摘要
目的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。方法培养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,4h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(50mg/L)组、LPS+exo(100mg/L)组和LPS+exo(200mg/L)组。加入外泌体后孵育24h,倒置相差显微镜观察BV2细胞的形态变化。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,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。结果 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(50mg/L)组、LPS+exo(100mg/L)组、LPS+exo(200mg/L)组的LDH、TNF-α、IL-1β和IL-6浓度,iNOS mRNA、CXCR5mRNA表达明显高于对照组,LPS+exo(50mg/L)组、LPS+exo(100mg/L)组、LPS+exo(200mg/L)组的LDH、TNF-α、IL-1β和IL-6浓度,iNOS mRNA、CXCR5mRNA表达明显高于LPS组,LPS+exo(100mg/L)组、LPS+exo(200mg/L)组的LDH、TNF-α、IL-1β和IL-6浓度,iNOS mRNA、CXCR5mRNA表达明显高于LPS+exo(50mg/L)组,LPS+exo(200mg/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,CXCR5mRNA表达明显低于LPS+exo(100mg/L)组,差异均有统计学意义(P<0.05)。结论本研究证实GBMexo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,�
Objective 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.Methods 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.Results 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-
作者
杨建凯
齐雪姣
焦保华
赵宗茂
吕中强
李琛
YANG Jian-kai;QI Xue-jiao;JIAO Bao-hua;ZHAO Zong-mao;LYU Zhong-qiang;LI Chen(Department of Neurosurgery,the Second Hospital of Hebei Medical University,Shijiazhuang050000,China;Neurological Laboratory of Hebei Province,Shijiazhuang 050000,China)
出处
《河北医科大学学报》
CAS
2018年第11期1311-1315,共5页
Journal of Hebei Medical University
基金
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)
