瘦素预处理的MSC体外调节SLE患者淋巴细胞亚群的初步研究
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摘要
目的:前期研究证实系统性红斑狼疮(SLE)患者血清异常升高的瘦素可加剧间充质干细胞(MSC)衰老,本研究旨在探讨瘦素预处理MSC对淋巴细胞亚群免疫调节的变化情况。方法:分离脐带MSC,并收集SLE患者外周血单个核细胞(PBMC),分为三组:PBMC组,MSC+PBMC,瘦素(100 ng/ml)预处理MSC 3 d+PBMC,1∶10共培养3 d,收集悬浮细胞。流式细胞仪检测淋巴细胞亚群:CD4~+CD25~+Foxp3~+调节性T(Treg)细胞、CD4~+IL-17~+Th17细胞、CD4~+CXCR5~+PD-1~+滤泡辅助T(Tfh)细胞、CD19~+B细胞,并检测CD3~+T细胞、CD19~+B细胞表面CD25、CD69平均荧光强度(MFI)。结果:与对照组相比,瘦素预处理的MSC对Treg细胞上调作用受损[(8.53±2.33)%vs(6.79±2.14)%,P<0.01],对Th17细胞下调亦受影响[(1.28±0.70)%vs(1.64±0.55)%,P<0.01],Tfh细胞比例有增加的趋势,但差异无统计学意义[(1.48±1.36)%vs(2.08±1.52)%,P=0.051]。与MSC组比较,瘦素预处理组T细胞活化分子CD25、CD69表达增加。而在B淋巴细胞活化指标,瘦素预处理后CD25 MFI较前有升高[(19.16±3.62)vs(21.05±2.36),P<0.05],但对CD69影响则无统计学差异。结论:瘦素体外作用于MSC,不仅使加剧其细胞衰老,亦损伤其对T、B淋巴细胞的免疫调节能力。
Objective:Our previous study demonstrated serum leptin in patients with systemic lupus erythematosus(SLE) accelerated the senescence of mesenchymal stem cells( MSC),the aim of this study is to observe the effects of leptin-treated MSC on immune cells. Methods:Umbilical cord derived MSCs were isolated,and peripheral blood mononuclear cells( PBMC) in SLE patients were obtained by density gradient centrifugation with Ficoll. Three groups were divided,PBMC only,the co-culture of PBMC and MSC treated with or without leptin( 100 ng/ml). The frequencies of immune cells were detected by flow cytometry,including CD4~+CD25~+Foxp3~+regulatory T( Treg) cells,CD4~+IL-17~+Th17 cells,CD4~+CXCR5~+PD-1~+T follicular helper( Tfh) cells,and CD19~+B cells. Results:The frequency of regulatory T cells was lower in leptin-treated MSC group,compared with MSC group. The frequency of Th17 cells was increased by MSC pretreated with leptin. The frequency of Tfh cells was elevated by leptin-treated MSC compared with MSC control,but there was no statistical significance. MSC pretreated with leptin also restored the activation of T cells evaluated by CD25 and CD69,compared with untreated MSC. In addition,leptin-treated MSC increased the median fluorescence intensity of CD25,but not CD69 on B cells. Conclusion:Leptin not only accelerated cell senescence of MSC in vitro,but also impaired the capacity of MSC modulating the immune cells.
Objective:Our previous study demonstrated serum leptin in patients with systemic lupus erythematosus(SLE) accelerated the senescence of mesenchymal stem cells( MSC),the aim of this study is to observe the effects of leptin-treated MSC on immune cells. Methods:Umbilical cord derived MSCs were isolated,and peripheral blood mononuclear cells( PBMC) in SLE patients were obtained by density gradient centrifugation with Ficoll. Three groups were divided,PBMC only,the co-culture of PBMC and MSC treated with or without leptin( 100 ng/ml). The frequencies of immune cells were detected by flow cytometry,including CD4~+CD25~+Foxp3~+regulatory T( Treg) cells,CD4~+IL-17~+Th17 cells,CD4~+CXCR5~+PD-1~+T follicular helper( Tfh) cells,and CD19~+B cells. Results:The frequency of regulatory T cells was lower in leptin-treated MSC group,compared with MSC group. The frequency of Th17 cells was increased by MSC pretreated with leptin. The frequency of Tfh cells was elevated by leptin-treated MSC compared with MSC control,but there was no statistical significance. MSC pretreated with leptin also restored the activation of T cells evaluated by CD25 and CD69,compared with untreated MSC. In addition,leptin-treated MSC increased the median fluorescence intensity of CD25,but not CD69 on B cells. Conclusion:Leptin not only accelerated cell senescence of MSC in vitro,but also impaired the capacity of MSC modulating the immune cells.
引文
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[20]Engela AU,Baan CC,Dor FJ,et al.On the interactions between mesenchymal stem cells and regulatory T cells for immunomodulation in transplantation[J].Front Immunol,2012,3:126.
[21]Sun Y,Deng W,Geng L,et al.Mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting Th17 cells[J].J Immunol Res,2015,2015:284215.
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[23]Anolik JH.B cell biology:implications for treatment of systemic lupus erythematosus[J].Lupus,2013,22(4):342-349.
