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目的研究制备、鉴定小鼠骨髓来源的高产量树突状细胞(dendritic cells,DC)的方法,为制备DC及体外研究提供依据。方法取BALB/c小鼠骨髓细胞,经重组小鼠巨噬细胞-粒细胞集落刺激因子(recombinant mouse granulocyte-macrophage colony stimulating factor,rmGM-CSF)、重组小鼠白细胞介素4(recomtinant mouse interleukin 4,rmIL-4)诱导、脂多糖(lipopolysacchrides,LPS)刺激制备高纯度DC。利用光学显微镜、扫描电镜对所制备细胞做形态学观察;流式细胞术检测特定表型标记分子表达情况;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、干扰素γ(interferon gamma,IFN-γ)的分泌量。从形态、表型标记、细胞因子分泌功能三方面对所制备细胞进行初步鉴定。结果所制备细胞具有典型的DC形态学特征,细胞表面高表达CD1a、CD11c、CD80和CD86,相对于普通DC细胞有大量的TNF-α和IFN-γ分泌。结论利用BALB/c小鼠骨髓细胞在细胞因子rmGM-CSF、rmIL-4诱导及LPS的刺激下成功制备出纯度较高的成熟DC。
Objective To study the preparation and identification of mouse bone marrow-derived dendritic cells (DCs) with high yield and to provide basis for the preparation of DC and in vitro studies. Methods Bone marrow cells of BALB / c mice were treated with recombinant mouse granulocyte-macrophage colony stimulating factor (rmGM-CSF), recombinant mouse interleukin-4 , rmIL-4), lipopolysacchrides (LPS) stimulated preparation of high purity DC. The morphology of the prepared cells was observed by light microscopy and scanning electron microscopy. Flow cytometry was used to detect the expression of specific phenotypic markers. The expression of tumor necrosis factor-alpha (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA) factor alpha, TNF-α), interferon gamma (IFN-γ) secretion. From the morphological, phenotypic markers, cytokine secretion function of the prepared cells were initially identified. Results The cells were characterized by typical morphological features of DCs. CD1a, CD11c, CD80 and CD86 were highly expressed on the surface of cells, showing a large amount of TNF-α and IFN-γ secretion compared with ordinary DC cells. Conclusion BALB / c mouse bone marrow cells were successfully prepared with higher purity of mature DCs induced by rmGM-CSF, rmIL-4 and LPS.