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浙江大学学报(医学版)  2020, Vol. 49 Issue (6): 758-764    DOI: 10.3785/j.issn.1008-9292.2020.12.11
原著     
趋化因子CXCL12可能参与白细胞介素17A对小鼠肺成纤维细胞的活化
王华英(),吕佳佩,陈丽萍,俞万钧*()
宁波大学附属人民医院 宁波市鄞州人民医院呼吸与危重症医学科, 浙江 宁波 315040
IL-17A activates mouse lung fibroblasts through promoting chemokine CXCL12 secretion
WANG Huaying(),LYU Jiapei,CHEN Liping,YU Wanjun*()
Department of Respiratory and Critical Care Medicine, People's Hospital Affiliated to Ningbo University, Yinzhou People's Hospital, Ningbo 315040, Zhejiang Province, China
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摘要:

目的: 研究IL-17A促进小鼠肺成纤维细胞活化及趋化因子CXCL12在其中的作用。方法: 以雄性BALB/c小鼠为研究对象,小鼠腹腔注射重组小鼠IL-17A后取小鼠肺组织。采用实时逆转录PCR和蛋白质印迹法检测小鼠肺组织α平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原蛋白的mRNA和蛋白表达,采用免疫组织化学法和实时逆转录PCR法测定肺组织中趋化因子CXCL12的表达。分离培养正常小鼠原代肺成纤维细胞,采用免疫荧光染色法和光学显微镜观察鉴定原代肺成纤维细胞。将分离的肺成纤维细胞与不同浓度的重组小鼠IL-17A共培养后收集细胞及上清液,采用实时逆转录PCR法测定细胞中α-SMA、Ⅰ型胶原蛋白和CXCL12的mRNA水平,采用ELISA法测定培养上清液中Ⅰ型胶原蛋白和CXCL12蛋白水平。结果: 与对照组比较,重组小鼠IL-17A处理后小鼠肺组织中α-SMA、Ⅰ型胶原蛋白的mRNA和蛋白水平均升高(均P < 0.01)。与不同浓度的重组小鼠IL-17A共培养后,小鼠肺成纤维细胞表达α-SMA、Ⅰ型胶原蛋白和CXCL12的mRNA明显增加(均P < 0.01),其培养基上清液中Ⅰ型胶原蛋白、CXCL12的浓度也明显升高(均P < 0.01),且呈剂量依赖性。结论: IL-17A能促进肺成纤维细胞的活化并转化为肌成纤维细胞,分泌Ⅰ型胶原蛋白明显增加,促进细胞外基质的沉积,从而导致肺纤维化的发生和发展,而活化的成纤维细胞分泌的趋化因子CXCL12可能参与了这个过程。

关键词: 白细胞介素17A趋化因子CXCL12成纤维细胞α平滑肌肌动蛋白Ⅰ型胶原蛋白小鼠    
Abstract:

Objective: To investigate the role of IL-17A in promoting the activation of lung fibroblasts and the secretion of chemokine CXCL12, and to explore the possible mechanism. Methods: Lung tissues of BALB/c mice were collected after intraperitoneal injection of recombinant mouse IL-17A (rmIL-17A). Real-time RT-PCR and Western blotting were used to detect the expression levels of α-smooth muscle actin (α-SMA) and collagen I in lung tissues, and immunohistochemical staining and real-time RT-PCR were used to determine the expression of CXCL12. Normal mouse primary lung fibroblasts were isolated and cultured, and identified by immunofluorescence staining with optical microscopy. Cells and supernatant of culture medium were collected after stimulation with rmIL-17A at different concentrations. mRNA levels of α-SMA, collagen I, and CXCL12 in the cells were determined by real-time RT-PCR, and the levels of collagen I and CXCL12 in the supernatant of culture medium were determined by ELISA. Results: The mRNA and protein levels of α-SMA and collagen I in the lung tissue of mice injected with rmIL-17A were significantly increased compared with the control group (all P < 0.01). The mRNA levels of α-SMA, collagen I and CXCL12 in mice primary lung fibroblasts were increased after stimulation of rmIL-17A at different concentrations (all P < 0.01), and the concentration of collagen Ⅰ and CXCL12 in the supernatants of culture medium were also increased in a dose-dependent manner (all P < 0.01). Conclusions: IL-17A can promote the activation of lung fibroblasts and translation into myofibroblast. The secretion of collagen is increased, which promote the deposition of extracullular matrix, and leads to the occurrence and development of lung fibrosis. CXCL12, a chemokine secreted by activated fibroblasts, may be involved in this process.

Key words: Interleukin-17A    Chemokine    CXCL12    Fibroblast    α-smooth muscle actin    TypeⅠ collagen    Mice
收稿日期: 2020-05-15 出版日期: 2021-01-14
CLC:  R363  
基金资助: 国家自然科学基金(81800040)
通讯作者: 俞万钧     E-mail: yingmeire@163.com;nbyuwanjun@163.com
作者简介: 王华英(1978-), 女, 博士, 副主任医师, 主要从事慢性气道炎症性疾病的免疫学发病机制研究; E-mail:yingmeire@163.com; https://orcid.org/0000-0003-3775-8897
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引用本文:

王华英,吕佳佩,陈丽萍,俞万钧. 趋化因子CXCL12可能参与白细胞介素17A对小鼠肺成纤维细胞的活化[J]. 浙江大学学报(医学版), 2020, 49(6): 758-764.

