GBM细胞来源外泌体促进小胶质细胞介导的炎症反应

doi:10.3969/j.issn.10073205.2018.11.017

河北医科大学学报

GBM细胞来源外泌体促进小胶质细胞介导的炎症反应

1.河北医科大学第二医院神经外科，河北石家庄 050000；2.河北省神经病学实验室，河北石家庄 050000

出版日期:2018-11-25 发布日期:2018-11-21
作者简介:杨建凯（1983-），男，河北石家庄人，河北医科大学第二医院主治医师，医学博士，从事神经系统肿瘤诊治研究。
基金资助:
河北省自然科学基金项目（H2018206232）；河北省医学科学研究重点课题（20180345）；河北省青年拔尖人才计划项目（BJ2018060）；河北医科大学第二医院科学研究基金项目（2h201814）

GBMexo promotes microglia mediated inflammatory response#br#

1.Department of Neurosurgery, the Second Hospital of Hebei Medical University, Shijiazhuang
050000, China; 2.Neurological Laboratory of Hebei Province, Shijiazhuang 050000, China

Online:2018-11-25 Published:2018-11-21

摘要/Abstract

摘要： ［摘要］〓
〖HTH〗目的〖HTSS〗〖KG*2〗探讨胶质母细胞瘤来源外泌体（GBM cellderived exosomes，GBMexo）在小胶质细胞活化、炎症网络激活和肿瘤化疗耐药性中的作用及潜在机制。
〖HTH〗方法〖HTSS〗〖KG*2〗培养U87胶质瘤细胞和BV2小胶质细胞，提取U87细胞来源外泌体（U87exo）并进行鉴定，荧光示踪验证细胞间的外泌体传递。各实验组用1 mg/L脂多糖（lipopolysaccharides，LPS）刺激BV2小胶质细胞建立体外炎症模型，对照组加入PBS，4 h后实验组加入不同浓度的U87exo，根据U87exo浓度不同分为4组： LPS组、LPS+exo(50 mg/L)组、LPS+exo(100 mg/L)组和LPS+exo(200 mg/L)组。加入外泌体后孵育24 h，倒置相差显微镜观察BV2细胞的形态变化。〖JP2〗收集各组BV2细胞和上清液，ELISA检测各组上清液中乳酸脱氢酶（lactate dehydrogenase，LDH）、肿瘤坏死因子α（tumor necrosis factorα,〖JP〗TNFα）、白细胞介素1β(interleukin 1β,IL1β)和白细胞介素6(interleukin 6,IL6)的浓度， RTPCR检测CXC趋化因子受体5（CXC chemokine receptor type 5,CXCR5）、诱导型一氧化氮合酶(inducible nitric oxide synthesis,iNOS) mRNA的表达水平，Western blotting检测各组小胶质细胞iNOS、CXCR5蛋白表达的变化。
〖HTH〗结果〖HTSS〗〖KG*2〗U87exo可以转移到BV2小胶质细胞中，且能够促进小胶质细胞活化和细胞形态改变。LPS组、LPS+exo(50 mg/L)组、LPS+exo(100 mg/L)组、LPS+exo(200 mg/L)组的LDH、TNFα、IL1β和IL6浓度，iNOS mRNA、CXCR5 mRNA表达明显高于对照组，LPS+exo(50 mg/L)组、LPS+exo(100 mg/L)组、LPS+exo(200 mg/L)组的LDH、TNFα、IL1β和IL6浓度，iNOS mRNA、CXCR5 mRNA表达明显高于LPS组，LPS+exo(100 mg/L)组、LPS+exo(200 mg/L)组的LDH、TNFα、IL1β和IL6浓度，iNOS mRNA、CXCR5 mRNA表达明显高于LPS+exo(50 mg/L)组，LPS+exo(200 mg/L)组的LDH、TNFα、IL1β和IL6浓度，iNOS mRNA表达明显高于LPS+exo(100 mg/L)组，CXCR5 mRNA表达明显低于LPS+exo(100 mg/L)组，差异均有统计学意义（P＜005）。
〖HTH〗结论〖HTSS〗〖KG*2〗本研究证实GBMexo能促进肿瘤微环境中小胶质细胞介导的炎症反应，增强肿瘤化疗耐药性，这将为进一步认识和治疗脑胶质瘤提供有力的依据。

