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

›› 2019, Vol. 39 ›› Issue (11): 1530-1536.

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

徐会友¹,马珂¹,赵飞²,张健²,江继鹏¹,王仁杰¹,陈旭义¹

1. 1.中国人民武装警察部队后勤学院 2.中国人民武装警察部队特色医学中心脑科中心
2. 中国人民武装警察部队特色医学中心脑科中心

收稿日期:2018-12-26 修回日期:2019-04-24 出版日期:2019-11-05 发布日期:2019-11-05
通讯作者: 陈旭义 E-mail:chenxuyi1979@126.com
基金资助:
脊髓损伤及脑损伤再生修复生物材料产品的研发;力学引导下神经元轴突微延伸远距离生长及髓鞘化移植修复脊髓损伤研究;基于纳米微粒化学发光技术的创伤标志物S100B检测方法的研究;中枢神经损伤再生修复生物材料产品的研发;葡萄籽原花青素改善抑郁症致认知障碍的表现遗传学机制;高原肺动脉高压相关miRNA及其调控靶点研究;LncRNA TCONS-00058579作为ceRNA-322/apelin在高原肺动脉高压发生中的作用机制研究

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

Received:2018-12-26 Revised:2019-04-24 Online:2019-11-05 Published:2019-11-05

摘要/Abstract

摘要： 目的探讨经脑源性神经营养因子干预后的大鼠骨髓间充质干细胞源性外泌体对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50 μg/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造成的氧化应激损伤的保护作用优于MSCs 源性外泌体。

关键词: 外泌体, 骨髓间充质干细胞, PC12, 氧化应激损伤

Abstract: 【ABSTRACT】: Objective: To explore the protective effect of Marrow Stem Cells (MSCs) derived exosomes on PC12 injured by hydrogen peroxide in rats after intervention by BDNF. Methods: Rat MSCs were extracted 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 microscopy. Western blotting was used to verified the surface marker proteins CD9 and CD63. PC12 cells were divided into control group, simple injured group, MSCs exosome group, BDNF-MSCs exosome group. PBS was added to the medium of the first two groups, and corresponding exosomes (exosome protein concentration 50 microns /ml) were added to the latter two groups to pretreat them for 12h, and the last 3 groups were established cell damage models of oxidative stress by H2O2 on PC12. 10h later cell viability was detected by CCK8 method, ROS activity and SOD value were detected by kit, and Bcl-2 and Bax protein expression were detected by Western Blotting. Result: CD90 expression on the cell surface of P3 generation MSCs was positive. Exosomes were successfully extracted , a large number of irregular spheres or saucer-shaped vesicles with diameters of 40-100 nm were observed under transmission electron microscopy. The membranes were clear and relatively complete, and CD9 and CD63 proteins were positively expressed 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. Conclusion: The protective effect of MSCs-derived exosomes after intervention by BDNF on oxidative stress injury induced by H2O2 on PC12 was better than that of MSCs-derived exosomes.

Key words: exosome, MSCs, PC12, oxidative stress

中图分类号:

R651

徐会友马珂赵飞张健江继鹏王仁杰陈旭义. 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[J]. , 2019, 39(11): 1530-1536.

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