摘要
目的探讨建立合适的大鼠骨髓源性未成熟树突状细胞(imDC)的培养方法,并对其生物学特性进行评价。方法分别以不同剂量的粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白介素(IL)-4诱导分化获得大鼠骨髓源性树突状细胞(DC),比较收获率及特异性标记蛋白(OX62)阳性的DC数量,获得最佳细胞因子浓度。并用流式细胞仪分析DC表型,混合淋巴细胞反应(MLR)检测其刺激同种T细胞增殖能力,ELISA法测定MLR上清IL-12、γ-免疫反应性纤维结合素(IFN-γ)水平。结果 GM-CSF 5 ng/ml可诱导出更高的收获率及OX62的阳性率。GM-CSF 5 ng/ml培养7 d的DC,表达中等水平的主要组织相容性(抗原)复合物Ⅱ类(MHC II)和低水平的CD86;其分泌少量IL-12;刺激同种T细胞增殖能力极低。脂多糖(LPS)刺激后MHCⅡ、CD86表达明显增加;分泌IL-12和IFN-γ增加;刺激同种T细胞增殖能力增强。结论 GM-CSF 5 ng/ml为诱导大鼠骨髓源性imDC的最佳剂量,培养7～9 d可以获得足够数量和纯度的imDC。
Objective To establish a suitable method for the generation of Lewis rat immature dendritic cells and e- valuate their biological characterization. Methods Bone marrow cells were developed under the concentration of granulocytemacrophage colonystimulating factor, and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization was analyzed with floweytometry, the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction （MLR）, and the levels of IL-12 secreted by DC and interfer- on-γ（IFN-γ） and IL-12 levels in 5-day mixed leukocyte culture supernatants were detected by ELISA. Results 5 % GM-CSF concentrations resulted in a higher number of cell yield and higher percentage of OX62 at day 7. Under the condition of GM-CSF（5 ng/ml）, DC at day 9 expressed an intermediate level of MHC Ⅱ and low level of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHC Ⅱ and CD86 and a stronger IL-2 and IFN-γ production and higher stimulatory capacity of allogeneic T cells. Conclusion GM-CSF at a concentration of 5 ng/ml is the best method to generate large number of Lewis rat immature dendritic cells, and this makes it possible to induce immunological tolerance by DC.
出处
《安徽医科大学学报》
CAS
北大核心
2013年第2期133-137,共5页
Acta Universitatis Medicinalis Anhui
基金
江苏省自然科学基金(编号:BK2007057)
关键词
大鼠
骨髓
未成熟树突状细胞
诱导
rat
bone marrow
immature dendritic cell
derivation
