沙晓燕?伍彬升?鲍俊杰?刘慧姝

【摘要】目的 探討蜕膜巨噬细胞对滋养细胞侵袭力的调控。方法 取20 ～ 35岁女性正常早孕（6 ～ 7+6周）行人工流产的蜕膜组织15 ～ 20 g。将蜕膜组织充分清洗后使用Collagenase和Dnase消化2次，每次30 min，细胞悬液过滤后通过CD14+免疫磁珠分选，并行免疫荧光鉴定。流式细胞仪分析巨噬细胞M1型（CD80+和CD86+）和M2型（CD163+和CD206+）。获得的巨噬细胞置于6孔板培养，24 h后收集细胞上清。巨噬细胞上清与滋养细胞系HTR-8/SVneo共培养24 h，实时定量PCR检测HTR-8/SVneo的水通道蛋白1（AQP 1）mRNA相对表达量变化，以及基质金属蛋白酶-2（MMP-2）和MMP-9 mRNA相对表达量的变化。应用实时无标记细胞分析技术（RTCA）检测人早孕蜕膜巨噬细胞上清对人滋养层细胞系HTR-8/SVneo侵袭力的影响。结果 成功建立人类早孕蜕膜巨噬细胞体外培养体系。流式细胞检测显示，人类正常早孕蜕膜组织中巨噬细胞以M2型为主，约占全部巨噬细胞的87%。巨噬细胞上清使HTR-8/SVneo的AQP 1和MMP-9 mRNA相对表达量升高（P均< 0.001）。RTCA结果显示巨噬细胞上清明显增强HTR-8/SVneo的侵袭力（P < 0.001）。结论 体外培养条件下，蜕膜巨噬细胞可能通过介导AQP1表达升高而增强滋养细胞的侵袭力。

【关键词】蜕膜巨噬细胞;水通道蛋白1;滋养细胞侵袭力;实时无标记细胞分析

Study of the regulation of trophoblast invasion by decidual macrophages Sha Xiaoyan， Wu Binsheng， Bao Junjie， Liu Huishu. Department of Obstetrics， Preterm Birth Prevention and Treatment Research Unit， Guang-zhou Women and Childrens Medical Center， Guangzhou Medical University， Guangzhou 510623， China

Corresponding author， Liu Huishu， E-mail： liuhuishu@ gwcmc. org

【Abstract】Objective To investigate the regulatory effect of decidual macrophages on the invasion of trophoblast. Methods Decidual tissues （15-20 g） were collected from clinically normal first trimester （6 - 7+6 weeks） pregnant women （20-35 years old） who underwent elective termination of pregnancy for non-medical reasons. Freshly-collected decidual tissues were rinsed with PBS and digested with collagenase Type IV and DNase I for 30 min twice. Macrophages were purified using a Ficoll-Hypaque gradient followed by CD14+ selection using magnetic beads， and identified by immunofluorescence. The percentage of M1 macrophages （CD80 and CD86） and M2 macrophages （CD163 and CD206） were analyzed by flow cytometry. The macrophages were cultured in a 6-well plate and the supernatant was collected 24 h later. The macrophage supernatant was co-cultured with trophoblast cell line HTR-8/SVneo for 24 h. The changes in the relative expression of AQP1 in HTR-8/SVneo as well as those of matrix metalloproteinase 2 （MMP2） and MMP9 were detected by real-time quantitative PCR. Real-Time Cell Analysis was used to evaluate the effect of macrophage supernatant on the invasiveness of HTR-8/SVneo. Results An in vitro cell culture system of human early pregnant decidual macrophages was established. Flow cytometry results showed that M2 macrophages were predominant in normal early pregnant decidual tissues， accounting for approximately 87%. Macrophage supernatant significantly up-regulated the expression levels of AQP 1 and MMP 9 in HTR-8/SVneo （both P < 0.001）. RTCA demonstrated that macrophage supernatant significantly enhanced the invasiveness of HTR-8/SVneo （P < 0.001）. Conclusion Decidual macrophages may enhance the trophoblast invasiveness by mediating the up-regulated expression of AQP 1 in vitro.

【Key words】Decidual macrophage;Aquaporin 1;Trophoblast invasion;Real-time cell analysis

胎盘滋养细胞侵袭力异常可引起流产、子痫前期/子痫、胎儿生长受限、妊娠滋养细胞疾病、胎盘植入等妊娠相关性疾病[1]。巨噬细胞在母胎界面免疫状态的形成和维持中发挥了重要的调控作用[2]。巨噬细胞如果发生分化异常、亚群平衡失调或功能异常时，也会导致流产、子痫前期、胎儿生长受限等病理妊娠及不良妊娠结局[3]。母胎界面巨噬细胞等免疫细胞可能调控滋养细胞的侵袭，但具体机制不清。多项研究显示，水通道蛋白1（AQP1）不仅调控多种细胞的水通透性，而且介导病理生理变化过程中的细胞迁移及侵袭[4]。……