Ⅰ型胶原蛋白对兔骨髓间充质干细胞成骨分化作用的实验研究

发布时间：2018-01-27 01:11

  本文关键词： 骨髓间充质干细胞 细胞培养 Ⅰ型胶原蛋白 成骨分化 兔　出处：《青岛大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的通过实验进一步探讨体外条件下分离、纯化兔骨髓间充质干细胞的有效方法,对所获细胞进行培养,观察细胞的生物学特性。以及观察Ⅰ型胶原蛋白对兔骨髓间充质干细胞成骨分化的作用。 方法实验于青岛大学医学院附属医院骨科研究所进行。首先采用密度梯度离心法分离兔骨髓间充质干细胞,用贴壁培养法对所获细胞进行培养、传代、纯化和扩增,观察细胞的生物学特性,倒置相差显微镜观察细胞的形态学特征,透射电镜观察细胞的超微结构,流式细胞术鉴定细胞表面标志物;将分离纯化的兔骨髓间充质干细胞培养于Ⅰ型胶原蛋白上面,对细胞的碱性磷酸酶表达、细胞矿化形成钙化结节的能力进行测定。 结果分离的细胞1天后完全贴壁,贴壁的细胞平均10d后形成细胞克隆,所获细胞形态均一,呈成纤维细胞样生长,细胞稳定表达骨髓间充质干细胞表面标志物。在Ⅰ型胶原蛋白上面培养后,细胞碱性磷酸酶显著表达,Von.Kossa染色可见矿化结节形成。 结论密度梯度离心法分离,贴壁培养法培养所得细胞,可以有效分离、纯化兔骨髓间充质干细胞。用此种方法获得的细胞具有骨髓间充质干细胞的特性。Ⅰ型胶原蛋白能够促进兔骨髓间充质干细胞的成骨分化。
[Abstract]:Objective to investigate the effective method of isolation and purification of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. To observe the biological characteristics of the cells and the effect of type I collagen on the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. Methods the experiment was carried out in the Institute of Orthopaedics, affiliated Hospital of Qingdao University Medical College. Firstly, bone marrow mesenchymal stem cells were isolated by density gradient centrifugation. The biological characteristics of cells were observed by purification and amplification, the morphological characteristics of cells were observed by inverted phase contrast microscope, the ultrastructure of cells was observed by transmission electron microscopy, and cell surface markers were identified by flow cytometry. The purified rabbit bone marrow mesenchymal stem cells were cultured on type I collagen. The expression of alkaline phosphatase and the ability of mineralization to form calcified nodules were measured. Results the isolated cells were completely adhered to the wall one day later, and the adherent cells formed cell clones after an average of 10 days. The obtained cells were homogeneous in morphology and grew like fibroblasts. The surface markers of bone marrow mesenchymal stem cells were stably expressed in the cells. After cultured on type 鈪，

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