骨髓间充质干细胞分泌半乳凝素1可抑制哮喘小鼠的气道炎症
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摘要
背景:课题组前期研究表明移植骨髓间充质干细胞能减轻哮喘气道炎症程度且能调节哮喘炎症因子水平,进一步体外研究提示骨髓间充质干细胞分泌的半乳凝素1能影响树突状细胞的免疫功能,而骨髓间充质干细胞分泌的半乳凝素1对哮喘的作用尚不明确。目的:观察骨髓间充质干细胞分泌的半乳凝素1对哮喘小鼠气道炎症的影响。方法:构建小鼠pLVX-gal-1-shRNA并感染骨髓间充质干细胞。取40只雌性BALB/c小鼠,随机分为正常对照组、哮喘模型组、骨髓间充质干细胞组、重组半乳凝素1蛋白组、干扰半乳凝素1表达的骨髓间充质干细胞组,对比各组小鼠肺泡灌洗液中炎症细胞总数及分类的差异,采用苏木精-伊红染色观察各组小鼠气道炎症变化。结果与结论:(1)针对小鼠半乳凝素1基因设计了4条RNA干扰序列,通过Real-timePCR验证249干扰位点的干扰序列对半乳凝素1基因干扰效果最佳,并以此进行后续的体内实验;(2)与正常对照组相比,哮喘模型组肺泡和气道周围有大量的炎症细胞聚集,以嗜酸性粒细胞为主;骨髓间充质干细胞组或重组半乳凝素1蛋白组气道及肺泡灌洗液中炎症细胞显著减少,而半乳凝素1基因干扰的骨髓间充质干细胞输注对哮喘小鼠气道及肺泡灌洗液炎症细胞聚集无明显影响;(3)结果表明骨髓间充质干细胞分泌的半乳凝素1对哮喘小鼠气道炎症有明显的抑制作用。
BACKGROUND: Our previous studies have demonstrated that bone marrow mesenchymal stem cells can ameliorate airway inflammation and regulate levels of inflammatory factors. Moreover, galectin-1 secreted by bone marrow mesenchymal stem cells can inhibit the immune function of dendritic cells in vitro. However, the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on asthma is unknown.OBJECTIVE: To investigate the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on airway inflammation of asthmatic mice.METHODS: A recombinant lentiviral vector, pLVX-gal-1-shRNA, was constructed for RNA interference of galectin-1 gene and then was transferred into bone marrow mesenchymal stem cells. Forty female BALB/c mice were equally randomized into normal control group,asthmatic group, bone marrow mesenchymal stem cells treatment group(cell treatment group), galectin-1 treatment group and galectin-1 interference group. The number of total inflammatory cells and differential cells in the mouse bronchoalveolar lavage fluid was determined.Furthermore, hematoxylin-eosin staining was used to compare airway inflammation among five groups.RESULTS AND CONCLUSION: Four short-hairpin RNA sequences targeting mouse galectin-1 mRNA were designed. A real time-quantitative polymerase chain reaction demonstrated that bone marrow mesenchymal stem cells were inhibited most by interference site249 RNA sequences which were later used in vivo study. Accumulation of inflammation cells, particularly eosinophils, around the airway and in the bronchoalveolar lavage fluid was evidence in asthmatic mice compared to the normal control group. However, bone marrow mesenchymal stem cells engraftment or protein of galectin-1 infusion significantly reduced inflammatory infiltration both in the airway and in the bronchoalveolar lavage fluid. Moreover, eosinophils in the bronchoalveolar lavage fluid and in the airway attenuated dramatically in the cell treatment group and galectin-1 treatment group. However, there was no effect on inflammation accumulation in the bronchoalveolar lavage fluid and airway by infusion of galectin-1-transfected bone marrow mesenchymal stem cells to asthmatic mice. These results indicate that galectin-1 secreted by bone marrow mesenchymal stem cells could alleviate airway inflammation in the asthmatic mouse.
