短发夹RNA沉默Tim3分子表达对哮喘小鼠外周血Th1和Th17细胞的影响

doi:10.7499/j.issn.1008-8830.2013.04.014

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摘要目的：建立小鼠支气管哮喘模型，采用T 细胞免疫球蛋白黏蛋白分子-3（Tim-3）特异短发夹RNA（shRNA）沉默外周血单个核细胞（PBMCs）中Tim-3的表达，探讨Tim-3对辅助性T细胞1 (Th1)和Th17细胞分化的影响。方法：用卵白蛋白(OVA)致敏并激发建立哮喘小鼠模型，分离哮喘小鼠PBMCs，用 Tim-3特异的shRNA 片段沉默哮喘小鼠PBMCs中高表达的Tim-3 基因。Real-time PCR 和 Western blot 检测 Tim-3 的表达，流式细胞分析技术（FACS）检测Th1和Th17的比例；ELISA 检测细胞培养上清液中干扰素γ（IFN-γ），白介素-4（IL-4），和白介素-17（IL-17）的水平。结果：哮喘组小鼠PBMCs中Tim-3 mRNA表达明显升高；使用特异Tim-3 shRNA沉默哮喘小鼠PBMCs后，沉默组PBMCs中Th1细胞比例显著升高，Th17细胞比例显著下降，与阴性对照组相比差异有统计学意义（P<0.01）；沉默组PBMCs培养上清液中IFN-γ明显升高，IL-17明显降低，与阴性对照组相比差异有统计学意义（P<0.05）, IL- 4水平无明显变化。结论：特异Tim-3 shRNA有效地沉默了Tim-3的表达，Tim-3表达的改变影响了T 细胞的分化。

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	陆小霞
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关键词 ：哮喘, Tim-3, RNA沉默, T细胞, 小鼠

Abstract：OBJECTIVE: To investigate the effects of down-regulating Tim-3 gene in the peripheral blood mononuclear cells (PBMCs) of an asthmatic mouse model by short hairpin RNA (shRNA) and to explore the effect of Tim-3 on Th1 and Th17 cell differentiation. METHODS: An asthmatic murine model was established by ovalbumin sensitization and challenge. PBMCs were isolated from asthmatic mice and transfected by shRNA targeting Tim-3 gene. The mRNA and protein expressions of Tim-3 were detected by quantitative PCR and Western blot. Flow cytometry analysis was performed to determine the levels of Th1 and Th17, and ELISA was performed to determine concentrations of IFN-γ, IL-4 and IL-17 in the supernatant. RESULTS: Tim-3 mRNA expression in PBMCs was significantly increased in asthmatic mice. The mRNA and protein expression of Tim-3 decreased significantly in the shRNA group. Compared with the negative groups, Th1 cell levels increased and Th17 cell levels decreased significantly in the asthmatic groups after Tim-3 shRNA interference. In the Tim-3 shRNA interference groups concentrations of IFN-γ increased significantly while IL-17 decreased significantly. CONCLUSIONS: Specific Tim-3 shRNA effectively silences the expression of Tim-3 and change in Tim-3 expression could affect T cell differentiation.

Key words： Asthma Tim-3 RNA interference T helper cells Mice

引用本文:

陆小霞,徐佳莉,董宗祈等. 短发夹RNA沉默Tim3分子表达对哮喘小鼠外周血Th1和Th17细胞的影响[J]. 中国当代儿科杂志, 2013, 15(4): 302-307.

LU Xiao-Xia,XU Jia-Li,DONG Zong-Qi et al. Small hairpin RNA silencing Tim-3 affects peripheral blood Th1 and Th17 cells differentiation in asthmatic mice[J]. CJCP, 2013, 15(4): 302-307.

