抑制糖原合酶激酶活性对老龄大鼠内皮祖细胞增殖的作用机制  

The mechanism of glycogen synthase kinase 3β inhibition on the proliferation of aging endothelial progenitor cells

作　　者：崔斌[1] 刘曦[1] 秦浙学[1] 陈剑飞[1] 李佳蓓[1] 于世勇[1] 黄岚[1] CUI Bin;LIU Xi;QIN Zhexue;CHEN Jianfei;LI Jiapei;YU Shiyong;HUANG Lan(Institute of Cardiovascular Disease of PLA,Xinqiao Hospital,The Third Military Medical University,Chongqing 400037,China)

机构地区：[1]第三军医大学新桥医院全军心血管内科研究所,重庆400037

出　　处：《西部医学》2018年第10期1426-1429,1433,共5页Medical Journal of West China

基　　金：国家自然科学基金(81100149)

摘　　要：目的探讨抑制糖原合酶激酶3β(GSK3β)活性对老龄大鼠内皮祖细胞(EPC)增殖的作用机制。方法密度梯度离心法分离培养老龄大鼠骨髓源性EPC,取对数生长期的EPC,分别加入表达催化GSK3β失活的GSK3β-KM基因的重组缺陷型腺病毒pMSCV-GSK3β-KM(基因转染组)或空病毒pMSCV-GFP(对照组)。采用镜下计数法及四氮唑溴盐比色法(MTT)测定EPC增殖能力,荧光激活细胞分离仪(FACS)分析各组EPC细胞周期变化。采用蛋白印迹法测定Wnt信号通路中磷酸化糖原合酶激酶3β(pGSK-3β)、β-连环蛋白(β-catenin)及细胞周期蛋白D1(cyclinD1)的蛋白表达。结果与对照组比较,基因转染组EPC数量显著增加(P<0.01);MTT法检测基因转染组EPC在490nm吸光度值与对照组比较差异有统计学意义(P<0.01);FACS分析显示基因转染组细胞周期S期比例较对照组显著增加(P<0.01);蛋白印迹法测定显示,与对照组比较,基因转染组pGSK-3β、β-catenin及cyclinD1的蛋白表达显著增加(P<0.01)。结论抑制EPC的GSK-3β活性可通过激活Wnt信号通路促进老龄大鼠EPC的增殖能力。Objective To investigate the mechanism of glycogen synthase kinase 3β（GSK3β）inhibition on the proliferation of aging endothelial progenitor cells（EPCs）.Methods Mononuclear cells（MNCs）were isolated from bone marrow in aging Wister rats by density gradient centrifugation combined.EPCs were cultured and transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β（GKS3β-KM）or green fluorescent protein.EPCs proliferation was assessed by cells count and 3-{4,5-dimethylthiazol-2 yl}-2,5-diphenyltetrazolium bromide（MTT）assay.The cell cycle of EPCs was measured by fluorescence-activated cell sorting（FACS）.The expression of phosphor-glycogen synthase kinase 3β（pGSK-3β）,β-catenin and cyclinD1 in EPCs were detected by western blot.Results GSK3β inhibition improved aging EPCs proliferation.Compared with control group,EPCs proliferation was enhanced in GKS3β-KM gene transfection group（GSKi group）.The number of EPCs was obviously increased in GSKi groups than that in control group.S phrase in cell cycle was increased in GSKi group in comparison with control group by FACS assay.The protein expression of pGSK-3β,β-catenin and cyclinD1 were significantly higher than that in control group.Conclusion GSK3β inhibition could improve aging EPCs proliferation by activating Wnt signal pathway.

关 键 词：糖原合酶激酶3Β 内皮祖细胞 增殖 老龄大鼠 基因转染 

分 类 号：R392.1[医药卫生—免疫学]