体外研究人脐带血间充质干细胞对大鼠枯否细胞极化的影响
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摘要
目的采用脂多糖(LPS)诱导大鼠枯否细胞(KCs)发生极化改变,之后用人脐带血间充质干细胞(huM SCs)与LPS诱导的KCs在Transwell内共培养,以观察huM SCs对KCs极化偏移的调节作用。方法实验分为KCs组、KCs+LPS组、KCs+LPS+MSCs组。对各组的白介素-4(IL-4)、肿瘤坏死因子(TNF-α)、白介素-10(IL-10)、白介素-6(IL-6)等上清因子采用ELISA法进行检测;诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、信号传导与转录激活因子3、6(STAT-3、STAT-6)、核因子kappaB(NF-κB)用Western bolt进行检测,同时用荧光实时定量PCR(qRT-PCR)对以上结果进行验证。结果 KCs+LPS组促炎因子TNF-α、IL-6分泌增加,KCs+LPS+MSCs组抑炎因子IL-10、IL-4分泌增加;而Western blot检测表明,KCs+LPS组中iN OS升高,NF-κB p65入核增高;而KCs+LPS+MSCs组高表达Arg-1,同时pS TAT-3、pS TAT-6表达增加。结论 huM SCs能诱导已经发生M1极化的KCs向M2表型偏移,考虑可能与huM SCs分泌细胞因子有关,起到一种免疫调节作用,huM SCs调节巨噬细胞极化的分子机制可能与通过JAK-STAT信号转导通路有关。
Objective LPS-stimulated Kupffer cells( KCs) and human umbilical cord blood mesenchymal stem cells( hu MSCs) were co-cultured in Transwell to observe the effect of hu MSCs on the polarization of KCs. Methods During this study,the model of hu MSCs in vitro co-culturing with KCs was built,and three groups were assigned,which were KCs group( normal group),KCs cells + LPS group( control group),KCs + LPS + MSCs group( experimental group). Among the three groups,IL-4,TNF-α,IL-10,IL-6 were detected by ELISA while inducible nitricoxide synthase( i NOS),arginase-1( Arg-1),phosphorylation reaction level of signal transducer and activator of transcription-3,6( STAT-3,STAT-6) and nuclear factor-κB( NF-κB) were detected by Western bolt. After that,qRT-PCT was adopted to check the previous results. Results It was found that both TNF-α and IL-6 secretion increased in the control group,while both IL-10 and IL-4 secretion increased in the experimental group. Western blot detection showed that the level of i NOS increased in the control group,NF-κB p65 increased in the nucleus,while the experimental group was high expression of Arg-1,p STAT-6,p STAT-3. Conclusion Our experiment's findings indicate that hu MSCs can induce alternative activation of KCs,and the activation pathway might be the JAK-STAT pathways.
Objective LPS-stimulated Kupffer cells( KCs) and human umbilical cord blood mesenchymal stem cells( hu MSCs) were co-cultured in Transwell to observe the effect of hu MSCs on the polarization of KCs. Methods During this study,the model of hu MSCs in vitro co-culturing with KCs was built,and three groups were assigned,which were KCs group( normal group),KCs cells + LPS group( control group),KCs + LPS + MSCs group( experimental group). Among the three groups,IL-4,TNF-α,IL-10,IL-6 were detected by ELISA while inducible nitricoxide synthase( i NOS),arginase-1( Arg-1),phosphorylation reaction level of signal transducer and activator of transcription-3,6( STAT-3,STAT-6) and nuclear factor-κB( NF-κB) were detected by Western bolt. After that,qRT-PCT was adopted to check the previous results. Results It was found that both TNF-α and IL-6 secretion increased in the control group,while both IL-10 and IL-4 secretion increased in the experimental group. Western blot detection showed that the level of i NOS increased in the control group,NF-κB p65 increased in the nucleus,while the experimental group was high expression of Arg-1,p STAT-6,p STAT-3. Conclusion Our experiment's findings indicate that hu MSCs can induce alternative activation of KCs,and the activation pathway might be the JAK-STAT pathways.
引文
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[15]Goenka S,Kaplan M H.Transcriptional regulation by STAT6[J].Immunol Res,2011,50(1):87-96.
[16]Ishii M,Wen H,Corsa C A,et al.Epigenetic regulation of the alternatively activated macrophage phenotype[J].Blood,2009,114(15):3244-54.
[2]Liaskou E,Wilson D V,Oo Y H.Innate immune cells in liver inflammation[J].Mediators Inflamm,2012,2012:949157.
[3]Zhang Q Z,Su W R,Shi S H,et al.Human ingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing[J].Stem Cells,2010,28(10):1856-68.
[4]Kim J,Hematti P.Mesenchymal stem cell-educated macrophages:a novel type of alternatively activated macrophages[J].Exp Hematol,2009,37(12):1445-53.
[5]Dayan V,Yannarelli G,Billia F,et al.Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction[J].Basic Res Cardiol,2011,106(6):1299-310.
[6]Li J,Zhang L,Xin J,et al.Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs[J].Hepatology,2012,56(3):1044-52.
[7]Fibbe W E,Nauta A J,Roelofs H.Modulation of immune responses by mesenchymal stem cells[J].Ann N Y Acad Sci,2007,1106:272-8.
[8]Locati M,Mantovani A,Sica A.Macrophage activation and polarization as an adaptive component of innate immunity[J].Adv Immunol,2013,120:163-84.
[9]Moses T B,Cheng L,Zhang Z,et al.Hepatitis B virus infection and immunopathogenesis in a humanized mouse model:induction of human-specific liver fibrosis and M2-like macrophages[J].PLo S Pathog,2014,10(3):e1004032.
[10]Wan J,Benkdane M,Alons E,et al.M2 Kupffer cells promote hepatocyte senescence an IL-6-dependent protective mechanism against alcoholic liver disease[J].Am J Pathol,2014,184(6):1763-72.
[11]Waterman R S,Tomchuck S L,Henkle S L,et al.A new mesenchymal stem cell(MSCs)paradigm:polarization into a pro-inflammatory MSCs1 or an immunosuppressive MSCs2 phenotype[J].PLo S One,2010,5(4):e10088.
[12]Biswas S K,Mantovani A.Orchestration of metabolism by macrophages[J].Cell Metab,2012,15(4):432-7.
[13]Wan J,Benkdane M,Teixeira-Clerc F,et al.M2 Kupffer cells promote M1 Kupffer cell apoptosis:a protective mechanism against alcoholic and non-alcoholic fatty liver disease[J].Hepatology,2014;59(1):130-42.
[14]Sica A,Mantovani A.Macrophage plasticity and polarization:in vivo veritas[J].J Clin Invest,2012,122(3):787-95.
[15]Goenka S,Kaplan M H.Transcriptional regulation by STAT6[J].Immunol Res,2011,50(1):87-96.
[16]Ishii M,Wen H,Corsa C A,et al.Epigenetic regulation of the alternatively activated macrophage phenotype[J].Blood,2009,114(15):3244-54.