人乳牙牙周膜干细胞的生物学特性研究

目的:体外分离纯化人乳牙牙周膜组织来源的干细胞,研究其生物学特性、表面标记物及多向分化能力。方法:用酶消化法和有限稀释法对正常人乳牙牙周膜组织进行原代培养,并通过免疫细胞化学方法和流式细胞仪对其来源进行鉴定,进而从增殖能力、克隆形成能力和多向分化能力等方面对乳牙牙周膜干细胞的生物学特性进行研究。结果:分离培养获得的人乳牙牙周膜干细胞在形态上与恒牙牙周膜干细胞相似,均为长梭形成纤维细胞样;免疫细胞化学和流式细胞仪分子表型鉴定显示乳牙牙周膜干细胞阳性表达间充质来源的表面标志STRO-1,CD146,CD29和CD90,造血系来源的标志物CD34为阴性;细胞周期,MTT和克隆形成率的检测结果显示,人乳牙牙周膜干细胞的增殖能力强于恒牙牙周膜干细胞;人乳牙牙周膜干细胞在体外诱导条件下可以向骨,脂肪细胞方向分化;同时RT-PCR检测发现,矿化诱导后细胞成骨相关基因(ALP,OCN,Col I)的表达上调,成脂诱导后细胞成脂相关基因(PPAR-γ,C/EBPα)的表达上调。结论:人乳牙牙周膜中确实存在间充质来源的干细胞,并且具有较强的增殖能力和多向分化能力,为牙周组织工程种子细胞提供了新的可能来源。

The study on the biological characteristics of stem cells derived from periodontal ligament of deciduous teeth

Objective： To isolate the stem cells from periodontal ligament of deciduous teeth （DePDLSCs） and observe the biological behaviors of DePDLSCs. Method： DePDLSCs were isolated from the periodontal ligament tissue of deciduous teeth by enzyme digestion and limited dilution method. DePDLSCs were identified by immunocytochemical staining and flow cytometry assay. Cell characteristics of DePDLSCs were compared with PePDLSCs （Periodontal ligament stem ceils from permanent teeth） in colony forming capacity and proliferative capacity. DePDLSCs were analyzed for their multi-lineage differentiation. Result： The results showed that DePDLSCs were fibroblast-like and presented with positive expression of mesenchymal surface markers STRO-1, CD146, CD29 and CD90, and negative expression of the hematopoietic cell marker CD34. The proliferative capacity of DePDLSCs was higher than PePDLSCs by cell cycle analysis, MTT and colony-forming assay. De-PDLSCs could be induced into osteoblast and adipocytes in vitro. Expression of mineralization-related genes including ALP, collagen type I （COL I ） and osteocalcin （OCN） were significantly enhanced in DePDLSCs osteogenic group compared to the control by RT-PCR, Furthermore, expression of fat-related genes including PPAR-γ and C/EBPot were up-regulated in DePDLSCs adipogenic ogenic group. Conclution： These results suggested that stem cells from the periodontal ligament of deciduous teeth were mesenchymal origined. They were highly proliferative and had multiple differentiation capacity. It provides a new potential source of seed cells for periodontal tissue engineering.