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目的建立成熟、完善的不明原因性习惯性流产(unexplainedrecurrentspontaneousabortion,URSA)患者子宫蜕膜树突状细胞(dendriticcell,DC)的体外培养体系。方法选取2008年7月至2009年4月就诊于本院的妊娠早期流产的URSA患者(孕7～14周),将其子宫蜕膜组织初步机械破碎,经混合酶(Ⅳ型胶原酶/DNA酶Ⅰ)消化法分离,梯度离心获得单个核细胞,以粒-巨噬细胞集落刺激因子(GM-CSF)和白介素4(IL-4)体外诱导培养,光镜观察,于第10天收集的悬浮细胞用FITC-HLA-DR、PE-Lin小鼠抗人抗体顺序标记,采用流式细胞术鉴定。结果从蜕膜组织可获取足够数量蜕膜树突状细胞,体外培养24h细胞聚集成大小不等葡萄串状,逐渐悬浮,长出突起,第9天悬浮细胞增多,单个悬浮为主,细胞仍小而圆、表面突起少等未成熟DC的形态特征,培养过程中夹杂有少许细长梭状巨噬细胞培养。流式细胞术鉴定培养到第10天的悬浮细胞中DC纯度达80%以上。结论混合酶消化合并梯度离心法能成功分离URSA患者蜕膜树突状细胞的前体细胞单个核细胞,经GM-CSF联合IL-4培养可以成功诱导具有未成熟特性的DC,为研究树突状细胞在URSA患者母胎免疫耐受中的作用奠定基础。
Objective To establish a well-established in vitro culture system of dendritic cells (DCs) from patients with unexplained recurrent spontaneous abortion (URSA). Methods URSA patients (from 7 to 14 weeks of gestation) who were diagnosed early abortion in our hospital from July 2008 to April 2009 were selected and their uterine decidua tissue was mechanically disrupted by mixed enzyme (type Ⅳ collagenase / DNA Enzyme Ⅰ) digestion, mononuclear cells were obtained by gradient centrifugation, induced with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) Suspension cells were labeled with FITC-HLA-DR and PE-Lin mouse anti-human antibody and identified by flow cytometry. Results A sufficient number of decidual dendritic cells were obtained from the decidua tissue. After cultured in vitro for 24 h, the cells were aggregated into clusters of clusters ranging in size from grape to grape, gradually growing and protruding. On day 9, the number of suspended cells increased, Small and round, less protruding surface morphological characteristics of immature DC, culture process mixed with a little long spindle macrophage culture. Flow cytometry identification of DCs cultured in suspension cells on day 10 reached more than 80% purity. Conclusion Mixed enzyme digestion combined with gradient centrifugation successfully isolated precursor cells of decidual dendritic cells from URSA patients. DCs with immature characteristics could be successfully induced by GM-CSF combined with IL-4. Dendritic cells lay the foundation for the role of immune cells in URSA patients.