犬牙周膜干细胞体外分离培养和鉴定的实验研究  

Isolation and identification of dog periodontal ligament stem cells

作　　者：常秀梅[1,2] 刘宏伟[3] 金岩[2] 刘源[2] 贺慧霞[2] 

机构地区：[1]广东医学院附属医院口腔科,广东湛江524001 [2]第四军医大学口腔医学院口腔组织病理教研室,陕西西安710032 [3]同济大学口腔医院口腔内科,上海200072

出　　处：《华西口腔医学杂志》2009年第1期79-83,共5页West China Journal of Stomatology

基　　金：国家高技术研究发展计划(863计划)资助项目(2002AA-205041)

摘　　要：目的体外分离培养犬牙周膜干细胞,并对其进行生物学鉴定。方法采用有限稀释法进行犬牙周膜细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、苏木精-伊红染色以及生长因子分化诱导等方法观察其生物学特性。结果1)克隆化分离培养的犬牙周膜干细胞呈集落状生长,增殖快,克隆形成率为0.95%。2)苏木精-伊红染色见细胞呈成纤维样,体积小、核大,有较多的双核细胞。3)碱性磷酸酶染色阳性。4)细胞表型组化鉴定波形丝蛋白、Ⅰ型胶原、Ⅲ型胶原阳性,间充质干细胞表面标志STRO-1染色阳性,角蛋白染色阴性。5)在含β-磷酸甘油钠、维生素C、地塞米松的矿化液中,细胞被诱导分化为成骨细胞,碱性磷酸酶活性增强,Ⅲ型胶原染色阴性,能形成矿化结节,细胞表达成骨细胞特异的骨涎蛋白,在含β-羟基乙醇的培养液中,被诱导分化为神经细胞样细胞。结论克隆化分离培养的犬牙周膜干细胞具有很强的克隆形成能力,具有间充质干细胞表型和多向分化潜能。Objective To isolate, culture and identify a dog periodontal ligament stem cells（PDLSC） line in vitro. Methods The adult dog periodontal ligament cells were isolated by limited dilution of culture cell for single cell clone. Cells originated from one of these clones were assessed through colony-forming efficiency and immunocyto- chemistry assay and alkaline phosphatase stain was used to identify the source of adult dog periodontal stem cells, at the same time, PDLSC were induced with mineralizatin solution and was found to have long protrude like an osteoblast. Differentiation of PDLSC were assessed, Mineralized potential was studied by Von-Kossa staining. Results The dog PDLSC expressed STRO-1, which was the marker of mesenchymal stem cells. Also Vimentin, osteoblast-like marker alkaline phosphatase and Collagen-Ⅰ expressed weakly. Cells were elonegenic, highly proliferative cells and capable of differentiating into osteoblasts/cementoblasts. Conclusion The evidence suggests that the cultured cells were stem cells from adult dog periodontal ligament.

关 键 词：牙周膜干细胞 分离 鉴定 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]