Toll样受体3对胚胎骨髓来源间充质干细胞免疫调节Th17细胞的作用

doi:10.3969/j.issn.2095-4344.2014.45.001

摘要/Abstract

摘要：

背景：课题组前期研究发现胚胎骨髓来源的间充质干细胞促进人Th17细胞增殖，但内在的调节机制尚需阐明。

目的：探讨Toll样受体3对胚胎骨髓来源间充质干细胞免疫调节Th17细胞的作用。

方法：应用免疫磁珠分离正常人CD4⁺ T细胞，单独或与胚胎来源骨髓间充质干细胞共培养4 d。实时定量PCR测定白细胞介素17、Toll样受体3及MyD88相关基因的表达水平。

结果与结论：相对于CD4⁺ T细胞单独培养组，间充质干细胞共培养组白细胞介素17 mRNA表达水平明显升高(3.59±0.11 vs. 1.14±0.08，P < 0.01)；进一步发现Toll样受体3 mRNA亦相应升高(3.10±1.60 vs. 0.94±0.01，P < 0.05)。而且，相对于CD4⁺T细胞单独培养组，胚胎骨髓来源间充质干细胞共培养组促进了MyD88的表达(2.29± 0.05 vs. 1.85± 0.31，P < 0.01)。研究结果表明Toll样受体3可能对胚胎骨髓来源间充质干细胞免疫调节Th17细胞发挥作用，为今后潜在的细胞治疗策略提供可能的实验依据。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

关键词: 干细胞, 骨髓干细胞, 免疫调节, TLR3, Th17细胞, 白细胞介素17, MyD88, 国家自然科学基金

Abstract:

BACKGROUND: Previous studies have found that embryonic bone marrow mesenchymal stem cells can promote human Th17 cell proliferation, but the inherent regulatory mechanisms still need to be elucidated.

OBJECTIVE: To investigate the role of Toll-like receptor 3 in the immunoregulation of Th17 cells by mesenchymal stem cells.

METHODS: Human CD4⁺ T cells from healthy donors were isolated by immunomagnetic bead method, and then cultured alone or co-cultured with embryonic bone marrow mesenchymal stem cells for 4 days. The mRNA expression level of interleukin-17, Toll-like receptor 3 and MyD88 was detected by real-time PCR.

RESULTS AND CONCLUSION: Compared with CD4⁺ T cell cultured alone group, the mRNA level of interleukin-17 was significantly higher in the co-culture group (3.59±0.11 vs. 1.14± 0.08, P < 0.01). Consistent with the expression of interleukin-17 mRNA, increased level of Toll-like receptor 3 mRNA was detected in the co-culture group compared with the CD4⁺ T cell cultured alone group (3.10±1.60 vs. 0.94± 0.01, P < 0.05). Furthermore, MyD88 in the co-culture group was significantly higher than that in CD4⁺ T cell cultured alone group (2.29±0.05 vs. 1.85±0.31, P < 0.01). Toll-like receptor 3 may be involved in the immunoregulation of Th17 cells by embryonic bone marrow mesenchymal stem cells, which provides experimental evidence for potential cell therapeutic strategy.

全文链接：

Key words: mesenchymal stem cells, toll-like receptor 3, Th17 cells, immunomodulation

中图分类号:

R394.2

郭振兴，陈振萍. Toll样受体3对胚胎骨髓来源间充质干细胞免疫调节Th17细胞的作用[J]. 中国组织工程研究, 2014, 18(45): 7217-7221.

Guo Zhen-xing, Chen Zhen-ping. Role of Toll-like receptor 3 in the immunoregulatory function of embryonic bone marrow mesenchymal stem cells on Th17 cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(45): 7217-7221.

