免疫组织荧光与流式细胞术检测脊髓损伤局部巨噬细胞亚群的比较

The comparison of immunohistochemical fluorescence and flow cytometry in detection of the infiltrated macrophage subtypes in injured spinal cord

目的比较免疫组织荧光(IHF)与流式细胞术(FCM)检测脊髓损伤(SCI)局部M1和M2型巨噬细胞亚群的优缺点。方法采用纽约大学脊髓损伤仪制做大鼠重物坠落SCI模型。术后7天,分别采用IHF和FCM检测损伤脊髓中M1和M2型巨噬细胞,并进行比较。结果两种方法均可有效地检测脊髓损伤局部浸润的巨噬细胞及其M1、M2亚群。IHF检测到的M1(CD68^+CCR7^+)和M2(CD68^+Arg1^+)细胞亚群的比率为2.79±0.21,FCM检测到的M1(CD68^+CD86^+)和M2(CD68^+CD163^+)细胞亚群的比率为2.46±0.20,两种方法检测到的结果没有统计学差异(P>0.05)。IHF可对待测的细胞进行定位,但在细胞计数和比例分析方面需要人工完成;而FCM则可以直接自动分析细胞数量和比例,但无法对其进行定位。结论 IHF和FCM在分析脊髓浸润的细胞表型方面各有优缺点,两者结合可有效地分析SCI局部M1和M2巨噬细胞亚群的数量和比例。

Objective To analyze and compare the advantages and disadvantages of immunohistochemical fluorescence （IHF） and flow cytometry （FCM） in detection of the infiltrated M1 and M2 macrophage subtypes in injured spinal cord. Methods The cont- usive spinal cord injury （SCI） model was established by using New York University Impactor. Seven days after SCI, the number and proportion of M1 and M2 macrophage subgroups in the inhured spinal cords were detected and compared by IHF and FCM. Results Both IHF and SCI could detect the infiltrated macrophages and M1 and M2 subtypes in the local injured spinal cord of rats. The ratio of MI （CD68^＋ CCR7^＋ ） to M2（CD68^＋ Argl^＋ ） detected by IHF was 2.79±0.21 , while the ratio of M1 （ CD68^＋ CD86^＋ ） to M2 （ CD68^＋ CD163^＋ ） detected by IHF was 2.46 ± 0.20 detected by FCM. There was no statistic difference in the results detected by the two methods （ P 〉 0.05 ）. IHF could be used to locate the detected cells. However, it was difficult to count ceils and analyze proportion automatically. Although FCM could do them automatically, it couldn＇t reveal the eel1 location. Conclusion There are advantages and limitations in testing the phenotype of the tissue infiltrated cells by IFH and FCM. It is the best way to analyze the number, percentage and position of M1 and M2 macrophage subgroups in the local injured spinal cords by combining IFH and FCM.