摘要
目的 探讨建立稳定可靠的婴幼儿血管瘤干细胞分离培养体系.方法 取增生期婴幼儿血管瘤标本,经胶原酶消化、CD133免疫磁珠分选,所得细胞接种于纤维连接蛋白铺层的96孔板,以含10％ FBS的EBM-2培养液培养.分别进行细胞形态学观察,细胞表面标记流式细胞检测,细胞成管试验,细胞诱导分化成脂、成骨试验以及裸鼠皮下成瘤试验.结果 从血管瘤组织分离纯化的CD133（＋）细胞,6h开始贴壁,细胞呈多突起的纺锤形和星形;CD133（＋）血管瘤干细胞阳性表达CD29（99.5％）、CD44（97.9％）、CD90（87.6％）和CD105 （98.5％）,几乎不表达CD31（0.2％）、CD34（0.1％）、CD45 （0.1％）和CD144（0.1％）;在体外可形成网状类似血管壁的结构,并可被诱导分化为成骨细胞和脂肪细胞;接种于裸鼠皮下可形成类似婴幼儿血管瘤的病灶.结论 该分离培养方法可获得血管瘤干细胞,为阐明血管瘤干细胞的特性及更广泛的应用奠定了良好的基础.
Objective To establish a reliable method of isolation and culture of infantile hemangioma stem cells （HemSCs）.Methods Proliferating infantile hemangioma specimens were digested with collagenase to form a single cell suspension.The HemSCs were isolated with anti-CD133 MicroBeads,and were incubated in fibronectin coated 96-well plates with EBM-2 （10% FBS）.HemSCs were identified by morphological characteristics,flow cytometry,cell tubule formation assay,osteoinductive and adipogenic differentiation assay,and subcutaneous tumor formation assay.Results This method enables the rapid isolation of HemSCs which demonstrated typical mesenchymal stem cell morphology in culture.CD133 （＋）HemSCs expressed CD29 （99.5%）,CD44 （97.9%）,CD90 （87.6%） and CD105 （98.5%）,but barely expressed CD31 （0.2%）,CD34 （0.1%）,CD45 （0.1%） and CD144 （0.1%）.These cells could differentiate into osteoblasts and adipocytes,and could form vascular wall like structure in vitro.When implanted into subcutaneous of the nude mice,the cells can develop into hemangioma like lesion histologically.Conclusions This technique can effectively isolate HemSCs from the proliferative hemangioma.These cells could be further used to reveal the charaeteristics of HemSCs,as well as for further study of widespread application.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2016年第4期293-298,共6页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金（81171827）
关键词
血管瘤
干细胞
细胞培养
模型
动物
Hemangioma
Stem cells
Cell culture techniques
Models,animal