图/表（结果） 4

参考文献

[1]Aggarwal S, Pittenger MF.Human mesenchymal stem cells modulate allogeneic immune cell responses.Blood. 2005; 105(4):1815-1822.
[2]Meisel R, Zibert A, Laryea M,et al.Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.Blood. 2004;103(12):4619-4621.
[3]Glennie S, Soeiro I, Dyson PJ,et al.Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells.Blood. 2005;105(7):2821-2827.
[4]Krampera M, Cosmi L, Angeli R, et al.Role for interferon- gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells.Stem Cells. 2006; 24(2): 386-398.
[5]Le Blanc K, Rasmusson I, Sundberg B,et al.Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet. 2004;363 (9419):1439-1441.
[6]Bartholomew A, Sturgeon C, Siatskas M,et al.Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.Exp Hematol. 2002;30(1):42-48.
[7]Zhang J, Li Y, Chen J,et al.Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice.Exp Neurol. 2005;195(1):16-26.
[8]Lazarus HM, Koc ON, Devine SM,et al.Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.Biol Blood Marrow Transplant. 2005;11(5):389-398.
[9]Guo Z, Zheng C, Chen Z,et al. Fetal BM-derived mesenchymal stem cells promote the expansion of human Th17 cells, but inhibit the production of Th1 cells.Eur J Immunol. 2009;39(10):2840-2849.
[10]Schirmer C, Klein C, von Bergen M,et al.Human fibroblasts support the expansion of IL-17-producing T cells via up-regulation of IL-23 production by dendritic cells.Blood. 2010;116(10):1715-1725.
[11]Eljaafari A, Tartelin ML, Aissaoui H,et al.Bone marrow-derived and synovium-derived mesenchymal cells promote Th17 cell expansion and activation through caspase 1 activation: contribution to the chronicity of rheumatoid arthritis.Arthritis Rheum. 2012;64(7):2147-2157.
[12]Fischer M, Ehlers M.Toll-like receptors in autoimmunity.Ann N Y Acad Sci. 2008;1143:21-34.
[13]Dominici M, Le Blanc K, Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317.
[14]Krishnan J, Selvarajoo K, Tsuchiya M,et al.Toll-like receptor signal transduction.Exp Mol Med. 2007;39(4):421-438.
[15]Wang Y, Xie J, Wang N,et al.Lactobacillus casei Zhang modulate cytokine and Toll-like receptor expression and beneficially regulate PolyI:C-induced immune responses in RAW264.7 macrophages.Microbiol Immunol. 2013;57(1): 54-62.
[16]Kamran N, Takai Y, Miyoshi J,et al.Toll-like receptor ligands induce expression of the costimulatory molecule CD155 on antigen-presenting cells.PLoS One. 2013;8(1):e54406.
[17]Wu J, Meng Z, Jiang M,et al.Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific.Immunology. 2010;129(3):363-374.
[18]St Paul M, Barjesteh N, Paolucci S,et al.Toll-like receptor ligands induce the expression of interferon-gamma and interleukin-17 in chicken CD4+ T cells.BMC Res Notes. 2012; 5:616.
[19]Bach NS, Låg M, Øvrevik J.Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells.Toxicol Lett. 2014;228(1):42-47.
[20]Saluja R, Delin I, Nilsson GP,et al.FcεR1-mediated mast cell reactivity is amplified through prolonged Toll-like receptor-ligand treatment.PLoS One. 2012;7(8):e43547.
[21]Mitchell D, Olive C.Regulation of Toll-like receptor-induced chemokine production in murine dendritic cells by mitogen-activated protein kinases.Mol Immunol. 2010;47 (11-12):2065-2073.
[22]McKernan DP, Dennison U, Gaszner G,et al.Enhanced peripheral toll-like receptor responses in psychosis: further evidence of a pro-inflammatory phenotype.Transl Psychiatry. 2011;1:e36.
[23]Chin AI, Miyahira AK, Covarrubias A,et al.Toll-like receptor 3-mediated suppression of TRAMP prostate cancer shows the critical role of type I interferons in tumor immune surveillance.Cancer Res. 2010;70(7):2595-2603.
[24]Meng L, Zhu W, Jiang C,et al.Toll-like receptor 3 upregulation in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats.Arthritis Res Ther. 2010; 12(3): R103.
[25]Liu Z, Guan Y, Sun X,et al.HSV-1 activates NF-kappaB in mouse astrocytes and increases TNF-alpha and IL-6 expression via Toll-like receptor 3.Neurol Res. 2013;35(7): 755-762.
[26]刘婧,刘水平,古力娜尔•库尔班,等. Toll样受体3信号通路在人类病毒性心肌炎心肌细胞凋亡及炎症反应中的作用[J].中国病理生理杂志,2013,29(3):404-407.
[27]Liotta F, Angeli R, Cosmi L,et al.Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling.Stem Cells. 2008;26(1):279-289.
[28]Tomchuck SL, Zwezdaryk KJ, Coffelt SB,et al.Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses.Stem Cells. 2008;26(1):99-107.
[29]Kattah MG, Wong MT, Yocum MD,et al.Cytokines secreted in response to Toll-like receptor ligand stimulation modulate differentiation of human Th17 cells.Arthritis Rheum. 2008; 58(6):1619-1629.
[30]Lombardi V, Van Overtvelt L, Horiot S,et al.Human dendritic cells stimulated via TLR7 and/or TLR8 induce the sequential production of Il-10, IFN-gamma, and IL-17A by naive CD4+ T cells.J Immunol. 2009;182(6):3372-3379.
[31]Tanaka J, Watanabe N, Kido M,et al.Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions.Clin Exp Allergy. 200;39(1):89-100.
[32]Fukata M, Breglio K, Chen A,et al.The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease.J Immunol. 2008;180(3):1886-1894.

