本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 軟骨細(xì)胞 組織工程 誘導(dǎo)　出處：《大連醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 軟骨缺損的修復(fù)一直是矯形外科的難題。早期開展的微骨折技術(shù)、骨膜軟骨膜移植、軟骨細(xì)胞移植等方法都存在諸多問題。上世紀(jì)80年代開始,組織工程的發(fā)展和應(yīng)用為軟骨缺損和修復(fù)帶來了新的希望。支架材料、生長因子、種子細(xì)胞是組織工程的三要素,隨著制造技術(shù)的進(jìn)步,支架材料的研究已取得突飛猛進(jìn)的進(jìn)展,已有人工骨應(yīng)用于臨床。而目前種子細(xì)胞是制約組織工程發(fā)展的瓶頸之一。骨髓間充質(zhì)干細(xì)胞因其成體干細(xì)胞特征和易獲取性,近年來倍受關(guān)注。但是,骨髓間充質(zhì)干細(xì)胞的分離、純化、鑒定以及多向分化條件和機(jī)理等目前都不十分清楚。不論是基礎(chǔ)研究還是臨床應(yīng)用,人們都希望獲得穩(wěn)定的、純化的、大量具有骨髓基質(zhì)干細(xì)胞生物學(xué)表型和功能的種子細(xì)胞,以便為基礎(chǔ)研究提供標(biāo)準(zhǔn)的細(xì)胞,為臨床應(yīng)用提供細(xì)胞庫,真正實現(xiàn)體外構(gòu)建組織器官的目的。為了達(dá)到以上目的,最佳途徑就是建立骨髓間充質(zhì)干細(xì)胞的細(xì)胞系。 目的:探索骨髓間充質(zhì)干細(xì)胞培養(yǎng)條件。觀察間充質(zhì)干細(xì)胞生物學(xué)行為和向軟骨誘導(dǎo)分化的能力。以供組織工程基礎(chǔ)研究及骨科臨床應(yīng)用。 方法:抽取5例健康志愿者骨髓,每例約4ml,利用含Hyclone胎牛血清的DMEM低糖培養(yǎng)液培養(yǎng),進(jìn)行細(xì)胞培養(yǎng)并探索條件。取正常骨髓做骨髓間充質(zhì)干細(xì)胞的原代培養(yǎng)和傳代培養(yǎng),對該細(xì)胞進(jìn)行流式細(xì)胞分析鑒定,根據(jù)臨床研究與應(yīng)用需求誘導(dǎo)分化,配制條件培養(yǎng)液。采用軟骨誘導(dǎo)條件培養(yǎng)基:完全培養(yǎng)基加地塞米松、TGF-β_1、維生素C,終濃度為, 5ug/L, 10mmol/L; BMSCs細(xì)胞進(jìn)行軟骨細(xì)胞誘導(dǎo)6, 12, 18, 24d,相差顯微鏡觀察,HE染色觀察細(xì)胞形態(tài);免疫細(xì)胞化學(xué)染色觀察軟骨細(xì)胞誘導(dǎo)II型膠原的表達(dá),原位雜交觀察軟骨細(xì)胞誘導(dǎo)II型膠原mRNA表達(dá) 結(jié)果:10%的血清最有利于細(xì)胞生長,Percoll細(xì)胞分離液分離細(xì)胞可以得到更純化的細(xì)胞,細(xì)胞首次換液時間為6天。對傳代培養(yǎng)細(xì)胞進(jìn)行流式細(xì)胞分析鑒定,細(xì)胞表達(dá)CD29, CD71, CD106,不表達(dá)CD34, CD45,表明培養(yǎng)細(xì)胞是間充質(zhì)干細(xì)胞。hBMSC細(xì)胞可以條件誘導(dǎo)為軟骨,具有成體干細(xì)胞的特征。結(jié)合細(xì)胞表面分子檢測結(jié)果,可以認(rèn)為BMSCs細(xì)胞為骨髓內(nèi)的間充質(zhì)質(zhì)干細(xì)胞。 結(jié)論:人骨髓間充質(zhì)干細(xì)胞優(yōu)化培養(yǎng)條件:10%胎牛血清、DMEM低糖培養(yǎng)基,首次換液時間為6天,密度梯度離心法分離細(xì)胞有利于獲得較純化的細(xì)胞,全骨髓貼壁分離法更有利于細(xì)胞生長;可培養(yǎng)出純化的人骨髓間充質(zhì)干細(xì)胞,細(xì)胞不表達(dá)造血系細(xì)胞表面特異分子,并具備多向分化的成體干細(xì)胞特征;人骨髓間充質(zhì)干細(xì)胞可以作為軟骨組織工程的種子細(xì)胞。
[Abstract]:Repair of cartilage defects has been a difficult problem in orthopaedic surgery. There are many problems in the early techniques of micro-fracture, periosteal chondrocyte transplantation, chondrocyte transplantation and so on. -20s. The development and application of tissue engineering bring new hope for cartilage defect and repair. Scaffold materials, growth factors and seed cells are the three elements of tissue engineering, with the progress of manufacturing technology. The research of scaffold materials has made rapid progress. Bone marrow mesenchymal stem cells (BMSCs) have attracted much attention in recent years because of their characteristics and accessibility of adult stem cells. At present, the isolation, purification, identification and multi-differentiation conditions and mechanism of bone marrow mesenchymal stem cells are not very clear. People hope to obtain stable and purified bone marrow mesenchymal stem cells, both in basic research and clinical application. A large number of bone marrow