971.2023.00.042

Funds：

内蒙古自治区自然科学基金资助项目（2021MS08114）；内蒙古自治区人民医院院内基金资助项目（2019YN10）

摘要：

目的 探究氧糖剥夺影响人脐静脉内皮细胞（HUVEC）紧密连接相关蛋白的机制。方法 本实验时间为2021年5月至2022年1月。将HUVEC分为正常组、氧糖剥夺6 h组和氧糖剥夺24 h组。正常组HUVEC进行正常培养；氧糖剥夺6 h组HUVEC制备氧糖剥夺模型，并在缺氧培养箱中培养6 h；氧糖剥夺24 h组HUVEC制备氧糖剥夺模型，并在缺氧培养箱中培养24 h。在倒置显微镜下观察HUVEC的形态并拍照，采用CCK-8检测孵育1、2、3、4 d细胞增殖水平。将HUVEC分为氧糖剥夺组、空质粒组、排斥导向分子（RGM）a质粒敲减组。氧糖剥夺组HUVEC制备氧糖剥夺模型，并在缺氧培养箱中培养24 h；空质粒组HUVEC在氧糖剥夺组基础上，转染空载体；RGMa质粒敲减组HUVEC在氧糖剥夺组基础上，转染慢病毒。采用Western blot法检测RGMa、紧密连接相关蛋白（Claudin-5和ZO-1）、基质金属蛋白酶（MMP）-9、CDC42。结果 与正常组相比，氧糖剥夺6 h组、氧糖剥夺24 h组细胞体肿胀或变形，光晕消失，细胞边缘不清晰，细胞体内出现空泡，突起缩短，连接断裂，视野内颗粒样物质增多，细胞变形。孵育1、2、3、4 d，正常组和氧糖剥夺6 h组细胞增殖水平比较，差异无统计学意义（P>0.05）；孵育1、2、3、4 d，氧糖剥夺24 h组细胞增殖水平均低于正常组（P<0.05）。空质粒组ZO-1、MMP-9低于氧糖剥夺组（P<0.05）;RGMa质粒敲减组RGMa、ZO-1、CDC42低于氧糖剥夺组，Claudin-5、MMP-9高于氧糖剥夺组（P<0.05）。结论 氧糖剥夺24 h可引起HUVEC增殖水平降低，氧糖剥夺引起HUVEC紧密连接相关蛋白改变的机制可能为RGMa通过CDC42而影响血脑屏障的紧密连接。

Abstract：

　Objective To explore the mechanism of oxygen-glucose deprivation affecting the tight junction-related proteins in human umbilical vein endothelial cells (HUVEC) . Methods This experiment time was from May 2021 to January HUVEC were divided into normal group, oxygen-glucose deprivation 6 h group and oxygen-glucose deprivation 24 h group. HUVEC in normal group was cultured normally; HUVEC in oxygen-glucose deprivation 6 h group were prepared oxygen glucose deprivation model and cultured in hypoxic incubator for 6 h; HUVEC in oxygen-glucose deprivation 24 h group was prepared oxygen-glucose deprivation model and cultured in hypoxia incubator for 24 h. The morphology of HUVEC was observed and photographed under an inverted microscope. CCK-8 was used to detect the cell proliferation level at 1, 2, 3 and 4 days of incubation. HUVEC were divided into oxygen-glucose deprivation group, empty plasmid group, rejection guidance molecule (RGM) a plasmid knockdown group. HUVEC in oxygen-glucose deprivation group was prepared oxygen-glucose deprivation model and cultured in hypoxic incubator for 24 h. HUVEC in empty plasmid group was transfected with empty vector on the basis of oxygen glucose deprivation group; HUVEC in RGMa plasmid knockdown group was transfected with lentivirus on the basis of oxygen glucose deprivation group. RGMa, tight junction associated protein (Claudin-5, ZO-1) , matrix metalloproteinase (MMP) -9 and CDC42 were detected by Western blot. Results Compared with normal group, the morphology of HUVEC in oxygen-glucose deprivation 6 h group and oxygen-glucose deprivation 24 h group changed significantly, the cell body was swollen or deformed, the halo disappeared, the cell edge was unclear, vacuoles appeared in the cell body, the process was shortened, the connection was broken, the granular material in the visual field increased, and the cells were deformed. There was no significant difference in cell proliferation between the normal group and the oxygen-glucose deprivation 6 h group at 1, 2, 3 and 4 days of incubation ( P > 0.05) . At 1, 2, 3, 4 days of incubation, the cell proliferation level in oxygen-glucose deprivation 24 h group was lower than that in normal group (P < 0.05) . ZO-1 and MMP-9 in the empty plasmid group were lower than those in the oxygen-glucose deprivation group (P < 0.05) . RGMa, ZO-1 and CDC42 in RGMa plasmid knockdown group were lower than those in oxygen glucose deprivation group, Claudin-5 and MMP-9 were higher than that in oxygen-glucose deprivation group ( P < 0.05) . Conclusion Oxygen-glucose deprivation for 24 h can cause a decrease in HUVEC proliferation. The mechanism of oxygen glucose deprivation causing changes in HUVEC tight junction-related proteins may be that RGMa affects the tight junction of BBB through CDC42.

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