人骨髓间充质干细胞向软骨诱导分化的基础研究

发布时间：2018-01-20 00:57

本文关键词： 骨髓间充质干细胞软骨细胞组织工程诱导　出处：《大连医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 软骨缺损的修复一直是矫形外科的难题。早期开展的微骨折技术、骨膜软骨膜移植、软骨细胞移植等方法都存在诸多问题。上世纪80年代开始,组织工程的发展和应用为软骨缺损和修复带来了新的希望。支架材料、生长因子、种子细胞是组织工程的三要素,随着制造技术的进步,支架材料的研究已取得突飞猛进的进展,已有人工骨应用于临床。而目前种子细胞是制约组织工程发展的瓶颈之一。骨髓间充质干细胞因其成体干细胞特征和易获取性,近年来倍受关注。但是,骨髓间充质干细胞的分离、纯化、鉴定以及多向分化条件和机理等目前都不十分清楚。不论是基础研究还是临床应用,人们都希望获得稳定的、纯化的、大量具有骨髓基质干细胞生物学表型和功能的种子细胞,以便为基础研究提供标准的细胞,为临床应用提供细胞库,真正实现体外构建组织器官的目的。为了达到以上目的,最佳途径就是建立骨髓间充质干细胞的细胞系。目的:探索骨髓间充质干细胞培养条件。观察间充质干细胞生物学行为和向软骨诱导分化的能力。以供组织工程基础研究及骨科临床应用。方法:抽取5例健康志愿者骨髓,每例约4ml,利用含Hyclone胎牛血清的DMEM低糖培养液培养,进行细胞培养并探索条件。取正常骨髓做骨髓间充质干细胞的原代培养和传代培养,对该细胞进行流式细胞分析鉴定,根据临床研究与应用需求诱导分化,配制条件培养液。采用软骨诱导条件培养基:完全培养基加地塞米松、TGF-β_1、维生素C,终浓度为, 5ug/L, 10mmol/L; BMSCs细胞进行软骨细胞诱导6, 12, 18, 24d,相差显微镜观察,HE染色观察细胞形态;免疫细胞化学染色观察软骨细胞诱导II型胶原的表达,原位杂交观察软骨细胞诱导II型胶原mRNA表达结果:10%的血清最有利于细胞生长,Percoll细胞分离液分离细胞可以得到更纯化的细胞,细胞首次换液时间为6天。对传代培养细胞进行流式细胞分析鉴定,细胞表达CD29, CD71, CD106,不表达CD34, CD45,表明培养细胞是间充质干细胞。hBMSC细胞可以条件诱导为软骨,具有成体干细胞的特征。结合细胞表面分子检测结果,可以认为BMSCs细胞为骨髓内的间充质质干细胞。结论:人骨髓间充质干细胞优化培养条件:10%胎牛血清、DMEM低糖培养基,首次换液时间为6天,密度梯度离心法分离细胞有利于获得较纯化的细胞,全骨髓贴壁分离法更有利于细胞生长;可培养出纯化的人骨髓间充质干细胞,细胞不表达造血系细胞表面特异分子,并具备多向分化的成体干细胞特征;人骨髓间充质干细胞可以作为软骨组织工程的种子细胞。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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