体外诱导小鼠骨髓细胞向未成熟树突状细胞分化的实验研究

发布时间：2018-04-10 14:20

本文选题：树突状细胞 + CD11c　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的:以小鼠骨髓细胞为前体,建立一种高效、简便的体外扩增、分离培养未成熟树突状细胞(imDC)的方法,从而为imDC的实验研究和临床应用奠定基础。方法:实验分为GM/IL-5、GM/IL-7和GM/IL-LPS三组:制备C57BL/6小鼠骨髓细胞,三羟甲基氨基甲烷-氯化铵缓冲液(Tris-NH4Cl)裂解红细胞后,以重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)和重组小鼠白细胞介素-4(rmIL-4)对其联合诱导培养,48 h后通过贴壁法去除悬浮的粒细胞及淋巴细胞,之后隔天半量换液一次,最后根据DC培养72 h后自动从塑料板分离的特性分别于第五天(GM/IL-5组)、第七天(GM/IL-7组)收集悬浮及疏松贴壁细胞,而GM/IL-LPS组则是于第六天半量换液后添加大剂量脂多糖(LPS)继续培养18 h后收集所得;将收获的三组细胞进行形态及功能学的鉴定,即:扫描电镜观察细胞形态、流式细胞术(FCM)测定细胞表面分子的表达、同种异体混合淋巴细胞反应(MLR)检测三组DC刺激同种异体T细胞增殖的能力。结果:扫描电镜下所收集的三组细胞均具有典型DC形态;流式结果表明细胞表面均高表达小鼠髓源性DC相对特异性标志CD11c,表达率达73%以上,GM/IL-5组DC细胞表面CD40、CD86、MHC-Ⅱ的表达率分别为34.46%、34.21%、45.16%,GM/IL-7组则分别为46.99%、68.79%、94.37%,GM/IL-LPS组为78.68%、89.84%、96.75%;MLR中GM/IL-5组DC刺激同种异体T细胞活化增殖的能力不如GM/IL-7、GM/IL-LPS组强(P0.05)。结论:此种方法能于体外定向诱导和扩增出大量具有典型DC形态、细胞表型及较高纯度的髓源性imDC,不仅具有降低实验成本、缩短培养时间等优点,而且能有效界定未成熟与成熟DC间的分化时限,这将为后续研究imDC在器官移植后诱导机体免疫耐受的临床应用奠定基础。
[Abstract]:Objective: to establish an efficient and simple method for isolation and culture of immature dendritic cells from mouse bone marrow cells in vitro, so as to lay a foundation for the experimental study and clinical application of imDC.Methods: the experiment was divided into three groups: C57BL/6 mice bone marrow cells were prepared by GM- / IL-5 GM- / IL-7 and GM/IL-LPS. The erythrocytes were lysed with trihydroxymethylaminomethane-ammonium chloride buffer solution (Tris-NH _ 4Cl).Recombinant mouse granulocyte macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4rmIL-4 (rmIL-4) were co-induced to remove suspended granulocytes and lymphocytes by adherent method for 48 h.Finally, suspension and loose adherent cells were collected according to the characteristics of DC cultured for 72 hours from plastic plates on the fifth day (GM- / IL-5 group) and on the 7th day (GM- / IL-7 group).In the GM/IL-LPS group, the cells were cultured for 18 hours after six and a half days of liquid exchange, and the three groups of cells were identified by morphology and function, that is, the morphology of the cells was observed by scanning electron microscope (SEM), and the cell morphology was observed by scanning electron microscope (SEM).Flow cytometry (FCM) was used to detect the expression of cell surface molecules, and MLR was used to detect the ability of DC to stimulate the proliferation of allogeneic T cells.Results: the three groups of cells collected under scanning electron microscope had typical DC morphology.The ability of T cells to activate and proliferate was not as strong as that of GM- / IL-7 / GM- / IL-LPS group.Conclusion: this method can induce and amplify a large number of myelogenous imDCs with typical DC morphology, cell phenotype and high purity in vitro, which can not only reduce the experimental cost, but also shorten the culture time.Moreover, it can effectively define the differentiation time between immature and mature DC, which will lay a foundation for further study on the clinical application of imDC in inducing immune tolerance after organ transplantation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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