引言

材料方法

设计：体外细胞学对比观察实验。

时间及地点：实验于清华大学第一附属医院完成。

材料：

实验方法：

胚胎骨髓来源间充质干细胞的分离：胚胎来源骨髓从孕15周胚胎获得，经本院伦理委员会批准，孕妇知情同意。胚胎来源骨髓应用淋巴细胞分离液(Ficoll)分离单个核细胞，通过分离黏附细胞而得到胚胎骨髓来源间充质干细胞。细胞接种于DF12完全培养基(40% MCDB-201，体积分数为2%胎牛血清，1×ITS，10^-⁸ mol/L地塞米松，50 U/mL青链霉素，10 μg/L人碱性成纤维细胞生长因子，10 μg/L人表皮生长因子和2 mmol/L L-谷胺酰胺)，在37 ℃、体积分数为5% CO₂孵箱培养。

胚胎骨髓来源间充质干细胞经过分析细胞表面标志物及诱导分化成脂肪、成骨、成软骨鉴定。

外周血CD4⁺T细胞的分离和共培养：肝素钠抗凝的健康人外周血采用Ficoll-Hypaque密度梯度离心法分离单个核细胞。CD4⁺T细胞由CD4-mAb磁珠(Miltenyi)分选，通过流式细胞仪鉴定，纯度>98%。CD4⁺T细胞单独或与胚胎骨髓来源间充质干细胞共培养(比例是10∶1)。共培养液包含IMDM、体积分数为20%胎马血清、50 U/mL青链霉素、 55 μmol/L 2-巯基乙醇、5 mg/L植物血凝素和5 μg/L人重组白细胞介素2，37 ℃、体积分数为5%CO₂孵箱培养4 d。

实时定量PCR(qRT-PCR)测定白细胞介素17、Toll样受体3、MyD88 mRNA的表达：离心收集1×10⁶ CD4⁺T或胚胎骨髓来源间充质干细胞，按照TRIzol一步法说明书(Invitrogen)抽提RNA，紫外分光光度仪检测浓度及纯度。依照M-MLV说明书反转录为cDNA。PCR引物序列见表1。依照SYBR Green RT-PCR试剂盒操作说明，于实时定量PCR仪(ABI)进行PCR扩增及检测。反应条件如下：95 ℃，10 min激活热启动酶；94 ℃，15 s，60 ℃，1 min，40循环。通过2^-^ΔΔ^Ct方法对白细胞介素17、Toll样受体3及MyD88进行相对定量。

