TLRs信号通路激活的MSCs外泌体对巨噬细胞极化的影响

doi:10.3877/cma.j.issn.2095-1221.2018.02.001

中华细胞与干细胞杂志(电子版) ›› 2018, Vol. 08 ›› Issue (02) : 65 -71. doi: 10.3877/cma.j.issn.2095-1221.2018.02.001

所属专题：文献；

论著

TLRs信号通路激活的MSCs外泌体对巨噬细胞极化的影响

冉凤英¹, 陈龙², 张斌强², 尚兵², 余飞², 陈炜², 陈琴华¹^,^†(

)

^1. 437100　咸宁，湖北科技学院药学院；442000　十堰，湖北医药学院附属东风医院实验中心
^2. 442000　十堰，湖北医药学院附属东风医院实验中心

收稿日期:2018-01-31 出版日期:2018-04-01
通信作者: 陈琴华
基金资助:
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)

Effect of MSCs exosomes activated by TLRs signaling pathway on the polarization of macrophages

Fengying Ran¹, Long Chen², Bingqiang Zhang², Bing Shang², Fei Yu², Wei Chen², Qinhua Chen¹^,^†()

^1. School of Pharmacy, Hubei University of Science and Technology, Xianning 437100, China; Medical Experimental Center, Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan 442000, China
^2. Medical Experimental Center, Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan 442000, China

Received:2018-01-31 Published:2018-04-01
Corresponding author: Qinhua Chen
About author:
Corresponding author: Chen Qinhua, Email: cqh77@163.com

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引用本文：

冉凤英, 陈龙, 张斌强, 尚兵, 余飞, 陈炜, 陈琴华. TLRs信号通路激活的MSCs外泌体对巨噬细胞极化的影响[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(02): 65-71.

Fengying Ran, Long Chen, Bingqiang Zhang, Bing Shang, Fei Yu, Wei Chen, Qinhua Chen. Effect of MSCs exosomes activated by TLRs signaling pathway on the polarization of macrophages[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2018, 08(02): 65-71.

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 ~ 200 nm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iNOS（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iNOS表达差异无统计学意义（P > 0.05）。qPCR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±0.073 vs 1.000±0.082，F = 12.315，P = 0.000、0.005、0.000）、IL-6（0.386±0.066、0.655±0.046、0.533±0.090 vs 1.000±0.204，F = 30.140，P = 0.001、0.006、0.016）。

结论

TLR3和TLR4信号通路激活的MSCs-Exo均能促使巨噬细胞向M2型极化。

关键词: 骨髓间充质干细胞, 外泌体, Toll样受体信号通路, 巨噬细胞

Objective

To investigate the effect of MSCs exosomes (mesenchymal stem cells derived exosome, MSCs-Exo) activated byToll-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 iNOS ) 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 qPCR, 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, MSCs-Exo 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 cytometry in the groups withthe macrophages activated by MSCs-Exo, MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways, and low expression ofiNOS (240.2±8.43 vs 308.8±9.88, P = 0.000)with the macrophages activated by MSCs-Exo,and invariant expression of iNOS (P > 0.05) with the macrophages activated by MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways.High expression of 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) and 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), low expression ofIL-1β (0.383±0.035, 0.640±0.143, 0.242±0.073 vs 1.000±0.082, F = 12.315, P = 0.000, 0.005, 0.000)and IL-6 (0.386±0.066, 0.655±0.046, 0.533±0.090 vs 1.000±0.204, F = 30.140, P = 0.001, 0.006, 0.016) were detected by qPCR.

Conclusion

Polarization of macrophages to M2 type can be stimulated by MSCs-Exo activated by TLR3 and TLR4 signaling pathway.

Key words: Mesenchymal stem cells, Derived exosome, Toll like receptor signaling pathway, Macrophages

表1 引物序列

基因名称	上游引物（5'-3'）	下游引物（5'-3'）	产物大小（bp）
GAPDH	ATGCTGGTGCTGAGTATGTC	AGTTGTCATATTTCTCGTGG	162
IL-1β	AATGACCTGTTCTTTGAGGCTGAC	CGAGATGCTGCTGTGAGATTTGAAG	115
CCL22	GGCAGGAAGGACCATACAAA	TCCAGAGGAGCAAGCAGATT	200
IL-6	AGAGACTTCCAGCCAGTTGC	AGTCTCCTCTCCGGACTTGT	85
IL-10	GAAGCTGAAGACCCTCTGGATACA	CCTTTGTCTTGGAGCTTATTAAAATCA	117