BACKGROUND: Our previous studies have demonstrated that bone marrow mesenchymal stem cells can ameliorate airway inflammation and regulate levels of inflammatory factors. Moreover, galectin-1 secreted by bone marrow mesenchymal stem cells can inhibit the immune function of dendritic cells in vitro. However, the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on asthma is unknown.OBJECTIVE: To investigate the effect of galectin-1 secreted by bone marrow mesenchymal stem cells on airway inflammation of asthmatic mice.METHODS: A recombinant lentiviral vector, pLVX-gal-1-shRNA, was constructed for RNA interference of galectin-1 gene and then was transferred into bone marrow mesenchymal stem cells. Forty female BALB/c mice were equally randomized into normal control group,asthmatic group, bone marrow mesenchymal stem cells treatment group(cell treatment group), galectin-1 treatment group and galectin-1 interference group. The number of total inflammatory cells and differential cells in the mouse bronchoalveolar lavage fluid was determined.Furthermore, hematoxylin-eosin staining was used to compare airway inflammation among five groups.RESULTS AND CONCLUSION: Four short-hairpin RNA sequences targeting mouse galectin-1 mRNA were designed. A real time-quantitative polymerase chain reaction demonstrated that bone marrow mesenchymal stem cells were inhibited most by interference site249 RNA sequences which were later used in vivo study. Accumulation of inflammation cells, particularly eosinophils, around the airway and in the bronchoalveolar lavage fluid was evidence in asthmatic mice compared to the normal control group. However, bone marrow mesenchymal stem cells engraftment or protein of galectin-1 infusion significantly reduced inflammatory infiltration both in the airway and in the bronchoalveolar lavage fluid. Moreover, eosinophils in the bronchoalveolar lavage fluid and in the airway attenuated dramatically in the cell treatment group and galectin-1 treatment group. However, there was no effect on inflammation accumulation in the bronchoalveolar lavage fluid and airway by infusion of galectin-1-transfected bone marrow mesenchymal stem cells to asthmatic mice. These results indicate that galectin-1 secreted by bone marrow mesenchymal stem cells could alleviate airway inflammation in the asthmatic mouse.
引文
[1]Ge X,Bai C,Yang J,et al.Effect of mesenchymal stem cells on inhibiting airway remodeling and airway inflammation in chronic asthma.J Cell Biochem.2013;114(7):1595-1605.
[2]Ge X,Bai C,Yang J,et al.Intratracheal transplantation of bone marrow-derived mesenchymal stem cells reduced airway inflammation and up-regulated CD4?CD25?regulatory T cells in asthmatic mouse.Cell Biol Int.2013;37(7):675-686.
[3]戈霞晖,白冲,陈若华,等.骨髓间充质干细胞移植对慢性哮喘小鼠气道炎症及气道重塑的影响[J].中国呼吸与危重监护杂志,2011,10(1):6-10.
[4]戈霞晖,白冲,陈若华,等.骨髓间充质干细胞对慢性哮喘小鼠血清IL-12和IL-4水平的影响[J].中国组织工程研究与临床康复杂志,2011,15(6):1030-1034.
[5]Zhang Y,Ge XH,Guo XJ,et al.Bone Marrow Mesenchymal Stem Cells Inhibit the Function of Dendritic Cells by Secreting Galectin-1.Biomed Res Int.2017;2017:3248605.
[6]沈璐,赖克方,姜华,等.不同激发方式对小鼠过敏性支气管哮喘模型的影响[J].中华哮喘杂志(电子版),2009,3(6):404-408.
[7]Rabinovich GA,Toscano MA.Turning'sweet'on immunity:galectin-glycan interactions in immune tolerance and inflammation.Nat Rev Immunol.2009;9(5):338-352.
[8]Barrionuevo P,Beigier-Bompadre M,Ilarregui JM,et al.Anovel function for galectin-1 at the crossroad of innate and adaptive immunity:galectin-1 regulates monocyte/macrophage physiology through a nonapoptotic ERK-dependent pathway.J Immunol.2007;178(1):436-445.
[9]Moore WC,Meyers DA,Wenzel SE,et al.Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program.Am J Respir Crit Care Med.2010;181(4):315-323.
[10]Gibson PG.Inflammatory phenotypes in adult asthma:clinical applications.Clin Respir J.2009;3(4):198-206.
[11]Wenzel SE.Asthma:defining of the persistent adult phenotypes.Lancet.2006;368(9537):804-813.
[12]impson JL,Scott R,Boyle MJ,et al.Inflammatory subtypes in asthma:assessment and identification using induced sputum.Respirology.2006;11(1):54-61.
[13]Halwani R,Al-Muhsen S,Al-Jahdali H,et al.Role of transforming growth factor-βin airway remodeling in asthma.Am J Respir Cell Mol Biol.2011;44(2):127-133.
[14]Castro M,Wenzel SE,Bleecker ER,et al.Benralizumab,an anti-interleukin 5 receptorαmonoclonal antibody,versus placebo for uncontrolled eosinophilic asthma:a phase 2b randomised dose-ranging study.Lancet Respir Med.2014;2(11):879-890.
