骨髓间充质干细胞诱导成类髓核细胞的体外研究.pdfVIP

目 录 中文摘要·······················································3 Abstract ······················································4 英文缩略词表 ·······················································5 前 言······················································6 骨髓间充质干细胞诱导成类髓核细胞体外研究 ··························8 引言······························································8 材料与方法 ························································8 结果······························································19 讨论 ······························································29 结论······························································35 参考文献 ··························································36 致谢 ······························································40 综述 ······························································41 2 万方数据 骨髓间充质干细胞诱导成类髓核细胞的体外研究 中文摘要 【目的】 1.分离、培养扩增和鉴定兔骨髓间充质干细胞 （BMSCs）； 2.通过体外诱导BMSCs 成类髓核细胞，寻找细胞体内移植的合适时间点； 【方法】 应用单纯贴壁培养法分离、培养、扩增兔BMSCs 和髓核细胞，并应用免疫细 胞化学方法检测BMSCs 表面相对特异性分子(CD90、 CD44、 CD45、CD34 )的表 达率以鉴定BMSCs；将接种有BMSCs 的Transwell 小室置于含有髓核细胞的培养 皿中，建立共培养模型；制定诱导BMSCs 生长曲线，用RT-PCR（reverse transcription PCR ）和 Western blot 检测其表达蛋白聚糖和Ⅱ型胶原，结合以上检测寻找最 佳共培养时间，为椎间盘组织工程种子细胞的更深入研究提供实验数据。 【结果】 应用贴壁培养法成功分离、培养、扩增兔BMSCs 和髓核细胞，免疫细胞化学 检测 BMSCs 表面相对特异性分子的阳性表达率显示：CD90 为 91.2%，CD44 为 93.5%，CD45 为4.4%，CD34 为5.5%。在共培养模型中，经细胞生长曲线检测、 RT-PCR 和Western blot 检测结果提示BMSCs 与髓核细胞共培养可诱导BMSCs 表 达蛋白聚糖和Ⅱ型胶原，在三代以内经诱导细胞生长曲线无明显改变，结合以上 检测认为细胞共培养15 天左右达到最佳状态，适合进行体内移植。 【结论】 1.采用单纯贴壁法可以成功分离出纯度较高的BMSCs； 2.在 Transwell 小室和培养皿构成共培养系统中，BMSCs 和髓核细胞共培养