小鼠心脏缺血再灌注模型中心肌树突状细胞的流式细胞评价  

Flow cytometric assessment of cardiac dendritic cells in cardiac ischemic-reperfusion injury of mice

作　　者：刘鸣[1] 王大英[1] 张文全[1] 徐佑龙[1] 金惠根[1] 刘宗军[1] LIU Ming;WANG Da ying;ZHANG Wen quan;XU You long;JIN Hui gen;LIU Zong jun(Department of Cardiology,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062,Chin)

机构地区：[1]上海中医药大学附属普陀医院心内科,上海200062

出　　处：《复旦学报（医学版）》2018年第4期485-489,544,共6页Fudan University Journal of Medical Sciences

基　　金：上海市卫计委重点学科建设项目(ZK2015A17);上海市卫计委青年科研项目(20134Y083)~~

摘　　要：目的通过小鼠心脏缺血再灌注模型明确心肌缺血时心肌树突状细胞(dendritic cell,DC)所占比例、动态变化及其与单核细胞的相对比例变化,以证明DC参与缺血损伤相关的免疫炎症反应。方法制作小鼠的心脏缺血再灌注模型,心脏缺血30 min后分别于再灌注1、2、4和7天时摘取小鼠心脏,制备心脏单个细胞悬液。同时加入CD45、CD11c和Ly6C抗体进行流式细胞检测,策略为选取CD45阳性炎症细胞进一步行DC标记CD11c、单核细胞标记Ly6C的细胞亚群分析。假手术组作为对照组。结果每只小鼠心脏制备所得的单个细胞悬液行细胞计数为(1~2)×106个。CD45阳性细胞占总细胞数的比例于再灌注1天时即达到高峰,逐渐恢复至对照组水平。CD45阳性细胞群中以CD11c和Ly6C标记细胞亚群的相对比例,以CD11c+Ly6C+DC动态变化最为明显,2、4、7天均有显著升高,7天时恢复至对照组水平;与此同时,CD11c+Ly6C-DC和CD11c-Ly6C+单核细胞的相对比例有短暂降低,CD11c-Ly6C-细胞的相对比例无显著变化。CD11c,Ly6C标记的各亚群细胞占心肌细胞数的比例在1~2天较对照组均有显著升高,7天时恢复至对照组水平。结论小鼠心脏缺血再灌注后1天时CD11c+Ly6C+DC明显增多,在短期内可消退,提示DC参与缺血损伤相关的免疫炎症反应。Objective To investigate cardiac dendritic cells （DCs） in inflammatory response after cardiac ischemic-reperfusion injury （IRI） by determining change of cardiac DC numbers and its proportion to monocytes in IRI mice. Methods Mice model was made by 30-minute cardiac ischemia followed by the reperfusion of 1, 2, 4 and 7 days, and single cell suspensions were prepared. After incubation with antibodies against CD45 （leukocyte marker）, CD11c （DC marker） and Ly6C （monocyte marker）, single cell suspensions were evaluated by flow cytometry. CD45＋ leukocytes and 4 subpopulations labelled by CD11c and Ly6C were analyzed. Sham-operated group was used as the control. Results The single cell suspension from a whole mouse heart yielded a total cell count of （1-2）×106. A marked increase of CD45＋ leukocytes was identified 1 day post reperfusion, which decreased to control level through 2 to 7 days post reperfusion. Proportion of CD11c＋Ly6C＋ DCs in CD45＋ leukocytes significantly increased from 1 to 4 days post reperfusion, and restored to the control level 7 days post reperfusion. A resultant decrease in proportions of CD11c＋Ly6C- DCs and CD11c-Ly6C＋ monocytes in CD45＋ leukocytes were noted. The proportion of CD11c-Ly6C- subpopulation demonstrated no major alteration. Moreover, in the CD45＋ leukocytes, the percentages of CD11c＋Ly6C＋, CD11c＋Ly6C-, CD11c-Ly6C＋ and CD11c-Ly6C- DCs subpopulation in total heart cells all peaked on 1 to 2 days, and restored to control level on 7 days. Conclusions The prominent change pattern of CD11c＋Ly6C＋ DCs after IRI in the mouse heart suggested a functional role in IRI related inflammation and warranted further investigation.

关 键 词：缺血再灌注 心肌树突状细胞 单核细胞 流式细胞术 小鼠 

分 类 号：R541[医药卫生—心血管疾病] R-332[医药卫生—内科学]