成年大鼠许旺细胞原代培养方法的比较

Comparative study on different culture methods to get stable primary adult Schwann cells

目的寻找一个稳定、快速、获取大量高纯度的成年许旺细胞的实验方法，为神经组织工程提供实验依据。方法制作成年SD大鼠两侧坐骨神经Wallefian变性模型。1周后取出变性神经．分别用胶原酶／中性蛋白酶双酶消化法、半植块单酶消化法、胰蛋白酶／胶原酶双酶消化法培养原代许旺细胞。S-100免疫细胞化学比较不同种方法的优劣。结果胶原酶／中性蛋白酶双酶消化法得到的细胞3～5d即可达融合．纯度达70％～80％；半植块单酶消化法培养3～5d后细胞从植块周围爬出的数量不均匀，10～12d后可达融合，但纯度低于40％；胰蛋白酶／胶原酶双酶消化法因消化后组织块易缠结．令细胞丢失过多，难以存活。结论本实验采用的三种方法中，胶原酶／中性蛋白酶双酶消化法为稳定快速获得成年大鼠许旺原代细胞的最佳方法。

Objective To seek a stable, rapid and effective method in order to obtain large number of pure adult Schwann cells （Scs） in vitro. Methods Bilateral sciatic nerves of each adult SD rat were crushed several times to establish the degeneration animal model. One week later,the degenerated nerves were detected and prepared to collagenase/dispase double enzyme digestion method,trypsin/collagenase double enzyme digestion method,single enzyme digestion for half-piece method,respectively. The S-100 immunocytochemlstry was used to detect the purity of Sos. Results The confluence of cells was happened within 3 - 5 days in eollagenase/dispase double enzyme digestion method. At time of confluenee,about 70% - 80% cells showed S-100 positive in this method. The quantities of cells from the degenerative nerve pieces cultured by single enzyme digestion were not uniform within 3 - 5 days,cells were confluent within 10 - 12 d and less than 40~ cells showed S-100 positive at time of confluence. Most of tissue pieces were tangled and cells were lost in trypsin/collagenase double enzyme digestion method. Only 3 - 5 cells in every 100 fields could survive in vitro,confluence is difficult. Conclusion The collagenase/dispase double enzyme digestion method was the most stable and effective method to obtain large number of pure adult Schwanu cells in vitro rapidly among the present three methods.