SN50定向诱导小鼠小胶质细胞分化促进缺氧损伤神经元修复的实验研究  

Effect of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice

作　　者：韩芳芳[1,2] 刘春龙 杨常青 孙元华[3] HAN Fangfang;LIU Chunlong;YANG Changqing;SUN Yuanhua(Medical Treatment Department,Luohe Medical College,Luohe Henan,462002,P.R.China;Clinical Medical College of Acupuncture Moxibustion and Rehabilitation,Guangzhou University of Chinese Medicine,Guangzhou Guangdong,510006,P.R.China;Department of Pediatrics,the Third Affiliated Hospital,Luohe Medical College,Luohe Henan,462002,P.R.China)

机构地区：[1]漯河医学高等专科学校医疗系,河南漯河462002 [2]广州中医药大学针灸康复临床医学院,广州510006 [3]漯河医学高等专科学校第三附属医院儿科,河南漯河462002

出　　处：《中国修复重建外科杂志》2020年第4期509-517,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：河南省医学科技攻关计划项目(201702341)。

摘　　要：目的探讨SN50定向诱导小鼠小胶质细胞分化对缺氧损伤神经元的修复作用及其机制。方法取新生BALB/c小鼠脑组织,采用差速贴壁结合震荡方法分离神经元和小胶质细胞。小胶质细胞首先采用免疫荧光染色鉴定特异性表达蛋白诱导型一氧化氮合酶(inducible nitric oxide synthetase,iNOS)和电离钙离子结合调节因子1(ionized calcium binding adapter molecule 1,Iba1)表达情况,实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测特异性表达基因iNOS、CD32和IL-10;然后采用SN50处理后,Western blot检测SN50对小胶质细胞NF-κB的调控,qRT-PCR检测小胶质细胞相关分化基因(iNOS、CD11b、IL-10、CD206)表达,流式细胞术检测SN50对小胶质细胞凋亡的影响,以上检测均以蒸馏水处理的小胶质细胞作为对照。神经元首先行缺氧损伤处理,分别于缺氧处理0、2、6、12、24、48 h后MTT检测神经元活力,另取缺氧处理12 h神经元经Western blot检测凋亡相关蛋白Bcl-2和半胱氨酸蛋白酶3(Caspase-3)的表达、流式细胞术检测神经元凋亡情况。最后,将缺氧损伤12 h神经元分别与无菌蒸馏水(对照组)和SN50(实验组)处理的小胶质细胞共培养24 h,MTT检测细胞活力、Western blot检测细胞凋亡相关蛋白(Bcl-2、Caspase-3)表达、流式细胞术检测细胞凋亡情况。结果免疫荧光染色和qRT-PCR鉴定震荡分离的细胞表达小胶质细胞特异性蛋白和基因。与正常小胶质细胞相比,SN50处理小胶质细胞后NF-κB蛋白及指示向M1型分化特异性基因iNOS、CD11b相对表达量降低(P<0.05),向M2型分化特异性基因IL-10和CD206相对表达量提高(P<0.05),但细胞凋亡率无明显改变(P>0.05)。与正常神经元相比,神经元缺氧处理后活力显著下降,Bcl-2、Caspase-3蛋白相对表达量降低,而cleaved-Caspase-3蛋白相对表达量提高,神经元凋亡率明显升高,差异均有统计学意义(P<0.05)。小胶质细胞与神经元共培养�Objective To explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice.Methods The microglia were isolated and purified from brain tissue of newborn BALB/c mice through differential velocity adherent and vibration technique.The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase(iNOS)and ionized calcium binding adapter molecule 1(Iba1)and real-time fluorescence quantitative PCR(qRT-PCR)for special expression genes[iNOS,CD32,and interlenkin 10(IL-10)].Then the microglia were cultured with SN50,and the expressions of nuclear factorκB(NF-κB),differentiation-related genes(iNOS,CD11b,IL-10,and CD206),and apoptosis were detected by Western blot,qRT-PCR,and flow cytometry,respectively.The hypoxia model of neuron was established,and the cell apoptosis was evaluated by MTT after 0,2,6,12,24,and 48 hours of anoxic treatment.The apoptosis related markers(Bcl-2 and Caspase-3)were measured by Western blot and flow cytometry.In addition,the neurons after anoxic treatment were cocultured with SN50 treated microglia(experimental group)and normal microglia(control group)for 24 hours.And the cell viability and apoptosis related markers(Bcl-2 and Caspase-3)were also measured.Results Immunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia.Compared with the normal microglia,the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased(P<0.05),the relative expressions of IL-10 and CD206 mRNAs significantly increased(P<0.05),and the cell apoptosis rate had no significant change(P>0.05).Compared with the normal neurons,the cell viability,the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased(P<0.05),while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased(P<0.05).In the co

关 键 词：小胶质细胞 神经元 NF-ΚB 细胞分化 缺氧损伤 小鼠 

分 类 号：R743.3[医药卫生—神经病学与精神病学]