Galectin-1处理的骨髓树突状细胞及白介素-10在GVHD中作用的实验研究

发布时间：2018-05-28 10:18

本文选题：移植物抗宿主病 + 树突状细胞　；参考：《昆明医科大学》2015年硕士论文

【摘要】：目的：探讨半乳糖凝集素1(Galectin-1)处理的骨髓树突状细胞(BM-DCgal)是否能上调Naive CD4+T细胞上细胞因子IL-10 (interleukin-10)的表达,以探索IL-10在移植物抗宿主病(graft-versus-host-disease, GVHD)中的作用。方法：1、从BALB/c小鼠的骨髓中制备骨髓前体细胞,通过GM-CSF和IL-4来诱导生成树突状细胞(dendritic cell, DC),同时加入Galectin-1刺激分化成熟。2、应用流式细胞术分选未经处理的BM-DC和经Galectin-1处理的骨髓树突状细胞BM-DCgal。3、从C57BL/6小鼠中制备脾细胞。4、应用免疫磁珠分选技术分选出Naive CD4+T细胞。5、BM-DC和BM-DCgal分别与免疫磁珠分选的Naive CD4+T细胞以及IL-10R (interleukin-10 receptor)抗体共培养。6、ELISA检测BM-DC、BM-DCgal共培养体系中IL-10的分泌情况。7、Real time-PCR检测共培养体系细胞表面IL-10的表达情况。8、采用流式细胞术检测共培养体系细胞表面标记IL-10、1L-17A、IFN-γ的表达情况。以此评估细胞分化功能特征。结果：1、成功制备和培养DC,并进行流式分选分选出DC。2、用免疫磁珠分选技术成功分选出Naive CD4+T细胞。3、Real time-PCR检测显示BM-DCgal共培养体系细胞表面IL-10的表达较BM-DC共培养体系细胞表面IL-10的表达有明显升高。4、流式细胞检测BM-DC, BM-DCgal共培养体系细胞表面IL-10,IFN-γ,IL-17A的表达有变化。BM-DCgal共培养组较之BM-DC共培养组细胞表面IL-10的表达升高而IFN-γ, IL-17A的表达降低。5、流式细胞术检测显示BM-DCgal与1L-10抗体中和处理的Naive CD4+T共培养后IL-10表达下调,共培养体系细胞表面标记IL-17A,IFN-γ的表达较之抗体中和之前升高。6、ELISA检测BM-DCgal分化过程中分泌大量IL-10,高于BM-DC的分泌。7、ELISA检测显示BM-DCgal与IL-10抗体中和处理的Naive CD4+T共培养后IL-10的分泌减少。结论：经Gelactin-1处理的BM-DC能上调Naive CD4+T细胞上IL-10的表达。IL-10在BM-DCgal诱导Naive CD4+T细胞向Trl方向分化中发挥重要作用,这为BM-DCgal诱导Naive CD4+T细胞向Tr1分化提供了理论依据,为BM-DCgal作为一类DC-tol用于防治GVHD提供了理论依据。
[Abstract]:Aim: to investigate whether bone marrow dendritic cells (BM-DCgal) treated with galactose-1Galectin-1 can up-regulate the expression of cytokine IL-10 interleukin-10 on Naive CD4 T cells in order to explore the role of IL-10 in graft-versus-host-disease (GVHD). Methods Bone marrow progenitor cells were prepared from bone marrow of BALB/c mice. Dendritic cells were induced by GM-CSF and IL-4, and Galectin-1 was added to stimulate differentiation and maturation. Flow cytometry was used to separate untreated BM-DC from bone marrow dendritic cells (BM-DCgal.3) treated with Galectin-1. Spleen cells were obtained from C57BL/6 mice. Using immunomagnetic bead sorting technique to separate Naive CD4 T cells. 5 BM-DC and BM-DCgal with immunomagnetic bead sorting Naive CD4 T cells and IL-10R interleukin-10 receptor antibody cocultured with. 6% Elisa to detect the secretion of IL-10 in BM-DCG BM-DCgal co-culture system. The expression of IL-10 was detected by flow cytometry. The expression of IL-101L-17An- 纬 was detected by flow cytometry. The function of cell differentiation was evaluated. Results: 1, DC2 was successfully prepared and cultured, and DC.2was selected by flow sorting. Naive CD4 T cells. 3P Real time-PCR analysis showed that the expression of IL-10 on the surface of BM-DCgal co-cultured cells was higher than that of BM-DC co-cultured cells. The results of immunomagnetic bead sorting technique showed that the expression of IL-10 on the surface of BM-DCgal co-cultured cells was higher than that of BM-DC co-cultured cells. The expression of IL-10 on the surface of BM-DCand BM-DCgal coculture system was detected by flow cytometry. The expression of IL-10IFN- 纬 -hIL-17A in BM-DCgal co-culture group was higher than that in BM-DC co-culture group, but the expression of IFN- 纬 and IL-17A was decreased by flow cytometry, and the expression of IFN- 纬 and IL-17A was decreased by flow cytometry. Cytopathological analysis showed that the expression of IL-10 was down-regulated after co-culture of Naive CD4 T treated with BM-DCgal and 1L-10 antibody. The expression of IL-17AtIFN- 纬 on the surface of co-cultured cells was higher than that before antibody neutralization. 6% Elisa was used to detect the secretion of IL-10 during the differentiation of BM-DCgal, which was higher than that of BM-DC. 7% Elisa showed that the secretion of IL-10 was decreased after co-culture of BM-DCgal and Naive CD4 T treated with IL-10 antibody. Conclusion: BM-DC treated with Gelactin-1 can up-regulate the expression of IL-10 on Naive CD4 T cells. IL-10 plays an important role in Trl differentiation of Naive CD4 T cells induced by BM-DCgal, which provides a theoretical basis for BM-DCgal induced Naive CD4 T cells to Tr1 differentiation. It provides a theoretical basis for the use of BM-DCgal as a class of DC-tol to prevent and cure GVHD.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7

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