【作者】 李鹏；

【导师】 齐志明；

【作者基本信息】 大连医科大学 ， 外科学， 2012， 硕士

【摘要】 目的:作为细胞生物学及组织工程学中的核心，滑膜干细胞隶属于成体间充质干细胞范畴。目前滑膜干细胞研究来源多为膝关节或颞下颌关节等关节囊内层的滑膜组织。作为滑膜组织体内另一分布--肌腱周围滑膜，其组织结构、生物化学性质及功能与关节内滑膜均相似，有可能成为滑膜干细胞另一有效组织来源。本实验将分离兔肌腱周围滑膜细胞，探讨经培养纯化后，获得腱周滑膜来源的滑膜干细胞的可行性及方法。进一步实验研究其定向成骨分化的能力，探讨其应用于骨质疏松及肌腱病治疗的可能性。方法:耳缘静脉注射空气处死实验用新西兰大白兔（约3月龄，1.5Kg）。借助显微镜无菌条件下暴露双侧跟腱，获取周围滑膜组织。剪碎后，II型胶原酶初步消化约1小时，织块培养法获得细胞。低密度种植，获得生长良好单克隆集落，通过局部消化、自然传代法获取纯化后细胞。取生长良好P3,P4代细胞，接种于24孔板，通过细胞计数法测定描绘SMSCs生长曲线，并计算细胞群体倍增时间。流式细胞仪测定细胞生长周期，计算增值指数等。使用CD44-FITC，CD45-FITC，CD90-FITC标记抗体，流式细胞仪测定SMSCs表面CD44，CD45，CD90标记性抗原。取第3代待80%-90%融合后细胞，更换成骨诱导液，进行成骨定向诱导分化，每天动态观察细胞生长形态变化，分别于培养10天后进行非特异性碱性磷酸酶（alkaline phosphatase,ALP)染色，3周后行Von Kossa’s矿化结节染色做成骨鉴定。结果:1.成功获取跟腱腱鞘内层薄层清亮滑膜组织。采用II型胶原酶消化结合组织块培养法，可获得贴壁生长良好的细胞，传代后形态均一，成纤维细胞状，生长曲线为S形，平均群体倍增时间为42.5小时，细胞增殖稳定。流式细胞仪标记性抗原检测结果CD44、CD90阳性，CD45阴性，符合一般滑膜干细胞表面标记特点；2.更换培养液向成骨方向诱导后，可见细胞形态随时间渐改变，由原来扁平纤维状，逐渐呈立体改变，细胞代谢旺盛，细胞外基质增多，10天后ALP检测显示阳性，3周后Von koss’a染色可见视野内细胞周围大量黑褐色矿化结节出现，成骨分化阳性。结论:1.肌腱周围分离滑膜组织简便易行，经分离培养、纯化后可获得具有间充质干细胞性质的SMSCs，并可能替代关节内滑膜来源的SMSCs，用于肌腱病等组织修复，且具有更强的优越性。2.肌腱周围SMSCs具有良好的成骨分化能力，可能应用于骨折及骨质疏松的治疗。更多还原

【Abstract】 Objective:Synovial membrane mesenchymal stem cell(SMSCs) is theimportant research topic of tissue engineering,SMSCs is acquired from the synoviallining layer of capsule of temporomandibular or knee jont, Another synovial membranesource,tenosynovium has the similar components, character of biochemistry andfunction，and the chance to become another source of SMSCs.We are trying to study themethod and feasibility of isolating the tenosynovium from rabbit achillestendon,culturing and obteining the purified SMSCs.further study the osteogenicdifferentiation potential in vitro,and discuss the possibility of application totendinopathy and osteoporosis.Methods:A3months old,1.5kilograms weight rabbit was killed by injecting airvia ear vein,with the help of surgery microscope, Synovial tissue was harvestedunder sterile conditions and microscopic magnification of the Achilles tendon sheath.The tissue was cut into small pieces,minced tissues were explant culture after digestingwith type II collagenase for one hour. Monoclone was obtained by selecting as primarySMSCs,under low planting density condition. The method of cytometry was used todraw a cellular growth curvethe at Passage3,4.Cell cycle analysis was performed byflow cytometry,Application of flow cytometry to detect the surface antigenCD44,CD45,CD90.The passage3cells were treated with inductive medium at,Cellmorphous was observed with an inverted microscope everyday.Osteogenicdifferentiation was tested by alkaline phosphatase and Von Kossa staining,separatelyon the tenth day,and3weeks later.Results:1.synovial tissue can be isolated from Achilles tendon sheath,after cultureand purification,the cells expanded steadyly and rapidly.Doubling time was42.5h.Surface antigen CD44and CD90wre positive,CD45is negative.After osteogenesis induction,the results for alkaline phosphatase and Von Kossa stainingwere postive.Conclusion:1.Synovial tissue is easy separated from Achilles tendon sheath,and SMSCs canbe isolated from it. Tenosynovium has the chance to take place of the joint synovium tobecome the new source of SMSCs,and is superior in tendinopathy.2.SMSCs from tenosynovium have the potential to differentiate into osteogenic invitro,and can be used for the therapy of osteoporosis更多还原