小鼠骨髓和脾来源树突状细胞的分离与扩增培养  

Isolation and purification of dendritic cells from mouse spleen and bone marrow and their expansion culture induced by cytokines in vitro

作　　者：杨洪艳[1,2] 张义国[1,2] 董子明 郑智敏[1,2] 吴皓 

机构地区：[1]河南医科大学病理生理学教研室 [2]郑州铁路中心医院

出　　处：《河南医科大学学报》1999年第1期19-22,共4页Journal of Henan Medical University

基　　金：河南医科大学回国人员科研启动基金;河南医科大学基础医学院科研基金;河南省教委重点攻关项目;河南省科委重点攻关项目

摘　　要：目的：探索树突状细胞（ＤＣ）及其前体的分离纯化及其体外扩增的方法。方法：无菌制备ＢＡＬＢ／Ｃ小鼠骨髓和脾细胞；先后用红细胞裂解液、抗鼠ＣＤ４、ＣＤ８、Ｂ细胞单抗（ＭｃＡｂ）和补体溶液，依次去除红细胞，Ｔ、Ｂ细胞，粒细胞和单核巨噬细胞等混杂细胞而获得纯化的ＤＣ及其前体；又在粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ）和白细胞介素（ＩＬ４）协同诱导下培育，ＤＣ前体分化发育成ＤＣ或郎罕细胞（ＬＣ）并扩增，同时阻抑巨噬细胞发育生长。结果：ＤＣ／ＬＣ细胞数增加，其形态在光镜下多为特征性星形，也有梭形和多角形；扫描电镜下观察其绝大多数为特征性星形，且有１～４级不等的树突状突起，其纯度高达９５％以上。ＤＣ／ＬＣ在功能上明显刺激同种异体混合淋巴细胞反应（ＭＬＲ）。结论：①结果所得细胞的形态和功能符合ＤＣ／ＬＣ；②建立连续排异结合ＧＭＣＳＦ＋ＩＬ４联合诱生培育的方法，可获得大量高纯度的ＤＣ／ＬＣ。Aims:To isolate and purify dendritic cells (DC) and their progenitors from the BALB/C mouse spleen and bone marrow in this experiment.Methods:spleen or bone marrow cell suspensions were prepared with aseptic technique,and then erythrocytes,T and B lymphocytes,granulocytes and monocyte macrophages from them were removed separately by treating successively with red cell lysing buffer,mouse anti CD4 and anti CD8 and anti B220 monoclonal antibodies,and rabbit complement buffer,and by semi adhesion of DCs to culture plates.Existing DCs and their progenitors were collected and induced in vitro culture by both granulocyte macrophage colony stimulating factor(GM CSF) and interleukin 4 (IL 4).In morphology DC were observed under inverted light microscope and examined by scanning electron microscope.Results: In the course of the conditioned culture,the DC progenitors differentiated and developed into immature Langerhans cells (LC) and mature DC,but not into macrophages because the growth of macrophages was blocked by IL 4 simultaneously.At early stage of maturation,freshly isolated cells were smaller,rounder and with less prominent processes,and after 24～72 hours,these cells become relatively large,and had a veiled,fusiform,polyateral and finally stellate appearance characteristic of 1～4 branches of 'dendritic' processes stemed from elaborate cell membrane.They had high motility and elongation and retraction of their processes along adherent surface were seen.In function they could stimulate proliferation of mixed allogeneic or syngeneic T lymphocytes.Conclusion:A large number of the cells with more than 95% of high purity,which have DC/LC characteristic of morphology and function,can be generated by a combination of fresh isolation and cytokines induced procedure established.

关 键 词：树突状细胞 细胞培养 肿瘤 骨髓 脾 小鼠 

分 类 号：R730.3[医药卫生—肿瘤] R392.12[医药卫生—临床医学]