兔肌腱周围滑膜干细胞的分离培养及成骨分化

发布时间：2018-02-07 14:18

本文关键词： 肌腱滑膜干细胞成骨　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective: to be the core of cell biology and tissue engineering. Synovial stem cells belong to the category of adult mesenchymal stem cells. At present, synovial stem cells are mainly derived from synovial tissue in the inner layer of the capsule of the knee joint or temporomandibular joint. Its tissue structure, biochemical properties and functions are similar to those of synovial membrane, which may be another effective tissue source of synovial stem cells. In this study, rabbit synovial cells around tendon were isolated and cultured and purified. To obtain the feasibility and method of synovial stem cells derived from peritendinous synovium, to further study the ability of directional osteogenic differentiation of synovial stem cells, and to explore the possibility of its application in the treatment of osteoporosis and tendon disease. Methods: new Zealand white rabbits (about 3 months old) were exposed to bilateral Achilles tendon under microscopically sterile condition to obtain the surrounding synovial tissue. After shearing, type II collagenase was initially digested for about 1 hour. The cells were obtained by tissue culture. The cells were cultured in low density, and grew well. The purified cells were obtained by local digestion and natural passage. The cells of P3 + P4 passage were obtained and inoculated on 24 well plate. SMSCs growth curve was measured by cell count method, cell population doubling time was calculated, cell growth cycle was measured by flow cytometry, increment index was calculated, and antibody was labeled with CD44-FITCU CD45-FITCtc CD90-FITC. Flow cytometry (FCM) was used to detect the CD44 + CD45 + CD90 labeled antigen on the surface of SMSCs. After the third passage, the cells were fused 80-90%, the osteoblast was replaced, the osteoblast was induced and differentiated, and the growth morphology of the cells was observed dynamically every day. After 10 days of culture, the bone was identified by Von Kossa's mineralized nodule staining after 3 weeks of nonspecific alkaline phosphatase (ALP) staining. Results:. 1. The thin layer of clear synovial tissue in the inner layer of Achilles tendon sheath was successfully obtained. By using type II collagenase digestion combined with tissue mass culture, the cells with good adherent growth were obtained, which were homogeneous in morphology, fibroblast-like and S-shaped in growth curve after passage. The mean population doubling time was 42.5 hours, and the cell proliferation was stable. The results of flow cytometry showed that CD44 + CD90 + CD45 was negative, which was consistent with the characteristics of surface marker of synovial stem cells. 2.After the change of culture medium was induced to osteogenesis, the morphology of the cells changed gradually with time, from the original flat fibrous, gradually changed stereoscopic, the cell metabolism was exuberant. 10 days after the proliferation of extracellular matrix (ECM), ALP showed positive results. 3 weeks later, Von koss'a staining showed that a large number of dark brown mineralized nodules appeared around the cells in the visual field, and osteogenic differentiation was positive. Conclusion:. 1. It is easy to isolate synovial tissue around tendon. SMSCs with mesenchymal stem cells can be obtained after isolation and culture, and it may replace SMSCsderived from synovial membrane in joint, which can be used for repair of tendon disease and has more superiority. 2. Peritendinous SMSCs has good osteogenic differentiation and may be used in the treatment of fracture and osteoporosis.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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