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目的:观察小鼠骨髓来源的树突状细胞(DCs)负载胰岛素B9-23多肽后其表型和功能的变化,探讨其对免疫状态的影响。方法:①应用10ng/ml的粒细胞巨噬细胞集落刺激因子(GM-CSF)及10ng/ml的白细胞介素(IL)-4诱导小鼠骨髓细胞分化为DCs,将其分为空白对照组、胰岛素B9-23多肽刺激组、脂多糖(LPS)刺激组;②流式细胞术检测DCs表面CD11c、CD80、CD86、CD40、MHC-Ⅱ类分子的表达;③CCK-8检测DCs刺激同种异体淋巴细胞增殖的能力;④ELISA检测DCs培养上清中IL-12及干扰素-γ(IFN-γ)的水平。结果:流式细胞术检测各组DCs,其CD11c的表达均在60%以上,与空白对照组相比,胰岛素B9-23多肽刺激组表达低水平的CD40以及中等水平的CD80、CD86、MHC-Ⅱ类分子,LPS刺激组表达较高水平的CD80、CD86、CD40、MHC-Ⅱ类分子,同时,胰岛素B9-23多肽组DCs刺激同种异体淋巴细胞增殖的能力显著增强(P<0.05),分泌较高水平的IL-12,但分泌较低水平的IFN-γ。结论:胰岛素B9-23多肽能够刺激产生半成熟的DCs,对进一步探讨应用胰岛素B9-23多肽负载DCs免疫NOD鼠诱导免疫耐受预防自身免疫性糖尿病的发生具有重要的意义。
OBJECTIVE: To observe the changes of phenotype and function of mouse bone marrow-derived dendritic cells (DCs) loaded with insulin B9-23 polypeptide and investigate their effects on immune status. Methods: ① The bone marrow cells were induced to differentiate into DCs with 10ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10ng / ml interleukin (IL) -4, which were divided into blank control group , Insulin B9-23 polypeptide stimulation group and lipopolysaccharide (LPS) stimulation group; ②The expression of CD11c, CD80, CD86, CD40 and MHC-Ⅱ molecules on DCs was detected by flow cytometry; ③CKS- Lymphocyte proliferation; ④ELISA was used to detect the levels of IL-12 and IFN-γ in the supernatant of DCs. Results: Flow cytometry showed that the expression of CD11c was above 60% in all DCs. Compared with the blank control group, the insulin B9-23 peptide stimulated group showed low levels of CD40 and moderate levels of CD80, CD86 and MHC- Ⅱprotein, LPS stimulated group expressed higher levels of CD80, CD86, CD40, MHC-Ⅱmolecules, at the same time, the ability of stimulating allogeneic lymphocyte proliferation by insulin B9-23 polypeptide group DCs was significantly enhanced (P <0.05) Secretion of higher levels of IL-12, but secretion of lower levels of IFN-γ. CONCLUSION: Insulin B9-23 polypeptide can stimulate the production of semi-mature DCs. It is of great significance to further explore the application of insulin B9-23 polypeptide-loaded DCs to immune NOD mice to induce immune tolerance and prevent the occurrence of autoimmune diabetes.