URSA患者子宫蜕膜树突状细胞的体外培养与鉴定  

Cultivation and idenification of decidual dendritic cell in URSA

作　　者：刘梅兰[1] 刘玉昆[1] 陈慧[1] 王蕴慧[1] 张睿[1] 刘颖琳[1] 张建平[1] 

机构地区：[1]中山大学孙逸仙纪念医院妇产科,广东广州510120

出　　处：《热带医学杂志》2011年第4期385-387,391,F0002,共5页Journal of Tropical Medicine

基　　金：广东省自然科学基金博士启动基金(06021369);教育部博士点新教师基金(20090171120075)

摘　　要：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 develop a method for the cultivation of human decidual dendritic cells from patients with an unexplained recurrent spontaneous abortion（URSA）.Methods Between July 2008 and April 2009,decidual tissues from URSA patients at week-7 to 14 of gestation were collected.The tissues were dispersed mechanically.Mononuclear cells were released by Ⅳ collagenase/DNA-Ⅰ digestion and the cells were subsequently isolated using density gradient centrifugation.DCs were then treated with GM-CSF and IL-4.The cell suspension was gently collected at day-10 after incubation.The cells were then stained with FITC-HLADR and PE-Lin,and analyzed by flow cytometric method.Results Decidual dendritic cells were obtained from the freshly collected decidual tissues..Microscopic study indicated that the adherent cells gradually form small colony in the suspension.At day-9,most of the cells were in single cell suspension.The cells were still in small and round morphology.The formation of dendritic processes was not apparent.The culture consisted of a mixture of small slender spindle-shaped macrophages and DCs.At day-10,the purity of DCs was above 80%.Conclusion The method using enzyme digestion and gradient centrifugation can successfully separate decidual dendritic cells from the tissues obtained from URSA patients.GM-CSF and IL-4 can promote the growth of the immature dendritic cells.The dendritic cells may be used the study the maternal immunity and immunological tolerance of the fetus in URSA patients.

关 键 词：URSA 树突状细胞 免疫耐受 蜕膜 培养和鉴定 

分 类 号：R392.9[医药卫生—免疫学]