stromal cells have biological phenotype and function of seed cells, in order to provide standard cells for basic research, and provide a cell bank for clinical applications. In order to achieve the above purpose, the best way is to establish the cell line of bone marrow mesenchymal stem cells. Objective: to explore the culture conditions of bone marrow mesenchymal stem cells and observe the biological behavior of mesenchymal stem cells and their ability to induce differentiation into cartilage for basic research of tissue engineering and clinical application in orthopedic department. Methods: bone marrow was extracted from 5 healthy volunteers (about 4 ml each) and cultured in low glucose DMEM medium containing Hyclone fetal bovine serum. The normal bone marrow was taken as primary culture and passage culture of bone marrow mesenchymal stem cells. The cells were identified by flow cytometry and induced differentiation according to clinical research and application needs. Preparation of conditioned medium: complete medium plus dexamethasone TGF- 尾 1, vitamin C, final concentration: 5ugr / L, 10 mmol / L; BMSCs cells were induced by chondrocytes for 6, 12, 18 and 24 days. The morphology of chondrocytes was observed by HE staining under phase contrast microscope. The expression of type II collagen induced by chondrocytes was observed by immunocytochemical staining, and the expression of type II collagen mRNA by chondrocytes was observed by in situ hybridization. Results it was found that 10% of the serum was most favorable to the cell growth and the isolation of Percoll cells, and the more purified cells could be obtained. The first time of cell exchange was 6 days. Flow cytometry analysis showed that the cells expressed CD29, CD71, CD106, and did not express CD34, CD45. The results showed that the cultured cells were mesenchymal stem cells. HBMSC cells could be induced into chondrocytes with the characteristics of adult stem cells and combined with the results of cell surface molecular detection. BMSCs cells can be considered as mesenchymal stem cells in bone marrow. Conclusion: the optimal culture condition of human bone marrow mesenchymal stem cells is 10% fetal bovine serum DMEM low sugar medium, the first time is 6 days. The method of density gradient centrifugation was helpful to obtain the purified cells, while the whole bone marrow adherent method was more favorable for cell growth. The purified human bone marrow mesenchymal stem cells could be cultured. The cells did not express the surface specific molecules of hematopoietic cells and had the characteristics of multi-differentiated adult stem cells. Human bone marrow mesenchymal stem cells can be used as seed cells for cartilage tissue engineering.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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