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  本文選題：移植物抗宿主病 + 樹突狀細胞��； 參考：《昆明醫(yī)科大學》2015年碩士論文

【摘要】：目的：探討半乳糖凝集素1(Galectin-1)處理的骨髓樹突狀細胞(BM-DCgal)是否能上調(diào)Naive CD4+T細胞上細胞因子IL-10 (interleukin-10)的表達,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、從BALB/c小鼠的骨髓中制備骨髓前體細胞,通過GM-CSF和IL-4來誘導(dǎo)生成樹突狀細胞(dendritic cell, DC),同時加入Galectin-1刺激分化成熟。2、應(yīng)用流式細胞術(shù)分選未經(jīng)處理的BM-DC和經(jīng)Galectin-1處理的骨髓樹突狀細胞BM-DCgal。3、從C57BL/6小鼠中制備脾細胞。4、應(yīng)用免疫磁珠分選技術(shù)分選出Naive CD4+T細胞。5、BM-DC和BM-DCgal分別與免疫磁珠分選的Naive CD4+T細胞以及IL-10R (interleukin-10 receptor)抗體共培養(yǎng)。6、ELISA檢測BM-DC、BM-DCgal共培養(yǎng)體系中IL-10的分泌情況。7、Real time-PCR檢測共培養(yǎng)體系細胞表面IL-10的表達情況。8、采用流式細胞術(shù)檢測共培養(yǎng)體系細胞表面標記IL-10、1L-17A、IFN-γ的表達情況。以此評估細胞分化功能特征。結(jié)果：1、成功制備和培養(yǎng)DC,并進行流式分選分選出DC。2、用免疫磁珠分選技術(shù)成功分選出Naive CD4+T細胞。3、Real time-PCR檢測顯示BM-DCgal共培養(yǎng)體系細胞表面IL-10的表達較BM-DC共培養(yǎng)體系細胞表面IL-10的表達有明顯升高。4、流式細胞檢測BM-DC, BM-DCgal共培養(yǎng)體系細胞表面IL-10,IFN-γ,IL-17A的表達有變化。BM-DCgal共培養(yǎng)組較之BM-DC共培養(yǎng)組細胞表面IL-10的表達升高而IFN-γ, IL-17A的表達降低。5、流式細胞術(shù)檢測顯示BM-DCgal與1L-10抗體中和處理的Naive CD4+T共培養(yǎng)后IL-10表達下調(diào),共培養(yǎng)體系細胞表面標記IL-17A,IFN-γ的表達較之抗體中和之前升高。6、ELISA檢測BM-DCgal分化過程中分泌大量IL-10,高于BM-DC的分泌。7、ELISA檢測顯示BM-DCgal與IL-10抗體中和處理的Naive CD4+T共培養(yǎng)后IL-10的分泌減少。結(jié)論：經(jīng)Gelactin-1處理的BM-DC能上調(diào)Naive CD4+T細胞上IL-10的表達。IL-10在BM-DCgal誘導(dǎo)Naive CD4+T細胞向Trl方向分化中發(fā)揮重要作用,這為BM-DCgal誘導(dǎo)Naive CD4+T細胞向Tr1分化提供了理論依據(jù),為BM-DCgal作為一類DC-tol用于防治GVHD提供了理論依據(jù)。
[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- 緯 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- 緯 -hIL-17A in BM-DCgal co-culture group was higher than that in BM-DC co-culture group, but the expression of IFN- 緯 and IL-17A was decreased by flow cytometry, and the expression of IFN- 緯 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- 緯 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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R457.7

【參考文獻】

相關(guān)期刊論文 前1條

1 王書紅;達萬明;靳海杰;靖域;顏光濤;;急性移植物抗宿主病病人血清IL-2、IL-8、IL-10及TNF-α水平變化的研究[J];中國實驗血液學雜志;2006年04期

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本文編號：1946329