主要观察指标：各组细胞白细胞介素17、Toll样受体3以及MyD88 mRNA的表达。

统计学分析：实验数据采用x(_)±s或(x(_)，95%的可信度区间)表示，2个培养组的差别采用配对t 检验。P < 0.05为差异有显著性意义。统计学处理应用SPSS 16.0统计软件完成。

全文链接：

讨论

Toll样受体主要存在于单核细胞和树突细胞，具有模式识别受体的功能，可以识别微生物的保守分子成分，启动机体的固有免疫系统，从而帮助机体清除病原体。Toll样受体的主要生物学功能是促进细胞因子的合成与释放，引发机体炎症反应。Toll样受体和配体结合后，通过MyD88依赖或非依赖途径，激活NF-κB或干扰素调节因子3，启动核内相关基因的表达，从而合成白细胞介素1、白细胞介素6、白细胞介素8、白细胞介素12、肿瘤坏死因子α和γ-干扰素等细胞因子，引起粒细胞、巨噬细胞等趋化聚集，诱发炎症反应，发挥早期免疫反应^[14-26]。目前研究表明Toll样受体在间充质干细胞中亦有较高表达，且可能对间充质干细胞的免疫调节发挥一定作用。Liotta等^[27]发现人骨髓来源的间充质干细胞高表达Toll样受体3和Toll样受体4，且TLR配体能够激活NF-κB，使白细胞介素6、白细胞介素8、CXCL10产量增加，并且能够阻断间充质干细胞抑制T细胞增殖的能力，这些资料表明间充质干细胞Toll样受体3和Toll样受体4的表达可能是阻断间充质干细胞免疫抑制活性的一个有效途径。Tomchuck等^[28]的研究亦表明人骨髓来源的间充质干细胞表达Toll样受体，并且经Toll样受体刺激剂后导致下游信号的激活，包括NF-κB、AKT和MAPK，产生一系列细胞因子，促进间充质干细胞的迁移和免疫调节。

新近，较多研究表明Toll样受体具有调节Th17细胞的功能。Kattah等^[29]研究发现Toll样受体4或者Toll样受体7/8配体刺激人单个核细胞后，上清能促进Th17细胞增殖，研究提示Toll样受体刺激后产生的细胞因子联合促进Th17细胞增殖。Lombardi等^[30]报道Toll样受体4和Toll样受体7/8导致人单核细胞来源的树突细胞产生多个炎性和促炎因子，包括白细胞介素10、白细胞介素12和白细胞介素23，从而导致Th17细胞增殖。Tanaka等^[31]研究发现Toll样受体3配体poly(I : C)激活的人树突细胞与异缘性CD4⁺T细胞培养后，促进了Th17细胞的分化。课题组前期的研究表明胚胎骨髓来源间充质干细胞促进Th17细胞的增殖，本次实验表明在白细胞介素17升高的同时，Toll样受体3的表达相应的升高，推测Toll样受体3在其中发挥了作用。

Toll样受体信号常通过髓样分化蛋白分子MyD88发挥作用，Fukata等^[32]发现在炎症性肠病中，Toll样受体信号通过MyD88对于肠道鼠效应T细胞反应是重要的，缺陷了MyD88的CD4⁺T细胞功能缺陷，尤其是Th17细胞的分化。课题组通过PCR定量研究发现MyD88的表达亦相应升高，提示MyD88可能在其中发挥作用。

既往研究发现胚胎骨髓来源的间充质干细胞促进人Th17细胞增殖。本次研究中，进一步发现胚胎骨髓来源间充质干细胞明显增加了CD4⁺T细胞Toll样受体3 mRNA的表达，并且与相应的白细胞介素17的升高呈一致性关系，而且，Toll样受体3下游通道MyD88的表达升高，提示其可能通过MyD88发挥了免疫调节Th17细胞增殖的作用，这将为今后间充质干细胞潜在的细胞治疗策略提供实验依据。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
全文链接：