表1 引物序列

基因名称	上游引物（5'-3'）	下游引物（5'-3'）	产物大小（bp）
GAPDH	ATGCTGGTGCTGAGTATGTC	AGTTGTCATATTTCTCGTGG	162
IL-1β	AATGACCTGTTCTTTGAGGCTGAC	CGAGATGCTGCTGTGAGATTTGAAG	115
CCL22	GGCAGGAAGGACCATACAAA	TCCAGAGGAGCAAGCAGATT	200
IL-6	AGAGACTTCCAGCCAGTTGC	AGTCTCCTCTCCGGACTTGT	85
IL-10	GAAGCTGAAGACCCTCTGGATACA	CCTTTGTCTTGGAGCTTATTAAAATCA	117

图1 倒置荧光显微镜下观察MSCs形态及成骨成脂诱导结果（油红O及茜素红S染色，×200）

图2 流式细胞术对MSCs表面标记物的鉴定结果

图3 透射电镜下MSCs-Exo的形态(200 nm，×30000)

图4 Western blot鉴定MSCs-Exo

图5 倒置相差显微镜下观察巨噬细胞形态（×400）

表2 FCM分析各组巨噬细胞表面蛋白CD206、Arg-1、iNOS、TNF-α的表达（±s）

分组	CD206	Arg-1	iNOS	TNF-α
M0组	56.0± 7.38	117.8±9.12	308.8±9.88	36.6±7.09
M1组	68.4±10.26	130.0±7.68	656.6±9.04^a	72.4±9.18^a
M2组	113.2± 6.30^a	163.8±8.81^a	320.4±5.94	28.6±5.27
MSCs-Exo组	107.2±6.87^a	135.2±6.87^a	240.2±8.43^a	27.0±5.65^a
TLR3MSCs-Exo组	102.4±9.83^a	130.2±7.59^a	320.6±7.33	24.6±5.02^a
TLR4MSCs-Exo组	112.0±9.24^a	203.4±9.07^a	321.6±8.41	25.6±4.15^a
F值	47.234	109.827	102.783	4.882
P值	0.000	0.000	0.000	0.013

表2 FCM分析各组巨噬细胞表面蛋白CD206、Arg-1、iNOS、TNF-α的表达（±s）

分组	CD206	Arg-1	iNOS	TNF-α
M0组	56.0± 7.38	117.8±9.12	308.8±9.88	36.6±7.09
M1组	68.4±10.26	130.0±7.68	656.6±9.04^a	72.4±9.18^a
M2组	113.2± 6.30^a	163.8±8.81^a	320.4±5.94	28.6±5.27
MSCs-Exo组	107.2±6.87^a	135.2±6.87^a	240.2±8.43^a	27.0±5.65^a
TLR3MSCs-Exo组	102.4±9.83^a	130.2±7.59^a	320.6±7.33	24.6±5.02^a
TLR4MSCs-Exo组	112.0±9.24^a	203.4±9.07^a	321.6±8.41	25.6±4.15^a
F值	47.234	109.827	102.783	4.882
P值	0.000	0.000	0.000	0.013

表3 qPCR分析各组巨噬细胞炎症因子IL-10、CCL22、IL-1β、IL-6的表达（±s）

分组	IL-10	CCL22	IL-1β	IL-6
M0组	1.000±0.130^a	1.000±0.111^a	1.000±0.082^a	1.000±0.204^a
M1组	0.475±0.098	0.196±0.050	1.915±0.100	1.584±0.230
M2组	1.967±0.353	12.082±2.250	0.042±0.009	0.613±0.063
MSCs-Exo组	1.233±0.136	2.277±0.744	0.383±0.035	0.386±0.066
TLR4MSCs-Exo组	1.390±0.155	1.642±0.443	0.242±0.073	0.533±0.090
TLR3MSCs-Exo组	2.426±0.343	1.570±0.209	0.640±0.143	0.655±0.046
F值	103.251	23.654	12.315	30.140
P值	0.000	0.000	0.001	0.000

表3 qPCR分析各组巨噬细胞炎症因子IL-10、CCL22、IL-1β、IL-6的表达（±s）

分组	IL-10	CCL22	IL-1β	IL-6
M0组	1.000±0.130^a	1.000±0.111^a	1.000±0.082^a	1.000±0.204^a
M1组	0.475±0.098	0.196±0.050	1.915±0.100	1.584±0.230
M2组	1.967±0.353	12.082±2.250	0.042±0.009	0.613±0.063
MSCs-Exo组	1.233±0.136	2.277±0.744	0.383±0.035	0.386±0.066
TLR4MSCs-Exo组	1.390±0.155	1.642±0.443	0.242±0.073	0.533±0.090
TLR3MSCs-Exo组	2.426±0.343	1.570±0.209	0.640±0.143	0.655±0.046
F值	103.251	23.654	12.315	30.140
P值	0.000	0.000	0.001	0.000

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