人牙周韧带细胞膜片的体外实验研究  

The investigation of human periodontal ligament cell-sheet in vitro

作　　者：张剑英[1] 付云[1] 俞少杰[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2013年第1期31-34,共4页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：广东省科技计划(2010B031100010)

摘　　要：目的观察体外培养人牙周韧带细胞(hPDLCs)的成骨、成脂能力;构建hPDLCs膜片并鉴定膜片细胞外基质的主要结构蛋白。方法通过酶联组织块法分离并纯化培养hPDLCs;免疫细胞化学法鉴定hPDLCs来源及干细胞标志物STRO-1表达情况;取第2～4代细胞分别用成骨诱导培养基和成脂诱导培养基培养,茜素红及油红O染色检测hPDLCs的成骨、成脂能力;制备成熟的hPDLCs膜片,苏木素-伊红(HE)及免疫组织化学染色法鉴定膜片细胞外基质主要结构蛋白Ⅰ型胶原、层黏蛋白及纤连蛋白的表达情况。结果经原代培养的hPDLCs抗波形蛋白染色阳性,干细胞标志物STRO-1染色阳性,证实hPDLCs来源于间充质并具有干细胞潜在分化能力;hPDLCs在诱导成骨、成脂分化培养14～16d后,茜素红及油红O染色结果阳性提示hPDLCs具有良好的成骨及成脂分化能力;HE染色及免疫化学染色发现hPDLCs膜片高表达细胞外基质主要结构蛋白Ⅰ型胶原、层黏蛋白及纤连蛋白。结论本研究从细胞分离培养,来源鉴定、多向分化潜能诱导及细胞膜片技术等方面证实PDLCs中存在成体干细胞,提示hPDLCs膜片能为牙周组织再生种子细胞提供新来源,可进一步深入研究。Objective To investigate human periodontal ligament cells＇ capacities of osteogenesis and adipogenesis, and to identify the expression of type-I collagen, laminin and fibronectin which are the main components of extracellular matrix in human periodontal ligament cell sheet through establish cell sheet model in vitro experiment. Methods Human periondontal ligament cells were purified and cultured by using enzymes digesting/tissue culture method. Immunocytochemistry was applied to identify the source and putative stem-cell marker STRO-1 of hPDLCs. The second to fourth passages of hPDLCs were cultured in the osteogenic medium and adipogenic medium for indicated days. Alizarin red and Oil red O staining method were applied to identify capabilities of osteogenesis and adipogenesis of hPDLCs. Haematoxylin-eosin staining and immunohistochemistry were applied to identify expression of type- I collagen, laminin and fibronectin in hPDLCs cell sheet. Results The cultured hPDLCs demonstrated extensive proliferation and could be expanded in vitro. Immunostaining demonstrated that the ceils expressed intermediate filament protein vimentin and the mesenchymal stem-ceU marker STRO-1. hPDLCs in the osteogenic medium formed alizarin red-positive nodules. When hPDLCs were incubated in adipogenic medium, the ceils developed into oil red O-positive lipid clusters and a significant high expression of type-I collagen, laminin and fibronectin in human periodontalligament cell sheet. Conclusions Our findings confirm that hPDLCs contained stem cells population from culture, source identification, expression of the mesenchymai stem cell marker STRO-1, multilineage differentiation potent under defined culture conditions. Our swdy indicates that the hPDLCs turn out to be an efficient source and cell sheet to be an excellent bio-material that can be used for periodontal tissue regeneration.

关 键 词：人牙周韧带细胞 细胞膜片 牙周组织再生 

分 类 号：R780.2[医药卫生—口腔医学]