链接本文:

http://www.zgddek.com/CN/10.7499/j.issn.1008-8830.2013.04.014 或 http://www.zgddek.com/CN/Y2013/V15/I4/302

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	［16］张志英，栾斌，冯晓霞.Galectin-9及其受体 Tim-3在哮喘小鼠肺组织中的表达［J］.中国当代儿科杂志，2011，13(5)：406-410.
	［5］Kearley J, McMillan SJ, Lioyd CM. Th2-driven, allergen-induced airway inflammation is reduced after treatment in vivo［J］. J Exp Med, 2007, 204 (6): 1289-1294.
	［6］North ML, Khanna N, Marsden PA, Grasemann H, Scott JA. Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma［J］. Am J Physiol Lung Cell Mol Physiol, 2009, 296(6): L911-L920.
	［17］Darcan-Nicolaisen Y,Meinicke H,Fels G, Hegend O, Haberland A, Kuhl A, et al. Small interfering RNA against transcription factor STAT6 inhibits allergic airway inflammation and hyperreactivity in mice［J］. Immunol, 2009, 182(12): 7501-7508.
	［18］Moriwaki A, Inoue H, Nakano T, Matsunaga Y, Matsuno Y, Matsumoto T, et al. T Cell treatment with small-interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a Murine Model of Asthma［J］. Am J Respir Cell Mol Biol, 2011, 44(4): 448-455.
	［7］Zhao Y, Yang J, Gao YD, Guo W.Th17 immunity in patients with allergic asthma［J］.Int Arch Allergy Immunol, 2010, 151(4): 297-307.
	［19］Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics［J］. Nat Methods, 2006, 3(9):701-706.
	［8］Agache I, Ciobanu C, Agache C, Anghel M. Increased serum IL-17 is an independent risk factor for severe asthma［J］. Respir Med, 2010, 104(8): 1131-1137.
	［9］Wilson RH, Whitehead GS, Nakano H, Free ME, Kolls JK, Cook DN. Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness［J］. Am J Respir Crit Care Med, 2009, 180(8): 720-730.
	［10］Nakae S, Komiyama Y, Nambu A, Sudo K, Iwase M, Homma I,et al. Antigen-Specific T Cell Sensitization Is Impaired in IL-17-Deficient Mice, Causing Suppression of Allergic Cellular and Humoral Responses［J］. Immunity, 2002, 17(3): 375-387.
	［11］胡斯明，罗雅玲，赖文岩.地塞米松对哮喘小鼠肺组织中转录因子RORγt mRNA表达的干预作用［J］. 细胞与分子免疫学杂志，2009, 25（12）: 1115-1118.
	［12］Nakae S, Iwakura Y, Suto H, Galli SJ. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17［J］. J Leukocyte Biol, 2007, 81(5): 1258-1268.
	［13］陈伟超，刘恩梅，邓昱，何云，陈杰华，刘玮，等.新生期卡介苗接种对实验性哮喘小鼠肺 Th17细胞和IL17的影响［J］.中国当代儿科杂志，2010，12（8）:650-653.
	［14］Kurschus FC, Croxford AL, Heinen AP, Wortge S, Lelo D, Waisman A. Genetic Proof for the transient nature of the Th17 phenotype［J］. Eur J Immunol, 2010, 40(12): 3336-3346.
	［15］Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease［J］. Nature, 2002, 415 (6871): 536-541.
	［16］张志英，栾斌，冯晓霞.Galectin-9及其受体 Tim-3在哮喘小鼠肺组织中的表达［J］.中国当代儿科杂志，2011，13(5)：406-410.
	［17］Darcan-Nicolaisen Y,Meinicke H,Fels G, Hegend O, Haberland A, Kuhl A, et al. Small interfering RNA against transcription factor STAT6 inhibits allergic airway inflammation and hyperreactivity in mice［J］. Immunol, 2009, 182(12): 7501-7508.
	［18］Moriwaki A, Inoue H, Nakano T, Matsunaga Y, Matsuno Y, Matsumoto T, et al. T Cell treatment with small-interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a Murine Model of Asthma［J］. Am J Respir Cell Mol Biol, 2011, 44(4): 448-455.
	［19］Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics［J］. Nat Methods, 2006, 3(9):701-706.