釉基质蛋白衍生物对人牙周膜干细胞分化、增殖的影响

Effect of enamel matrix derivatives on the differentiation and proliferation of human periodontal ligament stem cells

背景:釉基质衍生物已经在临床上用于治疗严重的牙周炎,发现其可促进牙周组织修复、再生,但其中的机制还未阐明。目的:探讨釉基质衍生物对牙周膜干细胞分化和增殖的作用。方法:分离培养人牙周膜干细胞,检测其克隆形成率、表面抗原表达情况,鉴定其多向分化潜能。将不同质量浓度的釉基质衍生物(20,50或100 mg/L)作用于牙周膜干细胞培养2,4周,用Trichrome和Von Kosa’s染色法检测牙周膜干细胞胶原合成及矿化结节形成情况,Real time RT-PCR方法检测成骨分化相关基因Ⅰ型胶原、骨钙素、RUX2的表达,MTT法和生长率法检测牙周膜干细胞的增殖情况。结果与结论:牙周膜干细胞呈梭形,其克隆形成率较牙周膜细胞高,表面抗原CD105,CD29,CD45,CD44的表达率分别为99.8%,99.7%,1.26%,98.8%,具备多向分化潜能。釉基质衍生物呈现一定的时间剂量效应关系促进牙周膜干细胞胶原合成及矿化结节形成,促进成骨分化相关基因Ⅰ型胶原、骨钙素、RUX2的表达,促进牙周膜干细胞的增殖,可能在牙周组织修复、再生中发挥作用。

BACKGROUND： The enamel matrix derivative has been used in the clinical treatment of severe periodontitis;however, the mechanism（s） by which enamel matrix derivative promotes periodontal regeneration is still obscure.OBJECTIVE： To explore the effects of enamel matrix derivatives on the differentiation and proliferation of periodontal ligament stem cells.METHODS： Periodontal ligament stem cells were isolated and identified from human teeth. Cloning forming efficiency, surface antigen expression and pluripotency were detected and identified. Enamel matrix derivatives with different concentrations（20, 50, 100 mg/L） were used to culture periodontal ligament stem cells for 2 and 4weeks. Collagen synthesis and mineralized nodule formation were detected using Trichrom staining and Von Kosa＇s staining, respectively; real-time RT-PCR was employed to detect expressions of collagen type I,osteocalcin, and RUX2; MTT and cell growth rate assay were used to detect the proliferation of periodontal ligament stem cells.RESULTS AND CONCLUSION： Periodontal ligament stem cells were spindle-shaped and showed a highercolony forming efficiency than periodontal ligament cells. The expressions of surface antigens of periodontal ligament stem cells-CD105, CD29, CD45, CD44 were respectively 99.8%, 99.7%, 1.26%, 98.8%, indicating periodontal ligament stem cells have the multilineage differentiation potential. Enamel matrix derivatives improve the collagen synthesis and mineralization nodule formation of periodontal ligament stem cells in a time-dose dependent manner. They also can improve the expression of osteogensis-related genes collagen type I, osteocalcin, RUX2 and proliferation of periodontal ligament stem cells.