白介素12A对CD4+CD25+Foxp3+Treg增殖分化的促进作用

doi:10.12092/j.issn.1009-2501.2019.05.003

中国临床药理学与治疗学 ›› 2019, Vol. 24 ›› Issue (5): 496-502.doi: 10.12092/j.issn.1009-2501.2019.05.003

白介素12A对CD4+CD25+Foxp3+Treg增殖分化的促进作用

张晓玲¹，官俏兵¹，杨毅²，郭丽³，韩晨阳²

¹嘉兴市第二医院神经内科，²药学部，³中心实验室，嘉兴 314001，浙江

收稿日期:2018-11-12 修回日期:2018-12-27 出版日期:2019-05-26 发布日期:2019-05-28
通讯作者: 韩晨阳，男，硕士，药师，研究方向：神经药理学。 Tel：0573-82795090 E-mail：691513770@qq.com
作者简介:张晓玲,女，硕士，主任医师，研究方向：神经免疫。 Tel：0573-82795090 E-mail：xiaoling-zhang66@163.com
基金资助:
浙江省自然科学基金项目(LYY19H280003)；浙江省科技厅项目（2017C37174）；浙江省卫生厅项目（2018KY804）

Effect of interleukin 12A on proliferation and differentiation of CD4+CD25+Foxp3+Treg

ZHANG Xiaoling¹，GUAN Qiaobin¹，YANG Yi², GUO Li³，HAN Chenyang²

¹ Department of Neurology, ²Pharmacy, ³Central Laboratory, the Second Hospital of Jiaxing, Jiaxing 314001, Zhejiang, China

Received:2018-11-12 Revised:2018-12-27 Online:2019-05-26 Published:2019-05-28

摘要/Abstract

摘要：

目的: 研究白介素12A（IL-12A）促进CD4+CD25-T细胞向CD4+CD25+Foxp3+Treg（iTreg）增殖分化的作用。方法: 采用免疫磁珠分选法分选出脐带血中CD4+CD25-T细胞，实验组和对照组均采用抗CD3和抗CD28单克隆抗体活化，并加入10 ng/mL的TGF-β1诱导，而实验组加入5 ng/mL的IL-12A同时诱导。细胞培养72 h后，采用流式细胞术（FACS）检测CD4+CD25+Foxp3+Treg（iTreg）细胞的比例，蛋白免疫印迹（Western-Blot）法检测细胞中Foxp3、Smad2、Smad3、Smad6的表达，实时荧光定量PCR检测TGF-β1、Foxp3、Smad2、Smad3、Smad6的mRNA表达。体外共培养诱导后iTreg和CD4+CD25-T细胞，CCK-8法评价淋巴细胞活性。体外加入5 ng/mL的IL-12A与iTreg、CD4+CD25-T细胞共培养检测IL-12A协同iTreg对于CD4+CD25-T细胞活性的影响。结果:FACS检测，IL-12A可以促进CD4+CD25-T向iTreg的分化，CCK-8结果显示，iTreg可以以浓度依赖方式抑制CD4+CD25-T细胞活性。而IL-12A可以协同iTreg对于CD4+CD25-T细胞活性的抑制作用。Western-Blot和RT-QPCR结果显示，iTreg分化过程中Foxp3、Smad2、Smad3、Smad6的表达水平增高，且相关mRNA的水平也增高。结论:IL-12A可以促进iTreg的诱导分化，对于效应T细胞的抑制有着一定的协同作用。

关键词: 白介素12A, 调节性T细胞, 效应T细胞, TGF-β1

Abstract:

