人牙囊细胞的体外分离、培养及表型鉴定

Culture and characterization of the dental follicle cells from human dental germ in vitro

目的 :建立人牙囊细胞 (dentalfolliclecells ,DFCs)的体外培养方法 ,为牙周组织工程研究提供可靠的种子细胞来源。方法 :分离人 8月龄胚胎下颌磨牙牙胚 ,胰酶消化后分离牙囊组织 ,采用胶原酶消化法和组织块法相结合原代培养人牙囊细胞 ,利用牙囊细胞和上皮根鞘细胞对胰酶的耐受性差别分离纯化 ,通过倒置显微镜及HE染色观察细胞形态及生长状况、透射电镜观察细胞的超微结构 ;采用免疫组化波形丝蛋白 (vimemtin ,Vim)、角蛋白(cytokeration ,CK)、Ⅰ型胶原 (typeⅠcollagen ,ColⅠ )、Ⅲ型胶原 (typeⅢcollagen ,ColⅢ )、纤维连接蛋白 (fibronectin ,FN)和早期矿化相关的标志骨桥蛋白 (osteopontin ,OPN)、骨涎蛋白 (bonesialapretion ,BSP)的表达情况对细胞做表型鉴定。结果 :原代培养的人牙囊细胞生长良好 ,但其中混有少量上皮根鞘细胞 ,经传代可完全分离 ,传至 11代仍保持原有活力 ;牙囊细胞为长梭型 ,成纤维样 ;电镜下见细胞核浆比例大 ,核仁明显 ,含有溶酶体、大量的粗面内质网和发达的高尔基体 ,含有大量的中间丝。电镜结果显示细胞代谢旺盛 ,增殖活性高 ,含有牙囊细胞特有的高密度电子颗粒 ,免疫组化Vim、ColⅠ、ColⅢ、FN、OPN、BSP呈阳性着色 ,抗角蛋白呈阴性着色。结论

Objective:To develop a culture model for human dental follicle cells and investigate it as seed cell for periodontal tissue engineering.Methed: Developing mamdibular germ from human embry were subjected to trypsin in Hank's balanced salt solution. After trypsinization, the dental follicle was enucleated from the tooth germ and seprarated from the associated epithelial root sheath. Pure dental follicle tissue were cultured in vitro. Cells were obtained from human dental dental follicle by collagenase digesting and purified it combining several methods. The cultured cells were identified by its morphological and ultrastructure observation by light microscope and TEM resperatively, and combined with immunostaining for expression vimemtin, typeⅠcollagen,type Ⅲ collagen,fibronectin,osteopontin and bone sialapretion.Result: The cultured human dental follicle cells were typical fibroblast cells in shape,and transmission electronic microscrope revealed that the cells contain electron-dense granules, aboundant rough endoplasmic reticulum, but did not form in desmosome . vimemtin, typeⅠcollagen,type Ⅲ collagen, fibronectin, osteopontin and bone sialapretion were present in dental follicle cells.Conclusion:The cells cultured in our research is human dental follicle cells, it may be useful as seeding cell for tissue engineering of periodontal tissue regeneration.