摘要
目的探讨Toll样受体(TLR)3和4信号通路激活的间充质干细胞外泌体(MSCs-Exo)对巨噬细胞极化的影响。方法差速贴壁法体外培养大鼠骨髓源MSCs,用外泌体提取试剂盒分别提取MSCs、TLR3信号通路激活的MSCs、TLR4信号通路激活的MSCs培养上清中的外泌体。用含10﹪FBS、10﹪L929条件培养基的RPMI-1640培养得M0型巨噬细胞,实验分6组:对照组及MSCs-Exo、TLR3信号通路激活的MSCs-Exo、TLR4信号通路激活的MSCs-Exo、LPS、IL-4+IL-13分别与M0型巨噬细胞共培养,48 h后收集各组巨噬细胞光镜下观察形态,流式和qPCR检测免疫表型(CD206、Arg-1、TNF-α、iNOS)及炎症因子(CCL22、IL-1β、IL-6、IL-10)表达的改变。组间比较采用单因素方差分析及独立t检验进行统计学分析。结果 MSCs鉴定符合间充质干细胞特性,MSCs-Exo为双层膜囊泡结构,直径在40~200nm之间,表达外泌体标志性蛋白CD9、HSP70;光镜下观察各组巨噬细胞形态,加MSCs-Exo及TLR3和TLR4信号通路激活的MSCs-Exo刺激的巨噬细胞呈长梭形,伪足较多;流式检测发现,加MSCs-Exo及TLR3和TLR4信号通路激活的MSCs-Exo刺激的巨噬细胞均高表达CD206(107.2±6.87、102.4±9.83、112.0±9.24 vs 56.0±7.38,F=47.234,P均<0.001)、Arg-1(135.2±6.87、130.2±7.59、203.4±9.07 vs 117.8±9.12,F=109.827,P=0.009、0.048、0.000);低表达TNF-α(27.0±5.65、24.6±5.02、25.6±4.15 vs 36.6±7.09,F=4.882,P=0.046、0.015、0.017),而MSCs-Exo刺激的巨噬细胞低表达i NOS(240.2±8.43 vs 308.8±9.88,P<0.001);TLR3和TLR4信号通路激活的MSCs-Exo刺激的巨噬细胞i NOS表达差异无统计学意义(P>0.05)。q PCR检测发现,加MSCs-Exo及TLR3和TLR4信号通路激活的MSCs-Exo刺激的巨噬细胞均高表达CCL22(2.277±0.744、1.570±0.209、1.642±0.443 vs 1.000±0.111,F=23.654,P=0.015、0.003、0.031)、IL-10(1.233±0.136、2.426±0.343、1.390±0.155 vs 1.000±0.130,F=103.251,P=0.048、0.000、0.008),低表达IL-1β(0.383±0.035、0.640±0.143、0.242±0.073vs 1.0
Objective To investigate the effect of MSCs exosomes（mesenchymal stem cells derived exosome, MSCs-Exo） activated by Toll-like receptors 3 and 4 signaling pathways on the polarization of macrophages. Methods After cultured MSCs sourced fromrat bone marrow by differential adherence, exosomesfrom MSCs, MSCs activated by TLR3 signaling and MSCs activated by TLR4 signaling were extracted from cell culture supernatant by exosome Extraction Kit. M0 macrophages cultured with RPMI-1640 containing 10﹪FBS and 10 ﹪ L929 medium,were divided into 6 groups： a control group, the MSCs-Exo, MSCs-Exo activated by TLR4 signaling pathways, MSCs-Exo activated by TLR3 signaling pathways, LPSand IL-4＋IL-13. After cultured for 48 h, morphology of macrophages was observed under an electronic microscope. The immunophenotype（CD206, Arg-1, TNF-α and i NOS） and difference of expression of inflammatory factors（CCL22, IL-1β, IL-6 and IL-10） of macrophages in each group were detected by flow and q PCR, respectively.Single factor analysis of variance and independent t test were used for statistical analysis. Results The cultured MSCs are identical with the characteristics of mesenchymal stem cells. The results showed that MSCs-Exo was a double layer membrane vesicle structure with the diameter of 40 ~ 200 nm, expressing ofexosomes protein CD9 and HSP70. The morphology of macrophages in each group was observed under an electronic microscope, Morphology of long carboxylic shape and many pseudopods were observed in groups withmacrophages stimulated by MSCs-Exo, MSCsExo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways. High expression of CD206（107.2±6.87, 102.4±9.83, 112.0±9.24 vs 56.0±7.38, F = 47.234, P = 0.000, respectively） and Arg-1（135.2±6.87, 130.2±7.59, 203.4±9.07 vs 117.8±9.12, F = 109.827, P = 0.009, 0.048, 0.000）, and low expression of TNF-α（27.0±5.65, 24.6±5.02, 25.6±4.15 vs 36.6±7.09, F = 4.882, P = 0.046, 0.015, 0.017）were respectively detected by flow
作者
冉凤英
陈龙
张斌强
尚兵
余飞
陈炜
陈琴华
Ran Fengying1,2,Chen Long2, Zhang Bingqiang2, Shang Bing2, Yu Fei2, Chen Wei2, Chen Qinhua1,2(1School of Pharmacy, Hubei University of Science and Technology, Xianning 437100, China; 2Medical Experimental Center, Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan 442000, Chin)
出处
《中华细胞与干细胞杂志（电子版）》
2018年第2期65-71,共7页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
基金
湖北省教育厅科学研究计划资助项目(D20172101)
湖北医药学院校基金立项资助项目(FDFR201614)
十堰市科学技术研究与开发项目(17Y49)