AIM: To study the role of interleukin 12A (IL-12A) in promoting proliferation and differentiation of CD4+CD25-T cells into CD4+CD25+Foxp3+Treg（iTreg）. METHODS: CD4+CD25-T cells in umbilical cord blood were selected by immunomagnetic beads. Both the experimental group and the control group were activated by anti-CD3 and anti-CD28 monoclonal antibodies, and induced by adding 10 ng/mL of TGF-1, while the experimental group was simultaneously induced by adding 5 ng/mL of IL-12A.After the cells were cultured for 72 h, the ratio of CD4+CD25+Foxp3+Treg (iTreg) cells was detected by flow cytometry (FACS). The expression of Foxp3, Smad2, Smad3 and Smad6 in the cells was detected by protein immunoblotting (Western-Blot), and the expression of TGF-β1 was detected by real time fluorescence quantitative PCR.iTreg and CD4+CD25-T cells were induced by co-culture in vitro, and lymphocyte activity was assessed by CCK-8.The co-culture of 5 ng/mL IL-12A with iTreg and CD4+CD25-T cells in vitro was used to detect the effect of IL-12A and iTreg on the activity of CD4+CD25-T cells.RESULTS:IL-12A could promote the differentiation of CD4+CD25-T into iTreg by FACS detection. CCK-8 showed that iTreg could inhibit the activity of CD4+CD25-T cells in a concentration-dependent manner. IL-12A can synergize the inhibitory effect of iTreg on CD4+CD25-T cell activity.Western-Blot and RT-QPCR results showed that Foxp3, Smad2, Smad3 and Smad6 expression levels were increased during iTreg differentiation, and related mRNA levels also was increased. CONCLUSION: IL-12A can promote the differentiation of iTreg and synergistic effect on the inhibition of effector T cells.

Key words: interleukin 12A, regulatory T cells, effect T cells, TGF-β1

中图分类号:

R965.2

张晓玲，官俏兵，杨毅，郭丽，韩晨阳. 白介素12A对CD4+CD25+Foxp3+Treg增殖分化的促进作用[J]. 中国临床药理学与治疗学, 2019, 24(5): 496-502.

ZHANG Xiaoling，GUAN Qiaobin，YANG Yi, GUO Li，HAN Chenyang. Effect of interleukin 12A on proliferation and differentiation of CD4+CD25+Foxp3+Treg[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(5): 496-502.

参考文献

［1］Fontenot JD, Gavin MA, Rudensky AY. Pillars article: foxp3 programs the development and function of CD4+CD25+ Regulatory T cells［J］. J Immunol, 2017, 198(3): 986-992.
［2］Ohl K, Tenbrock K. Regulatory T cells in systemic lupus erythematosus［J］.Eur J Immunol, 2015, 45(2):344.
［3］Nishikawa H, Sakaguchi S.Regulatory T cells in tumor immunity［J］.Int J Cancer,2010, 127(4):759-767.
［4］Gurung A, Kamble N, Kaufer BB, et al.Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens［J］.Plos Pathog, 2017, 13(12):e1006745.
［5］Neves F, Abrantes J, Almeida T,et al. Genetic characterization of interleukins (IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphs［J］. Innate Immun, 2015, 21(8):787-801.
［6］hman H, Natividad A, Bailey R, et al. Contribution of IL-12A and IL-12B polymorphisms to chlamydia trachomatis-specific cell-mediated immune responses［J］. Scand J Immunol, 2015, 81(3):209-218.
［7］Radhakrishnan S, Wiehagen KR, Pulko V, et al. Induction of a Th1 response from Th2-polarized T cells by activated dendritic cells: dependence on TCR:peptide-MHC interaction, ICAM-1, IL-12, and IFN-gamma［J］. J Immunol, 2007, 178(6):3583-3592.
［8］万俊, 罗英, 杨春平,等.变应性鼻炎患者外周血IL-35及EBI3 mRNA和IL-12A mRNA表达的研究［J］.临床耳鼻咽喉头颈外科杂志, 2014,28(13):952-954.
［9］Haribhai D, Chatila TA, Williams CB.Immunotherapy with iTreg and nTreg cells in a murine model of inflammatory bowel disease［J］.Methods Mol Biol,2016, 1422:197-206. DOI: 10.1007/978-1-4939-3603-8_19
［10］ Lu Y, Wang X, Gu J, et al. iTreg induced from CD39(+) naive T cells demonstrate enhanced proliferate and suppressive ability［J］. Int Immunopharmacol,2015, 28(2):925-930.
［11］Zhao C, Shi G, Vistica BP, et al. Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC［J］. Cell Immunol, 2014, 290(2):179-184.
［12］Beal AM, Ramoshernández N, Riling CR, et al. TGF-β induces the expression of Nedd4 family-interacting protein 1 (Ndfip1) to silence IL-4 production during iTreg cell differentiation［J］. Nature Immunol, 2011, 13(1):77-85.
［13］Filková M, Vernerová Z, Hulejová H, et al. Pro-inflammatory effects of interleukin-35 in rheumatoid arthritis［J］. Cytokine, 2015, 73(1):36-43.
［14］Tao YP, Wang WL, Li SY, et al. Associations between polymorphisms in IL-12A, IL-12B, IL-12Rβ1, IL-27 gene and serum levels of IL-12p40, IL-27p28 with esophageal cancer［J］. J Cancer Res Clin Oncol, 2012, 138(11):1891-900.
［15］Abarbanel DN, Seki SM, Davies Y, et al. Immunomodulatory effect of vancomycin on Treg in pediatric inflammatory bowel disease and primary sclerosing cholangitis［J］.J Clin Immunol,2013, 33(2):397-406.
［16］Zhao M, Zhang H, Liu X, et al. The effect of TGF-β on Treg cells in adverse pregnancy outcome upon toxoplasma gondii infection［J］. Front Microbiol, 2017, 8:901-912.
［17］Chakraborty S, Panda AK, Bose S, et al.Transcriptional regulation of FOXP3 requires integrated activation of both promoter and CNS regions in tumor-induced CD8+ Treg cells［J］. Sci Rep, 2017, 7(1):1628.
［18］Haribhai D, Luo X, Chen J, et al. TGF-β1 along with other platelet contents augments Treg cells to suppress anti-FVIII immune responses in hemophilia A mice［J］.Blood Adv, 2016, 1(2):139-151.
［19］欧阳寻丽, 袁博, 李寒, 等.成纤维样滑膜细胞通过IL-6下调类风湿关节炎患者外周血Treg ［J］ .中华微生物学和免疫学杂志,2018,38(8): 591-592.

