CAJ | 학술논문

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。
배경：간충질간세포시조직공정중상용적충자세포，이래원우인슬관절활막조직적간충질간세포능부작위골조직수복여재생중합괄적충자세포환수요진일보험증。 　　목적：관찰만기골관절염환자슬관절활막조직래원적간충질간세포체외향성골세포정향분화적잠능，대소유도세포적성골특성진행감정。 　　방법：무균조건하종행전슬관절치환적만기골관절염환자슬관절강내획취활막조직，Ⅰ형효원매소화분리획득유핵세포。이최괄밀도접충(200개유핵세포/cm2)，도선단세포극륭，사선출활막간충질간세포。기출배양기배양，전지제3대시개환위함지새미송、β-감유린산납화항배혈산적성골유도배양기유도배양。 　　결과여결론：체외분리배양적활막간충질간세포조기가형성규화양세포집락，극륭양생장。전대지제3대，세포정성섬유세포양생장，형태균일。활막간충질간세포성골유도후정전형적“포로석양”성골세포형태，유도지제7천감성린산매염색정강양성，감성린산매활성표체야재유도7 d시체최고봉；유도21 d천소홍염색가견대량개결절형성；동시반전록PCR검측도Ⅰ형효원、Runx2、골결합단백화골교단백적표체재유도후증가，21 d시표체최고。종만기골관절염환자슬관절활막조직분리획득적활막간충질간세포，재체외가성공유도분화위성골세포，소유도생성적세포구유전형적성골세포특성。제시활막간충질간세포유망성위골조직공정이상적충자세포래원，재골조직적재생수복중발휘중요작용。
BACKGROUND:Mesenchymal stem cells are commonly used in tissue engineering, while whether synovium-derived mesenchymal stem cells from human knee joints 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:cellpopulations were enzymatical y 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,β-glycerophosphate and ascorbic acid. RESULTS AND CONCLUSION:Nucleated cells from the synovial membrane formed single cel-derived colonies, 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 col agen type Ⅰ, Runx2, bone-binding protein and osteopontin, were al detected. These genes were expressed positively in osteogenic medium, and the mRNA expressions of col agen type Ⅰ, Runx2, bone-binding protein and osteopontin were enhanced significantly after 21 days. Our study demonstrates that synovium-derived mesenchymal stem cells isolated from knee joint of end-stage osteoarthritis patients could be induced into osteoblasts in vitro, and these induced cells have typical osteogenesis characteristics. Synovium-derived mesenchymal stem cells may play a role in the regenerative response during the process of bone injury, which are promising candidates for bone tissue engineering.