F344大鼠树突状细胞的分离与培养

Isolation and culture of F344 rat′s dendritic cells

目的 研究F344大鼠树突状细胞(DC)的分离与培养方法。方法 取大鼠四肢骨骨髓细胞悬液,经梯度离心法得到DC ,加入白细胞介素(IL) - 4、粒细胞巨噬细胞集落刺激因子(GM CSF) ,全RPMI 16 4 0培养,经倒置显微镜、电镜及流式细胞仪鉴定细胞纯度及成熟度。结果 细胞培养的第8天,经倒置显微镜、电镜观察DC出现典型形态;流式细胞仪检测第4天OX6 2阳性率为8 .92 %、主要组织相容性复合物(MHC) Ⅱ为2 0 . 90 %、CD86为16 98% ;第8天OX6 2阳性率为5 8. 0 7%、MHC Ⅱ为6 0 . 4 9%、CD86为6 2 . 94 % ;第15天OX6 2阳性率为84 . 6 8%、MHC Ⅱ为88 .0 3%、CD86为6 2 . 80 %。结论 用该方法得到的DC纯度可达80 %以上,为进一步研究其在肿瘤研究中的应用打下基础。

Objective Toestablisham et hodofisolating and culturing dendritic cells (DCs) from the bone marrow of F344 rats.Methods Dendritic cells obtained from the bone marrow ofF344rat′slimbsbysaline lavationwereculturedintheRPMI 1640 in vitro under the condition of granulocyte-ma crophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4).Dendrit ic cells were purified by centrifugation and identified by inversion microscopy, electron microscopy and flow cytometry.Resu lts After culture and induction,DCs displayed typical form with elongat ed dendritic processes viewed by inversion microscopy and electron microscopy.DC s expressed surface antigens,including OX62 8.92%,MHC-Ⅱ 20.9%,CD86 16.98% on t he 4th day;OX62 58.07%,MHC-Ⅱ 60.49%,CD86 62.94% on the 8th day;OX62 84.68%, MHC-Ⅱ 88.03%,CD86 62.80% on the 15th day.Conclusion The purity of DCs is up to 80% and over using this mothod,which presents the feasibility for further clinical use of DC in tumor immunotherapy.