[24]聂瑛洁,罗利梅,査艳,等.系统性红斑狼疮患者血浆降低间充质干细胞对B淋巴细胞的抑制作用[J].南方医科大学学报,2016,36(8):1090-1093.Nie YJ,Luo LM,Zha Y,et al.Plasma from patients with systemic lupus erythematosus inhibits suppressive activity of mesenchymal stem cells against lupus B lymphocytes[J].J South Med Univ,2016,36(8):1090-1093.
[25]Crop MJ,Baan CC,Korevaar SS,et al.Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells[J].Clin Exp Immunol,2010,162(3):474-486.
[2]Sun L,Akiyama K,Zhang H,et al.Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans[J].Stem Cells,2009,27(6):1421-1432.
[3]Liu Y,Yang R,Shi S.Systemic infusion of mesenchymal stem cells improves cell-based bone regeneration via upregulation of regulatory T cells[J].Tissue Eng Part A,2015,21(3-4):498-509.
[4]Ghannam S,Pene J,Moquet-Torcy G,et al.Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype[J].J Immunol,2010,185(1):302-312.
[5]Yang X,Yang J,Li X,et al.Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice[J].Lupus,2018,37(1):49-59.
[6]Cho KA,Lee JK,Kim YH,et al.Mesenchymal stem cells ameliorate B-cell-mediated immune responses and increase IL-10-expressing regulatory B cells in an EBI3-dependent manner[J].Cell Mol Immunol,2017,13:1-4.
[7]Sun LY,Zhang HY,Feng XB,et al.Abnormality of bone marrowderived mesenchymal stem cells in patients with systemic lupus erythematosus[J].Lupus,2007,16(2):121-128.
[8]Li X,Liu L,Meng D,et al.Enhanced apoptosis and senescence of bone-marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus[J].Stem Cells Dev,2012,21(13):2387-2394.
[9]Che N,Li X,Zhang L,et al.Impaired B cell inhibition by lupus bone marrow mesenchymal stem cells is caused by reduced CCL2expression[J].J Immunol,2014,193(10):5306-5314.
[10]Yao J Y,Chao K,Li M R,et al.Expression and significance of IL-21 in patients infected with hepatitis B virus at different stages[J].J Pract Med,31(7):1061-1064.
[11]Chen H,Shi B,Feng X,et al.Leptin and neutrophil-activating peptide 2 promote mesenchymal stem cell senescence through activation of the phosphatidylinositol 3-kinase/akt pathway in patients with systemic lupus erythematosus[J].Arthritis Rheumatol,2015,67(9):2383-2393.
[12]Xu WD,Zhang M,Zhang YJ,et al.Association between leptin and systemic lupus erythematosus[J].Rheumatol Int,2014,34(4):559-563.
[13]Lourenco EV,Liu A,Matarese G,et al.Leptin promotes systemic lupus erythematosus by increasing autoantibody production and inhibiting immune regulation[J].Proc Natl Acad Sci U S A,2016,113(38):10637-10642.
[14]Liu Y,Yu Y,Matarese G,et al.Cutting edge:fasting-induced hypoleptinemia expands functional regulatory T cells in systemic lupus erythematosus[J].J Immunol,2012,188(5):2070-2073.
[15]Yu Y,Liu Y,Shi FD,et al.Cutting edge:Leptin-induced ROR-gammat expression in CD4+T cells promotes Th17 responses in systemic lupus erythematosus[J].J Immunol,2013,190(7):3054-3058.
[16]Cras A,Farge D,Carmoi T,et al.Update on mesenchymal stem cell-based therapy in lupus and scleroderma[J].Arthritis Res T-her,2015,17:301.
[17]Valencia X,Yarboro C,Illei G,et al.Deficient CD4+CD25highTregulatory cell function in patients with active systemic lupus erythematosus[J].J Immunol,2007,178(4):2579-2588.
[18]Alunno A,Bartoloni E,Bistoni O,et al.Balance between regulatory T and Th17 cells in systemic lupus erythematosus:the old and the new[J].Clin Dev Immunol,2012,2012:823085.
[19]Blanco P,Ueno H,Schmitt N.T follicular helper(Tfh)cells in lupus:Activation and involvement in SLE pathogenesis[J].Eur JImmunol,2016,46(2):281-290.
[20]Engela AU,Baan CC,Dor FJ,et al.On the interactions between mesenchymal stem cells and regulatory T cells for immunomodulation in transplantation[J].Front Immunol,2012,3:126.
[21]Sun Y,Deng W,Geng L,et al.Mesenchymal stem cells from patients with rheumatoid arthritis display impaired function in inhibiting Th17 cells[J].J Immunol Res,2015,2015:284215.
[22]Liu R,Li X,Zhang Z,et al:Allogeneic mesenchymal stem cells inhibited T follicular helper cell generation in rheumatoid arthritis[J].Sci Rep,2015,5:12777.
[23]Anolik JH.B cell biology:implications for treatment of systemic lupus erythematosus[J].Lupus,2013,22(4):342-349.
[24]聂瑛洁,罗利梅,査艳,等.系统性红斑狼疮患者血浆降低间充质干细胞对B淋巴细胞的抑制作用[J].南方医科大学学报,2016,36(8):1090-1093.Nie YJ,Luo LM,Zha Y,et al.Plasma from patients with systemic lupus erythematosus inhibits suppressive activity of mesenchymal stem cells against lupus B lymphocytes[J].J South Med Univ,2016,36(8):1090-1093.
[25]Crop MJ,Baan CC,Korevaar SS,et al.Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells[J].Clin Exp Immunol,2010,162(3):474-486.