WANG Huaying,LYU Jiapei,CHEN Liping,YU Wanjun. IL-17A activates mouse lung fibroblasts through promoting chemokine CXCL12 secretion. J Zhejiang Univ (Med Sci), 2020, 49(6): 758-764.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.11        http://www.zjujournals.com/med/CN/Y2020/V49/I6/758

引物名称 引物序列(5′→3′)
α-SMA:α平滑肌肌动蛋白;collagen Ⅰ:Ⅰ型胶原蛋白.
CXCL12 正向:GGAGGATAGATGTGCTCTGGAAC
反向:AGTGAGGATGGAGACCGTGGTG
α-SMA 正向:CCCAGACATCAGGGAGTAATGG
反向:TCTATCGGATACTTCAGCGTCA
collagenⅠ 正向:TAAGGGTCCCCAATGGTGAGA
反向:GGGTCCCTCGACTCCTACAT
β-actin 正向:GTGACGTTGACATCCGTAAAGA
反向:GCCGGACTCATCGTACTCC
表 1  实时逆转录PCR分析引物序列
图 1  IL-17A组和对照组肺组织中Ⅰ型胶原蛋白和α平滑肌肌动蛋白表达的电泳图
图 2  IL-17A组和对照组肺组织中CXCL12、α平滑肌肌动蛋白和Ⅰ型胶原蛋白mRNA表达量比较
图 3  IL-17A组和对照组小鼠肺组织中CXCL12的表达
图 4  BALB/c小鼠原代肺成纤维细胞显微镜下表现
图 5  IL-17A对小鼠原代成纤维细胞中α平滑肌肌动蛋白、Ⅰ型胶原蛋白表达的影响
图 6  IL-17A对小鼠原代肺成纤维细胞表达趋化因子CXCL12的影响
1 DISTLER J , GY?RFI A H , RAMANUJAM M et al. Shared and distinct mechanisms of fibrosis[J]. Nat Rev Rheumatol, 2019, 15 (12): 705- 730
doi: 10.1038/s41584-019-0322-7
2 ZHANG J , WANG D , WANG L et al. Profibrotic effect of IL-17A and elevated IL-17RA in idiopathic pulmonary fibrosis and rheumatoid arthritis-associated lung disease support a direct role for IL-17A/IL-17RA in human fibrotic interstitial lung disease[J]. Am J Physiol Lung Cell Mol Physiol, 2019, 316 (3): L487- L497
doi: 10.1152/ajplung.00301.2018
3 SHU H K , YOON Y , HONG S et al. Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis[J]. PLoS One, 2013, 8 (11): e79768
doi: 10.1371/journal.pone.0079768
4 WILLIS B C , DUBOIS R M , BOROK Z . Epithelial origin of myofibroblasts during fibrosis in the lung[J]. Proc Am Thorac Soc, 2006, 3 (4): 377- 382
doi: 10.1513/pats.200601-004TK
5 BETTELLI E , KORN T , OUKKA M et al. Induction and effector functions of T(H)17 cells[J]. Nature, 2008, 453 (7198): 1051- 1057
doi: 10.1038/nature07036
6 EVASOVIC J M , SINGER C A . Regulation of IL-17A and implications for TGF-β1 comodulation of airway smooth muscle remodeling in severe asthma[J]. Am J Physiol Lung Cell Mol Physiol, 2019, 316 (5): L843- L868
doi: 10.1152/ajplung.00416.2018
7 DOS SANTOS T M , RIGHETTI R F , CAMARGO L et al. Effect of anti-IL17 antibody treatment alone and in combination with rho-kinase inhibitor in a murine model of asthma[J]. Front Physiol, 2018, 9:1183
doi: 10.3389/fphys.2018.01183
8 WANG H , PENG W , WENG Y et al. Imbalance of Th17/Treg cells in mice with chronic cigarette smoke exposure[J]. Int Immunopharmacol, 2012, 14 (4): 504- 512
doi: 10.1016/j.intimp.2012.09.011
9 WANG H , YING H , WANG S et al. Imbalance of peripheral blood Th17 and Treg responses in patients with chronic obstructive pulmonary disease[J]. Clin Respir J, 2015, 9 (3): 330- 341
doi: 10.1111/crj.12147
10 BONNER J C . Regulation of PDGF and its receptors in fibrotic diseases[J]. Cytokine Growth Factor Rev, 2004, 15 (4): 255- 273
doi: 10.1016/j.cytogfr.2004.03.006
11 JANSSENS R , STRUYF S , PROOST P . Pathological roles of the homeostatic chemokine CXCL12[J]. Cytokine Growth Factor Rev, 2018, 44:51- 68
doi: 10.1016/j.cytogfr.2018.10.004
12 DAUBEUF F , HACHET-HAAS M , GIZZI P et al. An antedrug of the CXCL12 neutraligand blocks experimental allergic asthma without systemic effect in mice[J]. J Biol Chem, 2013, 288 (17): 11865- 11876
doi: 10.1074/jbc.M112.449348
13 GONZALO J A , LLOYD C M , PELED A et al. Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of allergic airway disease[J]. J Immunol, 2000, 165 (1): 499- 508
doi: 10.4049/jimmunol.165.1.499
14 MAKINO H , AONO Y , AZUMA M et al. Antifibrotic effects of CXCR4 antagonist in bleomycin-induced pulmonary fibrosis in mice[J]. J Med Invest, 2013, 60 (1-2): 127- 137
doi: 10.2152/jmi.60.127
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