不同流式抗体分选小鼠原位肝癌模型中髓系来源抑制性细胞的比较  

Comparison of flow cytometry with different antibodies for enrichment of myeloid-derived suppressor cells from bone marrow of mouse models of orthotopic liver tumor

作　　者：赵文秀[1] 张正奇[1] 许雅苹[1] 吴端[1] 尹震宇[1] 王效民[1] 

机构地区：[1]厦门大学附属中山医院肝胆外科,福建省慢性肝病肝癌重点实验室,福建厦门361004

出　　处：《中国实验动物学报》2013年第4期42-46,I0002,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(81201894和81171976)

摘　　要：目的比较不同的流式抗体分选小鼠原位肝癌模型骨髓中的髓系来源抑制性细胞(myeloid-derived suppressor cells,MDSCs)。方法建立BALB/c小鼠原位肝癌移植模型,10d后分离荷瘤小鼠骨髓细胞,分别进行CD11b、Gr-1单色标记及CD11b和Gr-1双色标记后,进行流式分选。比较这三种方式分选的MDSCs纯度、活力,并检测分选的细胞精氨酸代谢酶1(arginase-1,Arg-1)和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达,并通过活性氧检测试剂盒检测MDSCs活性氧(reactive oxygen species,ROS)表达。结果三种方法分选到的细胞活力和纯度都大于90%。CD11b标记进行分选操作最为方便,易于统一,但纯度略差;Gr-1标记进行分选时,细胞群不好确定,但纯度高;双色标记进行分选纯度最高。分选的细胞高表达Arg-1、iNOS和ROS。结论三种抗体标记方法均能从小鼠原位肝癌模型骨髓中的分选到纯度高、活力好的MDSCs,而用CD11b单色标记操作方便,易于统一,可作为首选方法。MDSCs的成功分选,对于后续开展MDSCs的生物学和免疫学功能研究奠定了基础。Objective Accumulating evidences have demonstrated that myetoid-derived suppressor cells （ MD- SCs） play a pivotal role in tumor associated immune suppression. The main purpose of this study was to compare different antibodies used in flow cytometry to isolate MDSCs form bone marrow of mice models with orthotopic liver tumour. Methods The orthotopic hepatic tumor model was generated by direct intrahepatie injection of mouse hepatoma H22 cells （ 1 ~ 106 cells originating from the BALB/c mice）. The mice were sacrificed at 10 days after modeling. Mononuclear ceils were iso- lated from bone marrow and stained with CD1 l b, Gr-1 or double stained with CD11 b and Gr-1 antibnodies, and then sorted by flow cytometry. The expression of arginase-1 （Arg-1） and inducible nitric oxide synthase （iNOS） of isolated MDSCs were detected by flow cytometry. Cellular reactive oxygen species （ROS） detection assay kit was used to measure ROS pro- duction by MDSCs. Results The purity and viability of isolated MDSCs by using different antibodies was all greater than 90%. Enrichment of MDSCs with CDllb antibody was much easier, but with relatively low purity. MDSCs sorted by Gr-1 antibody have higher purity, but the cell population was hard to definite. MDSCs isolated by double staining had the highest purity. The isolated MDSCs expressed high levels of Arg-1, iNOS and ROS. Conclusions MDSCs with high purity and viability can be sorted from bone marrow of mice bering hepatic tumour by different labeling methods. Enrichment of MD- SCs with CDllb antibnody is easy to operate, unify and copy, thus, can be served as the first choice in sorting MDSCs. The successful sorting of MDSCs is important for the further studying the biological and immunological function of MDSCs.

关 键 词：髓系来源抑制性细胞 肝癌 荧光激活细胞分选 流式分选 抗体 骨髓 小鼠 

分 类 号：Q95-33[生物学—动物学]