晚期骨关节炎患者膝关节滑膜间充质干细胞的体外成骨分化  

Osteogenic differentiation of human synovium-derived mesenchymal stem cells from osteoarthritic knee joints

作　　者：芮云峰[1] 林禹丞 陈辉[1] 王宸[1] 

机构地区：[1]东南大学附属中大医院骨科,江苏省南京市210009

出　　处：《中国组织工程研究》2013年第45期7840-7846,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年基金项目(81201422);江苏省自然科学基金青年基金项目(BK2012334);东南大学基本科研业务费"创新基金"项目(3290002401);国家大学生创新训练计划(1210286090);中国博士后科学基金项目和江苏省博士后科学基金特别资助项目(2012M520983)~~

摘　　要：背景:间充质干细胞是组织工程中常用的种子细胞,而来源于人膝关节滑膜组织的间充质干细胞能否作为骨组织修复与再生中合适的种子细胞还需要进一步验证。目的:观察晚期骨关节炎患者膝关节滑膜组织来源的间充质干细胞体外向成骨细胞定向分化的潜能,对所诱导细胞的成骨特性进行鉴定。方法:无菌条件下从行全膝关节置换的晚期骨关节炎患者膝关节腔内获取滑膜组织,Ⅰ型胶原酶消化分离获得有核细胞。以最适密度接种(200个有核细胞/cm2),挑选单细胞克隆,筛选出滑膜间充质干细胞。基础培养基培养,传至第3代时改换为含地塞米松、β-甘油磷酸钠和抗坏血酸的成骨诱导培养基诱导培养。结果与结论:体外分离培养的滑膜间充质干细胞早期可形成葵花样细胞集落,克隆样生长。传代至第3代,细胞呈成纤维细胞样生长,形态均一。滑膜间充质干细胞成骨诱导后呈典型的"铺路石样"成骨细胞形态,诱导至第7天碱性磷酸酶染色呈强阳性,碱性磷酸酶活性表达也在诱导7 d时达最高峰;诱导21 d茜素红染色可见大量钙结节形成;同时反转录PCR检测到Ⅰ型胶原、Runx2、骨结合蛋白和骨桥蛋白的表达在诱导后增加,21 d时表达最高。从晚期骨关节炎患者膝关节滑膜组织分离获得的滑膜间充质干细胞,在体外可成功诱导分化为成骨细胞,所诱导生成的细胞具有典型的成骨细胞特性。提示滑膜间充质干细胞有望成为骨组织工程理想的种子细胞来源,在骨组织的再生修复中发挥重要作用。BACKGROUND： Mesenchymal stem cells are commonly used in tissue engineering, while whether synovium-derived mesenchymal stem cells from human knee joirlts can make a role in repair and regeneration of bone tissue as the appropriate seed cells need to be further verified. OBJECTIVE： To study the osteogenic differentiation potential of synovium-derived mesenchymal stem cells which were harvested from human knee joint with end-stage osteoarthritis in vitro. Meanwhile, to identify the osteogenic characteristics of these induced synovium-derived mesenchymal stem cells. METHODS： Cell populations were enzymatically released from the synovial membrane obtained from total knee arthroplasty. Nucleated cells were plated at an appropriate density （200 cells/cm2） for expansion at the maximum rate without colony-to-colony contact. Monoclone was obtained by selecting as primary synovium-derived mesenchymal stem cells. After primary cultured in Control medium and expanded to three passages, synovium- derived mesenchymal stem cells were subjected to in vitro assays to investigate their osteogenesis potential in osteogenic medium containing dexamethasone, -glycerophosphate and ascorbic acid. RESULTS AND CONCLUSION： Nucleated cells from the synovial membrane formed single cell-derivedcolonies, which were of polygon shape and star shape, uniform in size. After three passages, homogeneous populations of fibroblast-like cells were observed. Under appropriate culture conditions, synovium-derived mesenchymal stem cells were induced to differentiate to the osteocyte lineages which had typical ＂slabstone＂ appearance of osteoblasts. Osteogenesis was stained positively for alkaline phosphatase staining at day 7 and formed mineralized nodular structures at day 21, which was confirmed by Alizarin red staining. Alkaline phosphatase activity assay showed a rise after the osteogenesis induction and reached the peak at day 7. Expressions of osteocyte specific genes, such as collagen type ｜, Runx2, bone-binding protein and osteopo

关 键 词：干细胞 干细胞培养与分化 滑膜组织 间充质干细胞 滑膜干细胞 膝关节 骨关节炎 成骨诱导分化 成骨细胞 国家自然科学基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]