分泌型糖蛋白Reelin对N-甲基-D-天冬氨酸(NMDA)诱导的视网膜神经节细胞凋亡的抑制作用  

Secreted glycoprotein Reelin prevents N-methyl-D-aspartate-induced apoptosis of retinal ganglion cells

作　　者：王艳婷 金学民[2] 李晓华[1] 赵朝霞[1] WANG Yanting;JIN Xuemin;LI Xiaohua;ZHAO Zhaoxia(Department of Ophthalmology of Henan Provincial People’s Hospital,Zhengzhou 450000,Henan Province,China;Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,Henan Province,China)

机构地区：[1]河南省人民医院眼科,河南省郑州市450000 [2]郑州大学第一附属医院眼科,河南省郑州市450000

出　　处：《眼科新进展》2021年第6期518-521,共4页Recent Advances in Ophthalmology

基　　金：河南省自然科学基金项目(编号162300410296)。

摘　　要：目的探讨分泌型糖蛋白Reelin对N-甲基-D-天冬氨酸(NMDA)诱导的视网膜神经节细胞(RGC)凋亡的抑制作用。方法将大鼠RGC-5分为对照组、NMDA组、Reelin类似物组、Reelin阴性对照组和Reelin抑制物组。对照组细胞用含双抗的氨基酸双抗培养基培养,NMDA组在对照组细胞培养基中添加100μmol·L^(-1) NMDA后进行细胞培养,Reelin类似物组、Reelin阴性对照组和Reelin抑制物组在NMDA组细胞培养基的基础上利用Lipofectamine 2000将Reelin类似物、阴性对照和抑制物转染RGC-5,共同孵育24 h。用Annexin V FITC/碘化丙啶联合流式细胞仪检测细胞凋亡率,Western blot检测细胞内Reelin、磷酸化-Tau蛋白(p-Tau)、糖原合成酶激酶-3β(GSK-3β)和细胞周期素依赖蛋白激酶5(CDK5)表达情况。结果对照组RGC-5细胞凋亡率为(9.30±0.74)%,NMDA组为(25.40±1.13)%,NMDA组高于对照组,差异有统计学意义(t=6.311,P<0.001)。NMDA组RGC-5内Reelin蛋白表达水平低于对照组,p-Tau、GSK-3β和CDK5蛋白表达水平均高于对照组,差异均有统计学意义(均为P<0.001)。Reelin类似物组细胞凋亡率为(9.23±0.24)%,低于Reelin阴性对照组(14.33±1.02)%和Reelin抑制物组(26.81±1.15)%,各组细胞凋亡率差异均有统计学意义(均为P<0.05)。Reelin类似物组RGC-5内p-Tau、GSK-3β蛋白表达水平均低于Reelin阴性对照组和Reelin抑制物组,Reelin抑制物组RGC-5内p-Tau、GSK-3β蛋白表达水平均高于Reelin阴性对照组,差异均有统计学意义(均为P<0.05);CDK5蛋白表达水平在3组中基本一致,差异无统计学意义(P>0.05)。结论Reelin对NMDA诱导损伤的RGC具有保护作用,这种作用是通过下调GSK-3β表达抑制Tau蛋白磷酸化,进一步抑制细胞凋亡而实现的。Objective To investigate the inhibitory effect of secreted glycoprotein Reelin on N-methyl-D-aspartate(NMDA)-induced apoptosis of retinal ganglion cells(RGC).Methods RGC-5 of rats were divided into control group,NMDA group,Reelin analog group,Reelin negative control group and Reelin inhibitor group.Cells in the control group were cultured with double-antibody-containing amino acid double-antibody(DEME)medium,the NMDA group was cultured with DEME medium plus 100μmol·L^(-1) NMDA.Reelin analog group,Reelin negative control group and Reelin inhibitor group were cultured with DEME medium plus 100μmol·L^(-1) NMDA,and then RGC-5 cells were transfected with the Reelin analog,negative control and inhibitor using Lipofectamine 2000,respectively,for 24 hours.The apoptosis rates were detected by Annexin V FITC/propidium iodide combined with flow cytometry,while Western blot was used to detect the expression levels of Reelin,phosphorylated-Tau protein(p-Tau),Glycogen synthasc kinase-3β(GSK-3β),cyclin-dependent kinase 5(CDK5)protein.Results The apoptosis rate of RGC-5 cells in the control group was(9.30±0.74)%,the NMDA group was(25.40±1.13)%,and the apoptosis rate of NMDA group was higher than that of the control group,and the difference was statistically significant(t=6.311,P<0.001).The expression of Reelin protein in RGC-5 in the NMDA group was lower than that in the control group,and the expression levels of p-Tau,GSK-3βand CDK5 protein were higher than those in the control group,and the differences were statistically significant(all P<0.001).The apoptosis rate of the Reelin analog group was(9.23±0.24)%,which was lower than that of the Reelin negative control group(14.33±1.02)%and the Reelin inhibitor group(26.81±1.15)%,and the differences were statistically significant(both P<0.05).The expression levels of p-Tau and GSK-3βof RGC-5 in the Reelin analog group were lower than those in the Reelin negative control group and Reelin inhibitor group.The expression levels of p-Tau and GSK-3βin RGC-5 in the Reelin inhi

关 键 词：REELIN 青光眼 视网膜神经节细胞 Tau蛋白 细胞凋亡 

分 类 号：R774[医药卫生—眼科]