双重消减纯化体外培养许旺细胞

背景: 许旺细胞培养技术和纯化方法虽不断改良, 得到的培养物纯度仍然有限, 双重消减法是否可以培养高纯度许旺细胞。目的: 采用双重消减法对许旺细胞进行培养, 观察其生长情况并测定培养纯度。设计:对照动物实验。单位:首都医科大学附属北京同仁医院耳鼻咽喉头颈外科,北京市耳鼻咽喉科研究所。材料:实验于2006-03/07在北京市耳鼻咽喉科研究所免疫室和病理室完成。选用20只3天龄Wistar大鼠,雌雄不限,由中国医学科学院实验动物中心提供。方法:分离大鼠坐骨神经,以0.2%胶原酶消化后培养。①分组干预:细胞分为实验组及对照1,2,3组,每组5只。实验组应用双30min差速贴壁法和Ara-c纯化,对照组1,2分别用双30min差速贴壁法和Ara-c纯化处理,对照组3不进行纯化。②实验评估:倒置显微镜下观察许旺细胞生长情况;采用MTT法测量实验组和对照组3生长曲线,观察细胞增殖能力;采用免疫细胞化学染色对许旺细胞进行鉴定;计算许旺细胞培养纯度(每个视野S100阳性细胞占所有细胞的比例)。主要观察指标:①各组许旺细胞生长情况。②免疫细胞化学染色结果。③细胞生长曲线。④许旺细胞培养纯度。结果:①许旺细胞生长情况:培养5d时实验组许旺细胞形态及增殖能力良好,偶见成纤维细胞。对照组1、2在培养第5天时可见较多成纤维细胞,许旺细胞形态好,但较少呈漩涡状排列的生长区域。对照组3从培养第2天开始成纤维细胞迅速增殖,所占比例逐渐增大,许旺细胞生长受到影响,突起较短,少见漩涡状生长区域。②许旺细胞生长曲线:实验组许旺细胞于第3天进入对数增殖期,至第6天达平台期。对照组3较实验组提前1d进入对数增殖期。③免疫细胞化学鉴定结果:实验组经抗S100抗体染色,阳性的许旺细胞胞体及突起呈棕褐色,梭形或三角形,双极突起,漩涡状排列。成纤维细胞形态不规则,胞浆宽大,着蓝色。④许旺细胞计数:实验组S100阳性细胞所占比例高于对照组3(P<0.01)。结论:利用双重消减法得到较高纯度的许旺细胞培养物。

In vitro purification of Schwann cells with double negative selection

BACKGROUND: Although cultured technique and purified method of Schwann cells (SCs) are changed successively,purity is still limited. Whether double negative selections can culture high-purified SCs still needs a further study.OBJECTIVE: To investigate the effects of double negative selections on growth and purity of SCs.DESIGN: Controlled animal study.SETTING: Department of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University,and the Beijing Institution of Otolaryngology.MATERIALS: The experiment was carried out in the Department of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University and the Beijing Institution of Otolaryngology between May 2006 and July 2006. Twenty new-born Wistar rats of 3 days (without gender limitation) were provided by the Experimental Animal Center of the Chinese Medical Academy.METHODS: The sciatic nerves of rats were digested by 0.2% collagenase. ① Grouping: All the rats were divided into 4 groups with 5 each. The experimental group was depurated by differential adhesion and Ara-c for 30 minutes. SCs in the control group 1 and 2 were managed by differential adhesion and Ara-c for 30 minutes, respectively. Cells in the control group 3 were not purified. ② Evaluation: The cell growth was observed under invert microscope; growth curves were measured with MTT method in the experimental group and control group 3 to observe cell proliferation. In addition, the SCs were identified by immunocytochemistry; the culture purity was calculated (ratio of S100 positive cells in each sight).MAIN OUTCOME MEASURES: ① Growth of SCs; ② immunocytochemistry stain of SCs; ③ growth curve of SCs; ④ purity of SCs.RESULTS: ① Growth of SCs: At 5 days after incubation, the SCs proliferated well. SCs were spindle, bipolar and sometimes tripolar, with obvious nuclei and a little cytoplasm. There was more fibroblast cell in the group 3 than that in experimental group. ② Growth curve: The SCs in experimental group entered into the logarithm multiplication period at the third day. ③ Immunocytochemistry: The positive-stained SCs were brown in its body and process, and arranged like swirl. The FB with spread and anomaly shape was slight blue. ④ The ratio of anti-S100 positive cells was higher in experimental group than that in group 3 (P ＜ 0.01).CONCLUSION: With the double negative selection, high-purified SCs are obtained effectively.