BDNF干预MSCs源性外泌体减轻过氧化氢致PC12细胞氧化应激损伤  

Protective effect of BDNF intervention in MSCs-derived exosomes against PC12 cells injury by H2O2 oxidative stress

作　　者：徐会友 马珂 赵飞 张健 江继鹏 王仁杰 陈旭义 XU Hui-you;MA ke;ZHAO Fei;ZHANG Jian;JIANG Ji-peng;WANG Ren-jie;CHEN Xu-yi(Logistics University of People s Armed Police Forces;Characteristic Medical Center, Department of Neurosurgery,People s Armed Police Forces Medical Center, Tianjin 300162, China)

机构地区：[1]中国人民武装警察部队武警后勤学院,天津300162 [2]中国人民武装警察部队特色医学中心脑科中心,天津300162

出　　处：《基础医学与临床》2019年第11期1530-1536,共7页Basic and Clinical Medicine

基　　金：国家重点研发计划(2016YFC1101500);国家自然科学基金(11672332,11102235);天津市科技支撑重点项目(17YFZCSY00620);天津市自然科学基金项目(15JCYBJC28600,17JCDJC5400);天津市救援医学临床中心基金(15ZXLCSY00040)

摘　　要：目的探讨经脑源性神经营养因子(BDNF)干预后的大鼠骨髓间充质干细胞(MSCs)源性外泌体对H2O2损伤大鼠肾上腺嗜铬细胞瘤细胞的保护作用。方法全骨髓培养法提取大鼠MSCs,取P3代分为对照组及干预组(培养基中加入30 ng/mL BDNF),分别收集细胞上清,超速离心法分离并纯化外泌体,通过透射电镜观察鉴定外泌体形态;用Western blot鉴定外泌体表面标志蛋白CD9、CD63;实验将嗜铬细胞瘤细胞PC12分为4个组:对照组、H2O2组、MSCs外泌体组和BDNF-MSCs外泌体组,前2组培养基中加入PBS,后2组加入相应外泌体(MSCs外泌体组和BDNF-MSCs外泌体组外泌体蛋白浓度均为50μg/mL)均预处理12 h,后3组在培养基中加入H2O2(0. 521 mmol/L)对PC12建立氧化应激的细胞损伤模型;于10 h后以CCK-8法检测细胞活性,检测活性氧(ROS)及超氧化物歧化酶(SOD)值,Western blot检测Bcl-2与Bax蛋白质表达情况。结果 P3代MSCs细胞表面CD90表达阳性。成功提取出外泌体,透射电镜下可见大量直径40~100 nm的不规则球体或茶托形的囊泡结构,膜清晰,相对完整,Western blot显示CD9、CD63蛋白表达阳性。相比于单纯损伤组与MSCs外泌体组,BDNF-MSCs外泌体组细胞活性最高、SOD活性最高、ROS含量最低、Bcl-2/Bax值最高。结论经BDNF干预后的MSCs源性外泌体在H2O2对PC12细胞造成的氧化应激损伤的保护作用优于MSCs源性外泌体。Objective To explore the protective effect of Marrow Stem Cells (MSCs) derived exosomes on PC12 cells injured by hydrogen peroxide in rats after intervention by BDNF. Methods Rat MSCs were obtained by whole bone marrow adherent culture methods. The supernatant of cells was collected by adding BDNF into part of the medium for P3 generation and supernatant was also collected without BDNF intervention. Exosomes were isolated and purified by ultracentrifugation, and their morphology was observed and identified under transmission electron microscope. Western blot was used to verify the surface marker proteins CD9 and CD63. PC12 cells were divided into contral group, H 2O 2 group, MSCs exosome group and BDNF-MSCs exosome group. PBS was added into the medium of the first two groups, and corresponding exosomes (concentrations of exosome proteins in MSCs and BDNF-MSCs were both 50 μg/mL) were added into the latter two groups for 12 h, H 2O 2 (0.521 mmol/L)was added into the medium to establish the cell damage model of oxidative stress on PC12 cells in the latter three groups. 10 h later cell viability was detected by CCK8 method, ROS activity and SOD value were detected by kit, and Bcl-2 and Bax protein expression was detected by Western blot. Results CD90 expressed on the cell surface of P3 generation MSCs. Exosomes were successfully extracted, a large number of irregular spheres or saucer-shaped vesicles with diameters of 40-100 nm were observed. The membranes were clear and relatively complete, CD9 and CD63 proteins were examined by Western blot. Compared with simple injured group and MSCs exosome group, BDNF-MSCs exosome group had the highest cell viability, the highest SOD activity, the lowest ROS and the highest bcl-2 /Bax value. Conclusions The protective effect of MSCs-derived exosomes after intervention by BDNF on oxidative stress injury induced by H 2O 2 on PC12 cells is much more significant than that of MSCs-derived exosomes.

关 键 词：外泌体 间充质干细胞 PC12细胞 氧化应激损伤 

分 类 号：R741.05[医药卫生—神经病学与精神病学]