大鼠鼻窦黏膜干细胞的分离培养和成骨能力研究

目的明确大鼠鼻窦结构,尝试体外分离培养鼻窦黏膜干细胞,检测其多向分化能力,为口腔颌面部骨缺损修复治疗的体外研究寻找新的种子细胞。方法选用3月龄SD大鼠,进行口腔颌面部和鼻窦解剖,制作冠状位石蜡切片观察鼻窦形态和黏膜成分。取鼻窦内衬黏膜,改良酶消化法进行间充质干细胞(SMSCs)分离培养,检测其成克隆能力,使用流式细胞术进行间充质干细胞表面标志物鉴定。油红O染色检测SMSCs的成脂肪能力,茜素红染色和碱性磷酸酶(ALP)活性测试检验SMSCs的成骨能力,并与大鼠牙龈来源间充质干细胞(GMSCs)、大鼠骨髓来源间充质干细胞(BMSCs)比较。结果 SD大鼠具有结构清晰的上颌窦,窦腔内衬黏膜呈现典型的三层结构。使用该内衬黏膜能够成功分离培养出具有多向分化能力和成克隆能力且带有间充质干细胞表面标志物的SMSCs细胞。在成骨培养基诱导下,SMSCs胞外基质矿化能力与GMSCs和BMSCs相比差异无统计学意义(P0.05)。但14天时SMSCs的ALP活性显著大于GMSCs(P0.05)。结论 SD大鼠鼻窦内衬黏膜可成功分离培养出具有体外成骨能力的间充质干细胞,为口腔颌面部骨组织工程研究提供新的细胞选择。

Isolation and osteogenic differentiation of mesenchymal stem cells derived from rat sinus membrane

Objective: The present study aimed to investigate the anatomy of rat sinus to isolate mesenchymal stem cells (MSCs) from rat sinus membrane, and compare its differentiation capability with MSCs derived from other tissues. Materials and Methods: Eight Sprague-Dawley rats of 3 months were sacrificed. Paraffin sections of rat skulls with HE staining were prepared for anatomic structure observation. MSCs were isolated from the lining membrane of the rat sinus by modified enzyme digestion (SMSCs). The cell surface markers were characterized by flow cytometric analysis. The self-renewal capabillity was tested by colony-forming units (CFUs) assay. Its potential of osteogenic and adipogenic differentiation were analyzed by Alizarin red staining, alkaline phosphatase (ALP) activity and Oil red O staining, whose results were also compared with gingival and bone marrow derived MSCs (GMSCs and BMSCs). Results: An obvious structure of paranasal sinus and its three-layer lining membrane were detected on sections of rat skull. SMSCs with multi- lineage differentiation and colony-forming capability were isolated from rat sinus membrane. After certain days of osteogenic induction, the extra-cellular calcification showed no significant differences among SMSCs, GMSCs and BMSCs. But the ALP activity of SMSCs at 14 days was significant higher than that of GMSCs. Conclusion: SMSCs of comparable osteogenic potential can be isolated and cultured in vitro from the sinus membrane of Sprague-Dawley rats. This might provide a new cell resource for future studies on bone regeneration in oral and maxillo-facial surgery.