三色荧光标记鉴定早孕蜕膜及外周免疫细胞组成

Characterization of decidual and peripheral immune cells of the first-trimester pregnancy by 3-color flow cytometry

目的建立早孕妇女蜕膜免疫细胞高纯度的分离方法,流式细胞术多色荧光直接标记分析早孕蜕膜及外周血主要免疫细胞的构成比。方法经胶原酶消化、Percoll密度梯度离心、短期培养结合的方法分离纯化蜕膜免疫细胞,采用三色、双色及单色荧光直接标记流式细胞术分别检测早孕妇女和对照组中蜕膜、外周血CD3-CD56+CD16-和CD3-CD56+CD16+NK细胞、NKT和γδT细胞、T细胞和单核细胞的阳性率。结果与外周血相比,蜕膜免疫细胞以CD3-CD56+CD16-数量最多,约占免疫细胞总数的(67.02±18.33)%,T细胞占(11.05±7.22)%,单核细胞占(5.28±0.29)%,NKT细胞占(2.35±1.62)%;早孕妇女外周血T淋巴细胞较正常生育期妇女明显下降(P<0.05),而早孕妇女外周血中NK细胞、NKT细胞、单核细胞数量与对照组相比无显著差别。结论早孕妇女蜕膜中具有与外周血不同的免疫细胞组成,应用多色荧光标记的流式细胞术能够简单、直接地鉴定各免疫细胞亚群。

Objective： To establish a method to isolate and characterize efficiently the immune cells in decidua and periphery blood of human early pregnancy by using directly labeled 3-color flow cytometry. Methods： The decidual immune cells were isolated and purified by collagenase IV- digestion, discontinuous Percoll gradient centrifugation and short-period culture. CD3^- CD56^＋ CD16^- and CD3^-CD56^＋ CD16^＋ NK cell, NKT cell and γδT cell, T cell and monocyte were evaluated by directed labeled 3-color or 2-color or single color flow-cytometry from decidua and periphery blood. Results： CD3^- CD56^＋ CD16^- NK cell, T cell, monocyte and NKT cell accounted for（67.02± 18.33）%,（11.05±7.22）%, （5.28±0.29）%, （2.35± 1.62）% of decidual immune cell, respectively. T cell showed a significant decrease in periphery blood of pregnant women than in the control. Conclusions： Decidual immune cells consist of different lymphocyte subsets from those in peripheral blood. The 3-color cytometry can be successfully used to characterize the subsets of immune cells in decidua and periphery blood.