人脐带间充质干细胞复合海藻酸钙水凝胶支架材料构建组织工程软骨的实验研究

发布时间：2018-01-03 19:24

  本文关键词：人脐带间充质干细胞复合海藻酸钙水凝胶支架材料构建组织工程软骨的实验研究　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 脐带 间充质干细胞 软骨 海藻酸钙 组织工程

【摘要】：目的：体外分离培养人脐带间充质干细胞,并对其进行生物学鉴定及软骨方向诱导。构建海藻酸钙水凝胶,同时观察其生物相容性。检测人脐带间充质干细胞与海藻酸钙水凝胶构建组织工程软骨修复软骨缺损的效果。 方法：用酶消化法分离培养人脐带间充质干细胞,通过传代培养,扩增。倒置光学显微镜及原子力显微镜观察细胞形态。MTT检测细胞增殖曲线,流式细胞仪检测细胞周期及免疫表型。在低密度、高密度、微团的环境下对hUCMSCs向软骨方向诱导。诱导成软骨过程中应用倒置光学显微镜观察细胞结构的变化,并对不同组别进行甲苯胺蓝杂色、免疫组化检测。制备海藻酸钙水凝胶支架材料。将1UCMSCs与支架材料复合,体外成软骨诱导培养3周,扫描电镜观察细胞黏附情况,MTT法分析细胞增殖情况来评价其组织相容性。并通过原位植入兔股骨髁上软骨缺损,通过组织学等手段检测其软骨修复效果。 结果：采用酶消化法能有效分离纯化人脐带间充质干细胞。细胞增值能力强。流式细胞仪分析第3代细胞均强烈表达CD29、CD44、CD105,不表达CD34、CD45和HLA-DR。经成软骨诱导分化后,低密度诱导组及各对照组甲苯胺蓝杂色、Ⅱ型胶原免疫组化染色弱阳性；高密度诱导组甲苯胺蓝杂色、Ⅱ型胶原免疫组化染色阳性,微团诱导组染色均表现出强阳性。扫描电镜示细胞海藻酸钙水凝胶支架上吸附,生长良好。MTT示hUCMSCs在材料上增殖能力良好。hUCMSCs构建的组织工程软骨能够修复软骨缺损。 结论：本实验建立了一套体外稳定培养扩增人脐带间充质干细胞的方法,所培养的细胞成分单一,扩增迅速,生物学形状稳定；并能使其在体外采用不同环境下诱导向成软骨细胞分化。海藻酸钙水凝胶支架材料是一种具有良好生物相容性的支架材料。hUCMSCs与之构建组织工程软骨可修复软骨缺损。
[Abstract]:Objective: to isolate and culture human umbilical cord mesenchymal stem cells in vitro and to construct calcium alginate hydrogel. At the same time, the biocompatibility of human umbilical cord mesenchymal stem cells and calcium alginate hydrogel was observed. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured by enzyme digestion. The proliferation curves were observed by inverted optical microscope and atomic force microscope. Flow cytometry was used to detect cell cycle and immunophenotype. HUCMSCs was induced to cartilage in the environment of microclusters. The changes of cell structure were observed by inverted optical microscope during the process of cartilage formation, and toluidine blue hybrids were performed on different groups. Immunohistochemical examination. Calcium alginate hydrogel scaffold materials were prepared. 1UCMSCs were combined with scaffold materials and cultured in vitro for 3 weeks. The adhesion of cells was observed by scanning electron microscope (SEM). MTT method was used to evaluate the histocompatibility of the cells, and the cartilage repair effect was detected by in situ implantation of rabbit supracondylar cartilage defect. Results: human umbilical cord mesenchymal stem cells could be effectively isolated and purified by enzyme digestion. CD34, CD45 and HLA-DR were not expressed. After chondrogenic differentiation, toluidine blue heterochromatic staining and type 鈪，

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