荷瘤小鼠脾脏髓源抑制细胞的分选及鉴定  

Sorting and identification of myeloid-derived suppressor cells from tumor-bearing mouse spleen

作　　者：刁建军[1] 杨雪[2] 宋学东[2] 陈仕友 王胤[2] 王晓蓉[2] 罗春丽[2] 张尧[1] 吴小候[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科,400016 [2]重庆医科大学附属第一医院检验医学院,400016

出　　处：《免疫学杂志》2014年第12期1091-1095,共5页Immunological Journal

基　　金：国家自然科学基金(81272572)

摘　　要：目的研究荷瘤小鼠脾脏中髓源抑制细胞(myeloid derived suppresser-cell,MDSCs)的分选与鉴定方法。方法常规培养小鼠肾癌细胞(Renca细胞),于Balb/c小鼠皮下建立小鼠肾癌模型;分离小鼠脾脏制成单细胞悬液,磁珠分选Gr-1+CD11b+双阳性的MDSCs;台酚蓝染色检测细胞存活率;流式细胞仪(flow cytometry,FCM)测其细胞纯度;显微镜下观察细胞形态;免疫荧光测其表面Gr-1、CD11b荧光表达情况;RT-PCR检测Cox2和Arg-1的m RNA的表达情况;小鼠皮下成瘤实验观察MDSCs促肿瘤生长。结果成功建立小鼠肾癌模型,磁珠分选后经FCM检测Gr-1+CD11b+双阳性的MDSCs细胞可达92.3%,显著高于分选前(P<0.01);免疫荧光鉴定结果显示:分选后的细胞形态完整,Gr-1和CD11b的荧光表达于细胞膜且2种荧光可以融合;RT-PCR检测分选后的MDSCs细胞群的Cox2和Arg-1的m RNA相对表达量显著高于分选前(P<0.05);小鼠皮下成瘤观察到MDSCs组与实验对照组有显著差异(P<0.05)。结论用免疫磁珠从荷瘤小鼠脾脏中成功分选得到MDSCs,具有较高纯度和良好的生物活性,为后续实验提供了理想的细胞来源。To sort and identify myeloid-derived suppressor cells（MDSCs） from tumor-bearing mouse spleen,Mouse renal cancer cells（Renca cells） were routinely cultured, and a renal cancer model was then established subcutaneously in Balb/c mice. Spleens were harvested and made into single cell suspension, which was subsequently subjected to magnetic bead sorting for Gr-1＋CD11b＋double positive cells; phenol blue staining was used to test cell survival rate, flow cytometry（FCM） to measure the purity of MDSCs, and microscope to detect cell morphology; surface fluorescence expression of CD11 b and Gr-1 cell was detected by immunofluorescence. The m RNA expression of Cox2 and Arg-1 was measured by RT-PCR. Tumor growth assay in vivo was applied to evaluate tumor growth promoted by MDSCs. Data showed that Gr-1 and CD11 b double positive cells reached 92.3%after magnetic bead sorting, significantly higher than before separation（P 〈 0.01）; the fluorescence of Gr-1 and CD11 b expressed in cell membrane and able to fuse. RT-PCR revealed that the relative m RNA expressions of Cox2 and Arg-1 were remarkably higher than before sorting（P 〈 0.05）. Furthermore, tumor growth assay in vivo showed a statistical difference between MDSCs group and control（P 〈 0.05）. In conclusion, magnetic bead sorting successfully obtained MDSCs from tumor-bearing mouse spleen, and those MDSCs has a high purity and a good biological activity, providing an ideal source of cells for subsequent experiments.

关 键 词：MDSCS 脾脏 磁珠分选 鉴定 

分 类 号：R392[医药卫生—免疫学]