中文摘要：

目的：目前对树突状细胞的研究主要集中在人及小鼠系统，关于大鼠树突状细胞特性和功能的研究较少。体外扩增并纯化大鼠骨髓树突状细胞的方法，并对其进行形态学和免疫学鉴定。 方法：实验于2004—06／2005—06在第四军医大学西京医院耳鼻喉科实验室完成。实验材料：8～12周龄雌性SD大鼠由第四军医大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。实验方法：①参考Oliver方法，利用黏附法诱导分离SD大鼠骨髓细胞，获得高纯度的树突状细胞。采用扫描电镜和透射电镜观察培养6，8，10，12d与12d加内毒素情况下细胞形态，并采用流式细胞仪鉴定。②无菌条件下取出大鼠脾脏加RPMI1640培养液研磨并通过不锈钢筛网，收集未贴壁细胞，以RPMI1640培养液冲洗出的细胞作为反应细胞。取96孔培养板加入反应细胞，并按照1：480，1：240，1：120，1：60比例加入刺激细胞。采用ELX800型酶联免疫检测仪测定波长为490nm处吸光度值。 结果：①随培养时间的增长，细胞逐渐成熟，培养12d的细胞形态不规则，表面有大量树突状突起，培养12d加内毒素的细胞进一步成熟，体积增大，高表达CD80、CD86分子。②随着培养时间和树突状细胞数目的增加，树突状细胞刺激T细胞增殖的能力逐渐增强，加入内毒素后这种能力进一步加强。 结论：成功地建立了体外大量扩增大鼠骨髓树突状细胞方法，并可在培养过程中收获不同成熟状态树突状细胞。

英文摘要：

AIM： Currently, studies on dendritic cells are mainly on human and mice. Studies on rat dendritic cells are few. This article observes the method of amplifying large number of dendritic cells from rat bone marrow and identifies their morphological and immunological characteristics. METHODS： The experiment was conducted at the Otorhinolaryngology Laboratory of Xijing Hospital of Fourth Military Medical University of Chinese PLA from June 2004 to June 2005. Female SD rats aged 8-12 weeks were provided by Experimental Animal Center of Fourth Military Medical University of Chinese PLA. Animal intervention met the animal ethical standard. ①Bone marrow cells were collected from SD rats by adherence method according to Oliver method. Dendritic cells of high purity were harvested. Cell appearance was observed under scanning electron microscope and transmission electron microscope at days 6, 8, 10, 12 and after adding endotoxin at day 12, and then identified with flow cytometry. ②Rat spleen was obtained sterilely, grinded in RPMI1640 medium, checkout with stainless steel grit. Non-adhered cells were collected. Cells washed with RPMI1640 medium were considered as reactive cells, which were incubated in 96-well plate and cultured with stimulating cells at the proportion of 1：480,1：240,1：120,1：60. Absorbance at 490 nm was measured with ELX800 Enzyme-linked Immunosorbent Assay Appliance. RESULTS： ①With the prolongation of culture time, cells were gradually mature, irregular shape with a mass of dendrite processes at day 12. Cells cultured with endotoxin were further mature and increased volume, high expression of CD80 and CD86 at day 12. ②With the prolongation of culture time and increased count of dendritic cells, the ability of stimulating the proliferation of allogeneic T cells was enhanced, and became greater after adding endotoxin. CONCLUSION： A method to generate large number of dendritic cells from rat bone marrow is established. Dendritic cells at different maturation stages can be obtain