小鼠髓源性树突状细胞分离培养及鉴定  

Isolation,culture and identification of mouse bone marrow derived dendritic cells

作　　者：王卓[1] 李娴静 冷雪娇[1] WANG Zhuo;LI Xianjing;LENG Xuejiao(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Institute of Pharmaceutical Science,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]中国药科大学药物科学研究院,江苏南京211198

出　　处：《药学研究》2023年第2期89-94,共6页Journal of Pharmaceutical Research

基　　金：国家自然科学基金(No.82003288)。

摘　　要：目的建立小鼠髓源性树突状细胞分离培养及鉴定方法。方法取小鼠股骨和胫骨骨髓细胞,使用重组粒细胞-巨噬细胞集落刺激因子(recombinant granulocyte-macrophage colony-stimulating factor,rmGM-CSF)20 ng·mL^(-1)联合白细胞介素-4(interleukin 4,IL-4)10 ng·mL^(-1)诱导小鼠骨髓前体细胞分化为树突状细胞(dendritic cell)。体外培养10 d,使用流式细胞术鉴定细胞表面树突细胞特异性标志物CD11c、MHCⅡ、CD80、CD86、CD40及CCR7的表达。进一步使用OVA 257-264多肽处理细胞48 h,流式细胞术分析该多肽与MHC I形成的复合物的表达,验证该细胞的抗原提呈功能。结果依据本文方法,培养第10天每只小鼠可获得1.5×10^(7)~2×10^(7)个细胞。其中,38.4%细胞表达CD11c,CD11c阳性的细胞中有15.2%细胞表达MHCⅡ、32.3%细胞表达CD80、7.22%细胞表达CD86,但CD11c阳性的细胞中CD40阳性细胞仅4.15%,CCR7阳性细胞仅1.83%。LPS刺激24 h后,58.1%细胞表达CD11c,CD11c阳性的细胞中有39.8%细胞表达MHCⅡ,38.6%细胞表达CD80,43.1%细胞表达CD86,30.2%细胞表达CD40,10.2%细胞表达CCR7。使用该方法培养的树突细胞具有较强的抗原提呈能力。结论本试验成功建立体外培养树突细胞的方法,可为基于树突细胞的基础研究和转化应用提供实验基础。Objective To establish the isolation,culture and identification method of mouse bone marrow derived dendritic cells.Methods Bone marrow cells from murine femoral and tibial were taken,and the recombinant granulocyte-macrophage colony-stimulating factor(rmGM-CSF)20 ng·mL^(-1)and interleukin-4(IL-4)10 ng·mL^(-1)was added to induce the mouse myeloid precursor cells differentiates into dendritic cells(DC).The mouse myeloid precursor cells were cultured for 10 days,and then they were used to identify the expression of CD11c,MHC II,CD80,CD86,CD40,and CCR7 by flow cytometry.The cultured cells were further treated with OVA 257-264 polypeptide for 48 hours,and the expression of the complexes consisting of OVA 257-264 polypeptide and MHC I were detected,verifying the antigen presentation function of the cultured cells.Results According to the described method,1.5×10^(7)to 2×10^(7)cells per mouse were obtained on day 10.Through this method,38.4%of the cells expressed CD11c,and among CD11c positive cells,15.2%expressed MHCⅡ,32.3%expressed CD80 and 7.22%expressed CD86,while the CD11c-positive cells contained only 4.15%of CD40 and 1.83%of CCR7 positive cells.After 24 h of LPS treatment,58.1%of the total cells expressed CD11c,and the positive rates of MHCII,CD80,CD86,CD40 and CCR7 among CD11c expressed cells were 39.8%,38.6%,43.1%,30.2%and 10.2%,respectively.Functional analysis reveal that the cultured cells showed a strong antigen presentation capacity.Conclusion These experiment successfully established the method for DC culture in vitro,which can provide an experimental basis for the basic research and transformation application based on DCs.

关 键 词：小鼠骨髓细胞 树突状细胞 鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]