花姜酮对小鼠CD4~+CD25~+Treg细胞分化功能的影响及机制
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摘要
目的:建立小鼠CD4~+CD25~+Treg细胞的体外诱导及培养方法,同时探讨花姜酮(Zerumbone)对Treg细胞的分化、分泌功能的影响及其机制。方法:从BABL/c小鼠的脾脏分离、纯化CD4~+CD62L~+T细胞,加入转化生长因子(transforming growth factor,TGF)-β(5 ng/mL)、白细胞介素(interleukin,IL)-2(30μg/mL)促进CD4~+CD62L~+T细胞向Treg细胞分化。Treg细胞被分为5组:正常组、模型对照组、Zerumbone(1μmol/L)组、Zerumbone(10μmol/L)组、Zerumbone(30μmol/L)组。流式细胞术检测各组Treg细胞的比例,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)方法检测IL-10含量,Foxp3、IL-10 mRNA的表达用实时荧光定量聚合酶链反应(RT-PCR)方法检测。结果:本实验方法使Treg细胞的诱导比例明显升高(P<0.01)。与模型对照组相比,Zerumbone组的Treg细胞比例呈剂量依赖性升高(P均<0.05)。IL-10蛋白的表达与模型对照组比较也呈剂量依赖性升高(P均<0.05),Foxp3、IL-10 mRNA的表达同样升高(P<0.05),其中Zerumbone(30μmol/L)组升高最明显(P<0.01)。结论:在体外实验中,Zerumbone可以促进BABL/c小鼠体内的CD4~+CD62L~+T细胞向Treg细胞分化,并促进IL-10蛋白的表达,其机制可能与诱导CD4~+CD25~+Treg特异性转录因子Foxp3的表达有关。
Objective:To explore an induced and culture method for CD4~+CD25~+Treg cells in vitro,study the effect of Zerumbone about the differentiation and secretion functions of Treg cells and explore the mechanisms included.Methods:CD4~+CD62L~+T cells were isolated from BALB/c mice spleen and purified with magnetic bead methods.CD4~+CD62L~+T cells were co-cultured with transforming growth factor(TGF)-beta(5 ng/mL),interleukin(IL)-2(30μg/mL)for CD4~+CD25~+Treg polarization.The cultured CD4~+CD25~+Treg cells were divided into five groups:the normal group;the induced group,which were cultured with the above protocol;Zerumbone(1μmol/L)group;Zerumbone(10μmol/L)group;Zerumbone(30μmol/L)group.Flow cytometry was used to detect the proportion of CD4~+CD25~+Treg cells.The ELISA method was detected the levels of IL-10.Reverse transcriptase polymerase chain reaction(RT-PCR)was detected the level of IL-10 mRNA and Foxp3 mRNA.Results:The proportion of CD4~+CD25~+Treg cells cultured with the protocol were significantly higher compared with the normal group(P<0.05).The CD4~+CD25~+Treg cells proportion in Zerumbone(1μmol/L),Zerumbone(10μmol/L),Zerumbone(30μmol/L)groups were significantly increased compared with group model,there is dose dependent(P<0.05).The protein level of IL-10 was increased by Zerumbone and that was also dose-dependent.Zerumbone increased the expression of IL-10 mRNA and Foxp3 mRNA(P<0.05).Conclusion:Zerumbone can increase the differentiation of splenic CD4~+CD62L Zerumbone into CD4~+CD25~+Treg cells and induce the expressions of IL-10 protein in vitro.The results may be thought the activation of Foxp3 in CD4~+CD25~+Treg cells.
