维甲酸诱导小型猪牙周膜干细胞的体外成骨

背景：近期研究发现维甲酸对胚胎干细胞及多种成体干细胞具有成骨方向诱导的作用。目的：观察维甲酸对小型猪牙周膜干细胞体外成骨作用的影响。方法：采用组织块法获得小型猪牙周膜细胞,有限稀释法纯化小型猪牙周膜干细胞,免疫荧光法检测STRO-1、免疫细胞化学法检测波形蛋白、角蛋白鉴定小型猪牙周膜干细胞。CCK8法测定小型猪牙周膜干细胞增殖曲线,检测小型猪PDLSC克隆形成率。使用维甲酸对第3代小型猪牙周膜干细胞进行成骨诱导,茜素红染色检测矿化结节,免疫细胞化学方法检测成骨相关基因骨桥蛋白、骨钙素、Ⅰ型胶原、Ⅲ型胶原的表达。结果与结论：有限稀释法分离纯化小型猪牙周膜干细胞STRO-1,波形蛋白阳性表达,角蛋白阴性表达,克隆形成率为2.8%,维甲酸诱导14 d后,碱性磷酸酶染色阳性,诱导21 d后茜素红染色阳性,免疫细胞化学法检测骨桥蛋白、骨钙素、Ⅰ型胶原阳性表达,Ⅲ型胶原阴性表达。结果表明小型猪牙周膜干细胞能够被维甲酸诱导向成骨样细胞分化。

Retinoic acid induces osteogenic differentiation of periodontal ligament stem cells from miniature swine in vitro

BACKGROUND:Recent studies have found that retinoic acid can induce the osteogenic differentiation of embryonic stem cells and multiple adult stem cells.OBJECTIVE: To observe the effect of retinoic acid on osteogenic differentiation of porcine periodontal ligament stem cells (PDLSCs).METHODS:Porcine PDLCs were harvested by using outgrowth method; PDLSCs were isolated by limited dilution of culture cells for single cell clone. Immunofluorescence was used to detect the expression of STRO-1 and immunocytochemistry to detect the expression of vimentin and pan-Cytokeratin (PCK) of porcine PDLSCs. Cell counting kit8 (CCK8) was applied to evaluated cell proliferation of PDLSCs. The colony formation of porcine PDLSCs ratio was assayed. Third passage PDLSCs were induced with mineralized conditional medium containing retinoic acid, ascorbic acid and β-glycerophosphate. Mineralized nodules were studied by Alizarin red S staining. Osteopontin, osteocalcin, collagen typeⅠ and collagen type Ⅲ were detected by immunocytochemistry.RESULTS AND CONCLUSION:Porcine PDLSCs expressed STRO-1 and vimentin, and the result of pan-Cytokeratin was negative. Porcine PDLSCs colony formation ratio was 2.8%. PDLSCs induced by retinoic acid showed positive expression of alkaline phosphatase at 14 days and positive expression of Alizarin red S at 21 days. Osteopontin, osteocalcin, collagen typeⅠ were positive but collagen type Ⅲ was negative at 21 days. These findings indicate that retinoic acid can be an effective inducer of osteogenic differentiation of porcine PDLSCs.