本文關(guān)鍵詞： 肌腱 滑膜 干細(xì)胞 成骨　出處：《大連醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:作為細(xì)胞生物學(xué)及組織工程學(xué)中的核心，滑膜干細(xì)胞隸屬于成體間充質(zhì)干細(xì)胞范疇。目前滑膜干細(xì)胞研究來(lái)源多為膝關(guān)節(jié)或顳下頜關(guān)節(jié)等關(guān)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(gòu)、生物化學(xué)性質(zhì)及功能與關(guān)節(jié)內(nèi)滑膜均相似，有可能成為滑膜干細(xì)胞另一有效組織來(lái)源。本實(shí)驗(yàn)將分離兔肌腱周圍滑膜細(xì)胞，探討經(jīng)培養(yǎng)純化后，獲得腱周滑膜來(lái)源的滑膜干細(xì)胞的可行性及方法。進(jìn)一步實(shí)驗(yàn)研究其定向成骨分化的能力，探討其應(yīng)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(shí)驗(yàn)用新西蘭大白兔（約3月齡，1.5Kg）。借助顯微鏡無(wú)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(shí)，織塊培養(yǎng)法獲得細(xì)胞。低密度種植，獲得生長(zhǎng)良好單克隆集落，通過(guò)局部消化、自然傳代法獲取純化后細(xì)胞。取生長(zhǎng)良好P3,P4代細(xì)胞，接種于24孔板，通過(guò)細(xì)胞計(jì)數(shù)法測(cè)定描繪SMSCs生長(zhǎng)曲線，并計(jì)算細(xì)胞群體倍增時(shí)間。流式細(xì)胞儀測(cè)定細(xì)胞生長(zhǎng)周期，計(jì)算增值指數(shù)等。使用CD44-FITC，CD45-FITC，CD90-FITC標(biāo)記抗體，流式細(xì)胞儀測(cè)定SMSCs表面CD44，CD45，CD90標(biāo)記性抗原。取第3代待80%-90%融合后細(xì)胞，更換成骨誘導(dǎo)液，進(jìn)行成骨定向誘導(dǎo)分化，每天動(dòng)態(tài)觀察細(xì)胞生長(zhǎng)形態(tài)變化，分別于培養(yǎng)10天后進(jìn)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(zhǎng)良好的細(xì)胞，傳代后形態(tài)均一，成纖維細(xì)胞狀，生長(zhǎng)曲線為S形，平均群體倍增時(shí)間為42.5小時(shí)，細(xì)胞增殖穩(wěn)定。流式細(xì)胞儀標(biāo)記性抗原檢測(cè)結(jié)果CD44、CD90陽(yáng)性，CD45陰性，符合一般滑膜干細(xì)胞表面標(biāo)記特點(diǎn)； 2.更換培養(yǎng)液向成骨方向誘導(dǎo)后，可見細(xì)胞形態(tài)隨時(shí)間漸改變，由原來(lái)扁平纖維狀，逐漸呈立體改變，細(xì)胞代謝旺盛，細(xì)胞外基質(zhì)增多，10天后ALP檢測(cè)顯示陽(yáng)性，3周后Von koss'a染色可見視野內(nèi)細(xì)胞周圍大量黑褐色礦化結(jié)節(jié)出現(xiàn)，成骨分化陽(yáng)性。 結(jié)論: 1.肌腱周圍分離滑膜組織簡(jiǎn)便易行，經(jīng)分離培養(yǎng)、純化后可獲得具有間充質(zhì)干細(xì)胞性質(zhì)的SMSCs，并可能替代關(guān)節(jié)內(nèi)滑膜來(lái)源的SMSCs，用于肌腱病等組織修復(fù)，且具有更強(qiáng)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(yīng)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張正治,劉正津;滑膜細(xì)胞的來(lái)源及遺傳表型[J];解剖科學(xué)進(jìn)展;1998年02期

2 曹文;姚光慧;陳玉華;紀(jì)俐娜;黃輝;夏長(zhǎng)所;;富血小板血漿體外培養(yǎng)骨髓間充質(zhì)干細(xì)胞的增殖與膠原產(chǎn)生[J];中國(guó)組織工程研究與臨床康復(fù);2010年45期

3 龔忠誠(chéng);魏麗麗;吳楊;凌彬;劉慧;林兆全;龍星;;滑膜間充質(zhì)干細(xì)胞軟骨分化能力的實(shí)驗(yàn)研究[J];新疆醫(yī)科大學(xué)學(xué)報(bào);2010年01期

4 黃世峰;肖長(zhǎng)虹;顧為望;張嘉寧;陳德超;楊敏;;兔關(guān)節(jié)滑膜細(xì)胞的分離、培養(yǎng)和純化[J];中國(guó)人獸共患病學(xué)報(bào);2006年11期

本文編號(hào)：1494576