Objective:To explore an induced and culture method for CD4~+CD25~+Treg cells in vitro,study the effect of Zerumbone about the differentiation and secretion functions of Treg cells and explore the mechanisms included.Methods:CD4~+CD62L~+T cells were isolated from BALB/c mice spleen and purified with magnetic bead methods.CD4~+CD62L~+T cells were co-cultured with transforming growth factor(TGF)-beta(5 ng/mL),interleukin(IL)-2(30μg/mL)for CD4~+CD25~+Treg polarization.The cultured CD4~+CD25~+Treg cells were divided into five groups:the normal group;the induced group,which were cultured with the above protocol;Zerumbone(1μmol/L)group;Zerumbone(10μmol/L)group;Zerumbone(30μmol/L)group.Flow cytometry was used to detect the proportion of CD4~+CD25~+Treg cells.The ELISA method was detected the levels of IL-10.Reverse transcriptase polymerase chain reaction(RT-PCR)was detected the level of IL-10 mRNA and Foxp3 mRNA.Results:The proportion of CD4~+CD25~+Treg cells cultured with the protocol were significantly higher compared with the normal group(P<0.05).The CD4~+CD25~+Treg cells proportion in Zerumbone(1μmol/L),Zerumbone(10μmol/L),Zerumbone(30μmol/L)groups were significantly increased compared with group model,there is dose dependent(P<0.05).The protein level of IL-10 was increased by Zerumbone and that was also dose-dependent.Zerumbone increased the expression of IL-10 mRNA and Foxp3 mRNA(P<0.05).Conclusion:Zerumbone can increase the differentiation of splenic CD4~+CD62L Zerumbone into CD4~+CD25~+Treg cells and induce the expressions of IL-10 protein in vitro.The results may be thought the activation of Foxp3 in CD4~+CD25~+Treg cells.
引文
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[10]Fontenot JD,Gavin MA,Rudensky AY.Foxp3 programs the development and function of CD4+CD25+regulatory Tcells[J].Nat Immunol,2003,4(4):330-336
[11]Kipnis J,Avidan H,Caspi RR,et al.Dual effect of CD4+CD25+regulatory T cells in neurodegeneration:a dialogue with microglia[J].Proc Natl Acad Sci USA,2004,101(Suppl 2):14663-14669
[12]Kearley J,Barker JE,Robinson DS,et al.Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+regulatory T cells is interleukin 10 dependent[J].J Exp Med,2005,202(11):1539-1547
[2]Sakaguchi S,Setoguchi R,Yagi H,et al.Naturally arising CD4+regulatory t cells for immunologic self-tolerance and negative control of immune responses[J].Curr Top Microbiol Immunol,2006,305(1):51-66
[3]Kitayama T.Attractive reactivity of a natural product,zerumbone[J].Biosci Biotechnol Biochem,2011,75(2):199-207
[4]Sakaguchi S,Yamaguchi T,Nomura T,et al.Regulatory T cells and immune tolerance[J].Cell,2008,133(5):7757-7787
[5]Ji L,Zhan Y,Hua F,et al.The ratio of Treg/Th17 cells correlates with the disease activity of primary immune thrombocytopenia[J].PLoS One,2012,7(12):e50909
[6]Hori S,Nomura T,Sakaguchi S.Control of regulatory Tcell development by the transcription factor Foxp3[J].Science,2003,299(5609):1057-1061
[7]Shi HZ,Li S,Xie ZF,et al.Regulatory CD4+CD25+T lymphocytes in peripheral blood from patients with atopic asthma[J].Clin Immunol,2004,113(2):172-178
[8]Henderson WR Jr,Tang LO,Chu SJ,et al.A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model[J].Am J Respir Crit Care Med,2002,165(1):108-116
[9]Manjeet S,Sim MK.Atropine-and scopolamine-resistant subtypes of muscarinic receptors in the rabbit aorta[J].Eur J Pharmacol,1989,174(1):99-105
[10]Fontenot JD,Gavin MA,Rudensky AY.Foxp3 programs the development and function of CD4+CD25+regulatory Tcells[J].Nat Immunol,2003,4(4):330-336
[11]Kipnis J,Avidan H,Caspi RR,et al.Dual effect of CD4+CD25+regulatory T cells in neurodegeneration:a dialogue with microglia[J].Proc Natl Acad Sci USA,2004,101(Suppl 2):14663-14669
[12]Kearley J,Barker JE,Robinson DS,et al.Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+regulatory T cells is interleukin 10 dependent[J].J Exp Med,2005,202(11):1539-1547
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