大鼠嗅球嗅鞘细胞的体外分离培养及纯化  

In vitro isolation,culture,and purification of olfactory ensheathing cells from olfactory bulb of rats

作　　者：郭冕[1] 郑永日[1] 李青松[1] 王建交[1] 孙家行[1] 葛云龙[1] 赵岩[1] 

机构地区：[1]哈尔滨医科大学附属第二医院神经外科,黑龙江省哈尔滨市150086

出　　处：《中国组织工程研究与临床康复》2009年第40期7838-7842,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:差速贴壁+Thy1.1抗体及补体纯化法的应用较多,细胞换液过程中使用含血清的培养基主要针对成纤维细胞的去除,而对神经元的去除作用不明显。目的:根据神经元体外培养需要血清的特性,拟利用无血清培养基在体外建立一种分离培养嗅鞘细胞的简单方法。设计、时间及地点:细胞学体外观察,于2008-06/2009-01在哈尔滨医科大学动物实验中心完成。材料:成年SD大鼠10只,由哈尔滨医科大学实验动物中心提供。方法:在差速贴壁+Thy1.1抗体及补体纯化法的基础上,剪开大鼠颅骨,显露位于颅腔前方的嗅球,取出2只嗅球,在显微镜下去除嗅球表面的软脑膜和毛细血管及外周组织,保留富含嗅鞘细胞的嗅神经层和嗅球颗粒层,剪成1mm3小块分离获取单细胞悬浮液,调整细胞密度至1×107L-1,接种在用poly-l-lysine包被的培养瓶或培养板中,于37℃、体积分数为5%的CO2培养箱中培养,第3天用无血清DMEM/F12培养基换液培养。主要观察指标:嗅鞘细胞的形态学观察、生长曲线分析、免疫荧光染色结果。结果:培养前3d细胞数目无明显增加,甚至减少;然后细胞数目逐渐增多,13d细胞基本长满;传10代后细胞形态发生明显变化,胞浆内出现许多空泡,细胞增殖速度减慢或停止。纯化培养10d后,培养板内双极和三极细胞均呈GFAP,NGFRp75阳性。结论:在差速贴壁+Thy1.1抗体及补体纯化法的基础上,利用无血清DMEM/F12培养基进行换液培养可使嗅鞘细胞迅速增殖,是一种效率较高的体外培养方法。BACKGROUND：Differential adherent ＋ Thy1.1 antibody and complement-purification method have been frequently used in clinical application. Serum culture medium was used to remove fibroblasts but not neurons. OBJECTIVE：To establish a simple method for in vitro isolating and culturing olfactory ensheathing cells using serum-free culture medium. DESIGN,TIME AND SETTING：An in vitro cytological observation was performed at Animal Experimental Center of Harbin Medical University from June 2008 to January 2009. MATERIALS：A total of 10 adult SD rats were provided by Experimental Animal Center of Harbin Medical University. METHODS：According to adherent ＋ Thy1.1 antibody and complement-purification method,cranium was opened to expose olfactory bulb. Thereafter,two olfactory bulbs were obtained to remove cerebral pia mater,blood capillary,and peripheral tissues;additionally,olfactory nerve layer and olfactory bulb granular layer were sheared into 1-mm3 pieces for extract single-cell suspension. The cells were adjusted at the density of 1×107 /L and incubated with poly-l-lysine-coated culture bottle or culture plate in 5% CO2 incubator at 37 ℃. On the third day,cells were cultured with serum-free DMEM/F12 culture media. MAIN OUTCOME MEASURES：Morphological changes,growth curve,and immunofluorescence staining results of olfactory ensheathing cells. RESULTS：At day 3 prior to culture,cells were not increased obviously or even decreased. The number of cells was gradually increased and peaked at day 13 after culture. At the 10th passage,morphology was changed remarkably,showing a great majority of cavities in the cytoplasm and slow cell proliferation. Or even,the cell proliferation stopped. At day 10 after purification,GFAP-and NGFR p75-positive expressions were observed in the bipolar and the tri-polar cells. CONCLUSION：Based on differential adherent ＋ Thy1.1 antibody and complement-purification method,serum-free DMEM/F12 culture media rapidly promoted proliferation of olfactory ensheathing cells,thus this

关 键 词：嗅鞘细胞 分离 培养 纯化 

分 类 号：R394.2[医药卫生—医学遗传学]