摘要
目的:研究黑色素瘤细胞黏附分子(CD146)在视网膜血管内皮细胞中的功能及机制。方法:实验研究。于2021年1—6月构建氧诱导视网膜病变模型(OIR)小鼠16只作为实验组,以同日龄正常饲养小鼠16只作为对照组。采用免疫磁珠法分离视网膜血管内皮细胞;定量PCR分别检测2组小鼠mRNA的表达水平;免疫荧光染色检测小鼠视网膜上CD146的定位和表达。取人视网膜微血管内皮细胞(HRMEC)在1%O2环境中培养,Western blot检测HRMEC细胞和小鼠视网膜中CD146的蛋白表达水平。CD146小干扰RNA(siCD146)转染HRMEC作为siCD146转染组,无义小干扰RNA转染作为转染对照组(NC)。采用细胞活力测定实验(MTS)、细胞增殖实验(EDU)、流式细胞术分别检测siCD146转染组和NC细胞活力、细胞增殖和细胞周期;Transwell和划痕实验检测HRMEC的迁移能力;血管生成实验检测HRMEC的血管形成能力。OIR小鼠右眼玻璃体腔注射siCD146作为siCD146注射组,左眼注射无义小干扰RNA作为阴性注射对照组(NC),采用血管免疫荧光染色观察注射后OIR小鼠眼底新生血管的情况。采用独立样本t检验进行实验数据分析。结果:免疫荧光染色显示,CD146在小鼠视网膜血管上特异性表达。OIR小鼠视网膜血管内皮细胞中CD146 mRNA表达水平是正常小鼠的2倍(t=6.57,P=0.003)。与21%O2处理相比,1%O2处理的HRMEC中CD146蛋白表达升高(t=5.79,P=0.010)。与NC组细胞相比,siCD146转染组细胞迁移和血管生成能力均受到明显抑制(Transwell:siCD146-1:t=6.15,P=0.003;siCD146-2:t=3.88,P=0.005。划痕:siCD146-1:t=2.73,P=0.041;siCD146-2:t=4.10,P=0.006。血管生成:siCD146-1:t=2.81,P=0.048;siCD146-2:t=3.10,P=0.036)。Western blot结果显示:与NC相比,siCD146转染组的Ras同源家族成员A(RHOA)(siCD146-1:t=3.60,P=0.023;siCD146-2:t=4.12,P=0.015)、磷酸化p38蛋白(siCD146-1:t=2.89,P=0.044;siCD146-2:t=3.07,P=0.038)表达均下调。视网膜铺片结果显示:与NC相比,siCD146注射组小�
Objective:To investigate the functions and underlying mechanisms of melanoma cell adhesion molecule cluster of differentiation 146(CD146)in retinal vascular endothelial cells.Methods:In this experimental study,oxygen induced retinopathy(OIR)mouse model(n=16)was constructed from January to June 2021,and the age-matched mice were used as the control(n=16).Retinal vascular endothelia were isolated by the magnetic beads-based method.Real-time quantitative PCR was performed to detect the expression level mRNA in retinas and cells in the two groups.Immunofluorescent staining identified the expression and location of CD146 in retinas.Western blot assay determined the protein expressions in human retinal microvascular endothelial cell(HRMEC)and retinas.CD146 small interfering RNA(siCD146)transfected HRMEC was used as the transfection experimental group,while nonsense small interfering RNA transfection was used as the negative transfection control group(NC).MTS,EDU and flow cytometry were used to evaluate cell viability,cell proliferation,and cell cycle,respectively.Transwell and scratch assay were used to measure cell migration.Matrigel angiogenesis assay examined the ability of tube formation.And siRNAs were delivered into the retinas by intravitreal injection.Flat-mount of retina stained for isolectin B4(Ib4)analyzed the neovascularization and avascular region.Independent sample t-test was used for data analysis.Results:Immunofluorescent staining showed that CD146 was specifically expressed on retinal vessels.Compared to the normal mice,the level of CD146 mRNA in vascular retinal endothelial cells of OIR model mice were twice as high as those of normal mice(t=6.57,P=0.003).The protein expression of CD146 was also increased in hypoxia(1%O_(2))compared to the normoxia(21%O_(2))(t=5.79,P=0.010).CD146 inhibition by siRNA transfection inhibited HRMEC migration significantly(Transwell assay:siCD146-1:t=6.15,P=0.003;siCD146-2:t=3.88,P=0.005.Scratch assay:siCD146-1:t=2.73,P=0.041;siCD146-2:t=4.10,P=0.006).The matrigel angiogen
作者
林永
杨汝森
闫东升
陈晓燕
吕帆
Yong Lin;Rusen Yang;Dongsheng Yan;Xiaoyan Chen;Fan Lu(School of Ophthalmology and Optometry,School of Biomedical Engineering,Wenzhou Medical University,Wenzhou 325027,China)
出处
《中华眼视光学与视觉科学杂志》
CAS
CSCD
2023年第5期332-339,共8页
Chinese Journal Of Optometry Ophthalmology And Visual Science
基金
国家自然科学基金(81900818)
温州市基础性科研项目(Y20220144)
省部共建眼视光学和视觉科学国家重点实验室资助项目(J02-20190201)。