[1]	何阳，宋立莹，彭向东，方伟进，王春江，邓珍珍，樊志强，刘世坤. 发酵虫草菌粉对DEN致大鼠肝损伤的保护作用及TGF-β1/Smads通路的影响[J]. 中国临床药理学与治疗学, 2018, 23(9): 974-982.
[2]	吕平，彭万胜，董淮富. 新型抑制性细胞因子IL-35与支气管哮喘的研究进展[J]. 中国临床药理学与治疗学, 2018, 23(2): 230-234.
[3]	陆威，周炳荣，汤怡，樊一斌，陶小华. Th17细胞在痤疮发病中的作用及与痤疮严重度相关性的功能研究[J]. 中国临床药理学与治疗学, 2018, 23(1): 83-87.
[4]	韩超，杨柳，张秋玲，潘竞锵，徐俊. 黄芩苷对哮喘大鼠气道重塑作用的实验研究[J]. 中国临床药理学与治疗学, 2017, 22(7): 749-754.
[5]	沈忱，李运曼，夏念，徐丹，韩丹，陈仕杰. Treg细胞抑制基质金属蛋白酶-9对类风湿性关节炎伴急性脑缺血损伤的研究进展[J]. 中国临床药理学与治疗学, 2017, 22(7): 831-840.
[6]	胡坤媚，胡浩然，沈媛媛，卢毅宁，杨解人，韩军. 中药玉夏胶囊对自发性高血压大鼠肾纤维化的改善作用及TGF-β₁ /Smads信号通路的影响[J]. 中国临床药理学与治疗学, 2017, 22(4): 387-393.
[7]	俞万钧, 王华英, 翁跃颂, 宋嫣瑾, 顾霄, 应华娟. 短期与长期烟草烟雾暴露小鼠中CD₄^＋Foxp3^＋调节性T细胞的变化及意义[J]. 中国临床药理学与治疗学, 2014, 19(6): 601-606.
[8]	邹明智. 恩替卡韦与阿德福韦酯抑制乙型肝炎调节性T细胞的效价差异[J]. 中国临床药理学与治疗学, 2011, 16(6): 661-665.
[9]	王珊琳. 咪喹莫特对哮喘小鼠气道炎症和调节性T细胞的影响[J]. 中国临床药理学与治疗学, 2011, 16(1): 33-37.
[10]	陈存国, 李绍波, 王昕昕, 金小红, 童夏生, 赖小英. 特异性免疫治疗对哮喘大鼠白细胞介素-10和CD₄⁺CD₂₅⁺调节性T细胞的影响[J]. 中国临床药理学与治疗学, 2010, 15(8): 871-875.

白介素12A对CD4+CD25+Foxp3+Treg增殖分化的促进作用

Effect of interleukin 12A on proliferation and differentiation of CD4+CD25+Foxp3+Treg

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 10

编辑推荐

Metrics

本文评价