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  本文關鍵詞： 肌腱 滑膜 干細胞 成骨　出處：《大連醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的:作為細胞生物學及組織工程學中的核心，滑膜干細胞隸屬于成體間充質(zhì)干細胞范疇。目前滑膜干細胞研究來源多為膝關節(jié)或顳下頜關節(jié)等關節(jié)囊內(nèi)層的滑膜組織。作為滑膜組織體內(nèi)另一分布--肌腱周圍滑膜，其組織結(jié)構、生物化學性質(zhì)及功能與關節(jié)內(nèi)滑膜均相似，有可能成為滑膜干細胞另一有效組織來源。本實驗將分離兔肌腱周圍滑膜細胞，探討經(jīng)培養(yǎng)純化后，獲得腱周滑膜來源的滑膜干細胞的可行性及方法。進一步實驗研究其定向成骨分化的能力，探討其應用于骨質(zhì)疏松及肌腱病治療的可能性。 方法:耳緣靜脈注射空氣處死實驗用新西蘭大白兔（約3月齡，1.5Kg）。借助顯微鏡無菌條件下暴露雙側(cè)跟腱，獲取周圍滑膜組織。剪碎后，，II型膠原酶初步消化約1小時，織塊培養(yǎng)法獲得細胞。低密度種植，獲得生長良好單克隆集落，通過局部消化、自然傳代法獲取純化后細胞。取生長良好P3,P4代細胞，接種于24孔板，通過細胞計數(shù)法測定描繪SMSCs生長曲線，并計算細胞群體倍增時間。流式細胞儀測定細胞生長周期，計算增值指數(shù)等。使用CD44-FITC，CD45-FITC，CD90-FITC標記抗體，流式細胞儀測定SMSCs表面CD44，CD45，CD90標記性抗原。取第3代待80%-90%融合后細胞，更換成骨誘導液，進行成骨定向誘導分化，每天動態(tài)觀察細胞生長形態(tài)變化，分別于培養(yǎng)10天后進行非特異性堿性磷酸酶（alkaline phosphatase,ALP)染色，3周后行Von Kossa's礦化結(jié)節(jié)染色做成骨鑒定。 結(jié)果: 1.成功獲取跟腱腱鞘內(nèi)層薄層清亮滑膜組織。采用II型膠原酶消化結(jié)合組織塊培養(yǎng)法，可獲得貼壁生長良好的細胞，傳代后形態(tài)均一，成纖維細胞狀，生長曲線為S形，平均群體倍增時間為42.5小時，細胞增殖穩(wěn)定。流式細胞儀標記性抗原檢測結(jié)果CD44、CD90陽性，CD45陰性，符合一般滑膜干細胞表面標記特點； 2.更換培養(yǎng)液向成骨方向誘導后，可見細胞形態(tài)隨時間漸改變，由原來扁平纖維狀，逐漸呈立體改變，細胞代謝旺盛，細胞外基質(zhì)增多，10天后ALP檢測顯示陽性，3周后Von koss'a染色可見視野內(nèi)細胞周圍大量黑褐色礦化結(jié)節(jié)出現(xiàn)，成骨分化陽性。 結(jié)論: 1.肌腱周圍分離滑膜組織簡便易行，經(jīng)分離培養(yǎng)、純化后可獲得具有間充質(zhì)干細胞性質(zhì)的SMSCs，并可能替代關節(jié)內(nèi)滑膜來源的SMSCs，用于肌腱病等組織修復，且具有更強的優(yōu)越性。 2.肌腱周圍SMSCs具有良好的成骨分化能力，可能應用于骨折及骨質(zhì)疏松的治療。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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