[15]Lin YD,Fan XL,Zhang H,et al.The genes involved in asthma with the treatment of human embryonic stem cell-derived mesenchymal stem cells.Mol Immunol.2018;95:47-55.
[16]Lepelletier Y,Lecourt S,Renand A,et al.Galectin-1 and semaphorin-3A are two soluble factors conferring T-cell immunosuppression to bone marrow mesenchymal stem cell.Stem Cells Dev.2010;19(7):1075-1079.
[17]Sioud M,Mobergslien A,Boudabous A,et al.Mesenchymal stem cell-mediated T cell suppression occurs through secreted galectins.Int J Oncol.2011;38(2):385-390.
[18]Ge XN,Ha SG,Greenberg YG,et al.Regulation of eosinophilia and allergic airway inflammation by the glycan-binding protein galectin-1.Proc Natl Acad Sci U S A.2016;113(33):E4837-4846.
[2]Ge X,Bai C,Yang J,et al.Intratracheal transplantation of bone marrow-derived mesenchymal stem cells reduced airway inflammation and up-regulated CD4?CD25?regulatory T cells in asthmatic mouse.Cell Biol Int.2013;37(7):675-686.
[3]戈霞晖,白冲,陈若华,等.骨髓间充质干细胞移植对慢性哮喘小鼠气道炎症及气道重塑的影响[J].中国呼吸与危重监护杂志,2011,10(1):6-10.
[4]戈霞晖,白冲,陈若华,等.骨髓间充质干细胞对慢性哮喘小鼠血清IL-12和IL-4水平的影响[J].中国组织工程研究与临床康复杂志,2011,15(6):1030-1034.
[5]Zhang Y,Ge XH,Guo XJ,et al.Bone Marrow Mesenchymal Stem Cells Inhibit the Function of Dendritic Cells by Secreting Galectin-1.Biomed Res Int.2017;2017:3248605.
[6]沈璐,赖克方,姜华,等.不同激发方式对小鼠过敏性支气管哮喘模型的影响[J].中华哮喘杂志(电子版),2009,3(6):404-408.
[7]Rabinovich GA,Toscano MA.Turning'sweet'on immunity:galectin-glycan interactions in immune tolerance and inflammation.Nat Rev Immunol.2009;9(5):338-352.
[8]Barrionuevo P,Beigier-Bompadre M,Ilarregui JM,et al.Anovel function for galectin-1 at the crossroad of innate and adaptive immunity:galectin-1 regulates monocyte/macrophage physiology through a nonapoptotic ERK-dependent pathway.J Immunol.2007;178(1):436-445.
[9]Moore WC,Meyers DA,Wenzel SE,et al.Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program.Am J Respir Crit Care Med.2010;181(4):315-323.
[10]Gibson PG.Inflammatory phenotypes in adult asthma:clinical applications.Clin Respir J.2009;3(4):198-206.
[11]Wenzel SE.Asthma:defining of the persistent adult phenotypes.Lancet.2006;368(9537):804-813.
[12]impson JL,Scott R,Boyle MJ,et al.Inflammatory subtypes in asthma:assessment and identification using induced sputum.Respirology.2006;11(1):54-61.
[13]Halwani R,Al-Muhsen S,Al-Jahdali H,et al.Role of transforming growth factor-βin airway remodeling in asthma.Am J Respir Cell Mol Biol.2011;44(2):127-133.
[14]Castro M,Wenzel SE,Bleecker ER,et al.Benralizumab,an anti-interleukin 5 receptorαmonoclonal antibody,versus placebo for uncontrolled eosinophilic asthma:a phase 2b randomised dose-ranging study.Lancet Respir Med.2014;2(11):879-890.
[15]Lin YD,Fan XL,Zhang H,et al.The genes involved in asthma with the treatment of human embryonic stem cell-derived mesenchymal stem cells.Mol Immunol.2018;95:47-55.
[16]Lepelletier Y,Lecourt S,Renand A,et al.Galectin-1 and semaphorin-3A are two soluble factors conferring T-cell immunosuppression to bone marrow mesenchymal stem cell.Stem Cells Dev.2010;19(7):1075-1079.
[17]Sioud M,Mobergslien A,Boudabous A,et al.Mesenchymal stem cell-mediated T cell suppression occurs through secreted galectins.Int J Oncol.2011;38(2):385-390.
[18]Ge XN,Ha SG,Greenberg YG,et al.Regulation of eosinophilia and allergic airway inflammation by the glycan-binding protein galectin-1.Proc Natl Acad Sci U S A.2016;113(33):E4837-4846.
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