微囊化兔雪旺细胞体外培养及相关研究

发布时间：2018-03-06 04:13

本文选题：微囊化　切入点：雪旺细胞　出处：《南昌大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：探讨从成年大白兔周围神经分离、纯化、培养获取大量雪旺细胞的有效手段，观察微囊化兔雪旺细胞体外培养过程中的形态与增殖特征，为神经损伤后修复提供实验基础。方法：利用成年兔发生Wallerian变性的双侧坐骨神经，经改良后反复差速贴附法进行培养，快速分离纯化雪旺细胞，通过相差显微镜下观察雪旺细胞状态计数和S-100细胞免疫组化染色相结合鉴定，，观察其生长并绘制生长曲线，按照细胞倍增时间公式计算其倍增时间。随后，海藻酸钠-氯化钡一步成囊法包裹生长良好的第三代雪旺细胞，继续培养观察，运用MTT法测定微囊包裹的雪旺细胞和单纯雪旺细胞的增殖反应。结果：发现利用发生Wallerian变性后的兔双侧坐骨神经，经改良后差速贴壁培养方法能够明显抑制成纤维细胞的生长，获得大量雪旺细胞，经S-100蛋白免疫组化鉴定，雪旺细胞纯度达95％以上，细胞数量达1×10~6／ml以上；绘制其生长曲线，并计算得出体外培养的第三代雪旺细胞体外倍增时间为3d；MTT实验检测：经微囊包裹的雪旺细胞增殖速度明显弱于未经包裹的单纯雪旺细胞对照组，两者之间差别有统计学意义(P＜0.05)。结论：本培养方法能够获得大量高纯度的雪旺细胞，微囊包裹后的雪旺细胞体外生长良好，增殖速度较单纯雪旺细胞而言有所减慢。
[Abstract]:Objective: to investigate the effective means of isolating, purifying and culturing a large number of Schwann cells from the peripheral nerves of adult rabbits, and to observe the morphological and proliferative characteristics of microencapsulated Schwann cells in vitro. To provide experimental basis for nerve repair after nerve injury. Methods: the sciatic nerves of adult rabbits with Wallerian denaturation were cultured with modified differential attachment method. Schwann cells were isolated and purified quickly. By observing Schwann cell state count and S-100 cell immunohistochemical staining under phase contrast microscope, the growth of Schwann cell was observed and the growth curve was drawn. The doubling time was calculated according to the formula of cell doubling time. The third generation Schwann cells were encapsulated with sodium alginate and barium chloride. The proliferation of Schwann cells wrapped in microcapsules and Schwann cells were determined by MTT method. Results: it was found that the modified differential adherent culture method could inhibit the growth of fibroblasts and obtain a large number of Schwann cells by using the bilateral sciatic nerve denatured by Wallerian, which was identified by S-100 protein immunohistochemistry. The purity of Schwann cells was over 95%, and the number of Schwann cells was more than 1 脳 10 ~ (10) / ml. The results showed that the multiplication time of the third generation Schwann cells in vitro was 3 d. MTT assay showed that the proliferation rate of Schwann cells encapsulated by microencapsulation was significantly lower than that of unencapsulated Schwann cells (P < 0.05). Conclusion: a large number of Schwann cells with high purity can be obtained by this method. The microencapsulated Schwann cells grow well in vitro and the proliferation rate is slower than that of simple Schwann cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329

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