大鼠骨髓来源树突状细胞体外培养体系的优化

Optimization of the in vitro culture system for bone marrow-derived dendritic cells in rats

目的探讨优化大鼠骨髓来源树突状细胞(DC)体外培养体系的方法。方法应用重组大鼠粒细胞-巨噬细胞集落刺激因子(recombinant rat GM-CSF,rrGM-CSF)20μg/L和重组大鼠白介素(recombinant rat interleukin,rrIL)-410μg/L诱导分化Lewis大鼠骨髓单个核细胞,获得未成熟DC(immature DC,imDC)。第6日加入终含量为100μg/L的脂多糖(lipopolysaccharides,LPS)刺激细胞成为成熟DC(mature DC,mDC)。用倒置相差显微镜观察细胞形态,流式细胞术鉴定细胞表型、双抗夹心酶联免疫吸附测定法(ELISA)检测DC培养上清液中白介素(IL)-12、IL-10的含量,并以Brown Norway大鼠脾脏T淋巴细胞为反应细胞,做混合淋巴细胞反应检测,观察DC刺激T淋巴细胞增殖能力。结果经细胞形态学、流式细胞术及混合淋巴细胞反应三方面鉴定,证实所培养细胞为DC。体外培养第6日,见大量增殖细胞集落,细胞高表达OX62。LPS刺激细胞后,细胞伪足样突起明显增多,细胞逐渐呈半悬浮、悬浮生长,细胞表面高表达主要组织相容性抗原复合体(MHC)Ⅱ类分子和共刺激分子(CD40、CD86)。与imDC比较,mDC分泌IL-10和IL-12显著增加,刺激同种异体T淋巴细胞增殖的能力显著提高。结论通过采用调整rrGM-CSF和rrIL-4的用量等方法优化体外培养体系诱导大鼠骨髓单个核细胞,可分化出大量骨髓源性DC。

Objective To investigate the optimization of the in vitro culture system for bone marrow-derived dendritic cells（DC）in rats.Methods Immature DC from bone marrow cells of Lewis rats were induced and differentiated in vitro by 20 μg/L recombinant rat granulocyte macrophage colony stimulating factor（rrGM-CSF）and 10 μg/L recombinant rat interleukin（rrIL）-4.On day 6,100 μg/L of lipopolysaccharide was added and the cells were cultured for another 48 h to generate mature DC.The morphological features were observed by invert optical microscope.The immune phenotypes of DC were detected by flow cytometry（FACS）.The contents of interleukin（IL）-12 and IL-10 in the DC supernatant were detected by direct sandwich enzyme-linked immune absorbent assay（ELISA）.Mixed lymphocyte reaction（MLR）culture was performed to observe the proliferation of T lymphocyte cells by the DC stimulation,while Brown Norway rat splenetic T lymphocyte cells were used as responders.Results The cultured cells were identified as DC by cytomorphology,FACS and MLR.After culture for six days,the cultured cells displayed the typical morphology of dendritic cells,with high expression of OX62（the marker of rat DC）.After LPS stimulation,most cells in culture had more dendrite-like characters on the surface and expressed major histocompatibility complex（MHC）class Ⅱ,CD40 and CD86 abundantly.They stimulated allogeneic T cell responses effectively in MLR.Compared with immature DC,mature DC produced significantly more IL-12 and IL-10 to stimulate the proliferation of T lymphocyte cells.Conclusion The improved culture system from bone marrow cells of rats can induce plenty of bone marrow-derived DC in rats through adjusting the doses of rrGM-CSF and rrIL-4.