改良Nash差速贴壁联合阿糖胞苷法体外分离培养大鼠嗅球及嗅黏膜源性嗅鞘细胞*☆

背景：目前常用的嗅鞘细胞培养方法有差速贴壁法、化学抑制法、抗体亲和吸附法、补体法等，各自均存在优缺点，单一使用某种方法时细胞纯化率较低。 目的：拟采用改良Nash差速贴壁+阿糖胞苷法体外分离纯化大鼠嗅球及嗅黏膜来源的嗅鞘细胞。 设计、时间及地点：细胞学体外对照观察，于2007-11/2008-05在武汉大学人民医院骨科实验室和消化内科实验室完成。 材料：10周龄Sprague-Dauley大鼠10只，由武汉大学人民医院实验动物中心提供，阿糖胞苷由武汉大学人民医院制备。 方法：完整取大鼠双侧嗅球及剪取鼻中隔后1/3嗅黏膜，剪碎后胰酶消化，制成单细胞悬液，按1×109 L-1密度接种于未包被的25 cm2玻璃培养瓶中，18~20 h后将细胞悬液转移至另一未包被的25 cm 2玻璃培养瓶中（第1次差速贴壁），24 h后再将细胞悬液转移至经多聚右旋赖氨酸包被的25 cm2塑料培养瓶或经多聚右旋赖氨酸包被的6孔培养板中（第2次差速贴壁），接种培养48 h后半量换液以去除杂细胞。在差速贴壁培养后二三天，加入3.0~5.0 pmol/L阿糖胞苷去除残余成纤维细胞。 主要观察指标：嗅鞘细胞的形态观察、分裂增殖情况、免疫荧光染色鉴定结果及纯度测定。 结果：体外培养24 h嗅球源性嗅鞘细胞即可贴壁，而嗅黏膜源性嗅鞘细胞多在培养四五天后贴壁，两种来源的嗅鞘细胞形态相似，以双极或梭形细胞为主，少量为3极及多突起形多级细胞，同时夹杂扁平、煎鸡蛋形细胞。纯化培养10 d的嗅鞘细胞，胶质纤维酸性蛋白、神经生长因子受体p75免疫荧光染色及神经生长因子受体p75+hoechs免疫荧光双染后大部分双极或多极细胞膜、胞体、突起呈阳性，细胞纯度可达90%以上。 结论：改良Nash差速贴壁+阿糖胞苷法可在体外成功分离培养出高纯度的嗅球及嗅黏膜源性嗅鞘细胞，嗅球源性嗅鞘细胞贴壁时间及分裂增殖程度优于嗅黏膜源性嗅鞘细胞。

In vitro isolation and culture of olfactory ensheathing cells from rat olfactory mucosa and olfactory bulbs using modified Nash differential adhesion and arabinosylcytosin method

BACKGROUND: At present, commonly used culture method of olfactory ensheathing cells contain the differential adhesion method, chemical inhibition method, antibody affinity adsorption and anti-complement method. Each method has its advantage or disadvantage. Using a method alone can obtain a low purified rate. OBJECTIVE: To in vitro harvest and purify rat olfactory ensheathing cells derived from olfactory bulb and olfactory mucosa using modified Nash differential adhesion and arabinosylcytosin method. DESIGN, TIME AND SETTING: The cytology in vitro controlled study was performed at the Laboratories of Department of Orthopaedics and , Renmin Hospital of Wuhan University from November 2007 to May 2008. MATERIALS: Ten Sprague-Dawley rats aged 10 weeks old were obtained from Animal Experimental Center, Renmin Hospital, Wuhan University. Arabinosylcytosin was supplied by Renmin Hospital of Wuhan University. METHODS: Rat bilateral olfactory bulbs and olfactory mucosa at 1/3 nasal septum were obtained, sliced, digested in trypsin, and made into monoplast suspension. At 1×109/L, cells were incubated in uncoated 25 cm2 culture flask. At 18-20 hours, cell suspension was moved into another uncoated 25 cm2 culture flask (the first differential adhesion). At 24 hours, cell suspension was moved into a poly-D-lysine-coated 25 cm2 culture flask or poly-D-lysine-coated 6-well culture plate (the second differential adhesion). At 48 hours, parenchyma cells were removed after a half of medium was changed. At 2-3 days after differential adhesion culture, 3.0-5.0 pmol/L arabinosylcytosin was added to remove remaining fibroblasts. MAIN OUTCOME MEASURES: Morphology, proliferation, and purification of olfactory ensheathing cells; Identification of olfactory ensheathing cells using immunofluorescence. RESULTS: Olfactory bulb-derived olfactory ensheathing cells adhered to the wall at 24 hours. Olfactory mucosa-derived olfactory ensheathing cells adhered to the wall at 4-5 days. The morphology of the two kinds of cells was similar. Most of them were bipolar and spindle-shaped, and few were tripolar and flat. At 10 days, glial fibrillary acidic protein and nerve growth factor receptor 75 immunofluorescence and nerve growth factor p75+hoechs immunofluorescence showed many positive dipolar or mutipolar cell membrane, cell body and processes, resulting in an over 90% purified rate. CONCLUSION: High-purification olfactory bulb and olfactory mucosa-derived olfactory ensheathing cells can be collected by modified Nash differential adhesion and arabinosylcytosin method. The adherence time and proliferation degree of olfactory bulb-derived olfactory ensheathing cells are better than olfactory mucosa-derived olfactory ensheathing cells.