关键词: 神经胶质瘤, 小神经胶质细胞, 外泌体

Abstract: ［Abstract］〓Objective〖HTSS〗〓To investigate the role and mechanism of exosomes derived from glioblastoma multiforme cell(GBMexo) in facilitating microgliaelicited activation of inflammatory network and chemotherapeutic resistance.
〖HTH〗〖WTHZ〗Methods〖HTSS〗〓U87 and BV2 cells were cultured, and exosomes derived from U87(U87exo) was isolated. We labeled U87exo with GFP, and added them to BV2 cell culture medium for monitoring cargo delivery. Except for PBS control group, BV2 microglias were treated with 1 mg/L lipopolysaccharides(LPS) and U87exo at different concentrations, while they were divided into LPS group, LPS+exo(50 mg/L) group, LPS+exo(100 mg/L) group and LPS+exo(200 mg/L) group. Morphological changes of BV2 microglias in the five groups were observed after 24 h under phasecontrast microscoy. The BV2 cells and the cell cultured supernatant were harvested for detection of lactate dehydrogenase(LDH), tumor necrosis factorα(TNFα), interleukin 1β(IL1β） and interleukin 6(IL6) by ELISA, inducible nitric oxide synthesis(iNOS) and CXC chemokine receptor type 5(CXCR5) mRNA level by RTPCR, as well as protein expression changes by western blotting.
〖HTH〗〖WTHZ〗Results〖HTSS〗〓U87exo were transfered into BV2 cells and promoted microglia activation and morphological changes. Compared with control group, the levels of LDH, TNFα, IL1β, IL6, iNOS mRNA and CXCR5 mRNA were higher in LPS group, LPS+exo(50 mg/L) group, LPS+exo(100 mg/L) group and LPS+exo(200 mg/L) group(P＜005). Compared with LPS group, the levels of LDH, TNFα, IL1β, IL6, iNOS mRNA and CXCR5 mRNA were higher in LPS+exo(50 mg/L) group, LPS+exo(100 mg/L) group and LPS+exo(200 mg/L) group(P＜005). Compared with LPS+exo(50 mg/L) group, the levels of LDH, TNFα, IL1β, IL6, iNOS mRNA and CXCR5 mRNA were higher in LPS+exo(100 mg/L) group and LPS+exo(200 mg/L) group(P＜005). Compared with LPS+exo(100 mg/L) group, the levels of LDH, TNFα, IL1β, IL6 and iNOS mRNA were higher in LPS+exo(200 mg/L) group(P＜005), while CXCR5 mRNA were lower in LPS+exo(200 mg/L) group(P＜005).
〖HTH〗〖WTHZ〗Conclusion〖HTSS〗〓These results proved that GBMexo facilitated microgliaelicited activation of inflammatory network in microenvironment as well as chemotherapeutic resistance, which will provide a powerful basis for further understanding and treatment of glioma.

Key words: glioma, microglia, exosome

杨建凯1，齐雪姣2，焦保华1，赵宗茂1，吕中强1，李〓琛1. GBM细胞来源外泌体促进小胶质细胞介导的炎症反应[J]. 河北医科大学学报, doi: 10.3969/j.issn.10073205.2018.11.017.

YANG Jiankai1, QI Xuejiao2, JIAO Baohua1, ZHAO Zongmao1, LYU Zhongqiang1, LI Chen1. GBMexo promotes microglia mediated inflammatory response#br#[J]. Journal of Hebei Medical University, doi: 10.3969/j.issn.10073205.2018.11.017.

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GBM细胞来源外泌体促进小胶质细胞介导的炎症反应

GBMexo promotes microglia mediated inflammatory response#br#

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