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目的探讨Wnt通路在记忆性T细胞(TM)增殖活化中发挥的调控作用。方法建立同种异体(BALB/c-C57BL/6)小鼠皮肤移植模型,采用免疫磁珠方法分选培养C57BL/6小鼠未成熟树突状细胞(imDC)、成熟树突状细胞(mDC)和TM,流式细胞术鉴定。在96孔板或TranswellTM中用imDC和负载BALB/c小鼠抗原的mDC与TM进行混合淋巴细胞反应(MLR)。氚标记的胸腺嘧啶核苷(3H-TdR)检测TM的增殖情况。ELISA检测培养上清中白细胞介素2(IL-2)、IL-10、γ干扰素(IFN-γ)的水平。实时定量PCR检测imDC和mDC中Wnt基因的表达情况。双荧光素酶报告基因检测MLR中TM的β联蛋白/T细胞因子(β-catenin/TCF)信号通路转录活性。结果与对照组相比,在96孔板中负载BALB/c小鼠抗原的mDC显著诱导TM的增殖,上调IL-2和IFN-γ,下调IL-10的表达。mDC中Wnt3a和Wnt5a的mRNA表达水平显著高于imDC。mDC与TM共培养可显著增强TM的β-catenin/TCF信号通路转录活性。而在TranswellTM中培养的细胞基本未见细胞增殖。结论负载供者抗原的受者小鼠mDC可通过直接接触的方式有效激活受者效应型TM的增殖,Wnt通路的β-catenin/TCF转录激活可能是介导TM活化增殖的重要信号通路。
Objective To investigate the regulatory role of Wnt pathway in the proliferation and activation of memory T cells (TM). Methods Skin allograft (BALB / c-C57BL / 6) mice model was established. Immunomagnetic beads were used to culture immature immature dendritic cells (imDC) and mature dendritic cells ) And TM, flow cytometry identification. Mixed lymphocyte reaction (MLR) with imDC and mDC loaded with BALB / c mouse antigens and TM in 96-well plates or Transwell ™. Tritiated thymidine (3H-TdR) was used to detect TM proliferation. The levels of interleukin 2 (IL-2), IL-10 and interferon gamma (IFN-γ) in the culture supernatants were detected by ELISA. Real-time quantitative PCR was used to detect the expression of Wnt gene in imDC and mDC. Dual luciferase reporter assay detects TM β-catenin / TCF signaling pathway transcriptional activity in MLR. Results Compared with the control group, mDC loaded with BALB / c mouse antigens in 96-well plates significantly induced TM proliferation, up-regulated IL-2 and IFN-γ, and down-regulated IL-10 expression. The mRNA expression levels of Wnt3a and Wnt5a in mDC were significantly higher than that of imDC. The co-culture of mDC and TM significantly enhanced the transcriptional activity of TM β-catenin / TCF signaling pathway. However, cells cultured in TranswellTM showed little cell proliferation. CONCLUSION: Recipient mouse mDCs loaded with donor antigen can effectively activate the proliferation of recipient-type TM by direct contact. The β-catenin / TCF transcriptional activation of Wnt pathway may be an important signaling pathway that mediates TM activation and proliferation.