以差速贴壁与化学药物并胰酶限时消化法纯化新生大鼠嗅鞘细胞:优于单一差速贴壁和化学药物法吗?

Differential attachment,chemicals and trypsinization to purify olfactory ensheathing cells:Comparison with differential attachment or chemicals alone

背景:目前国内外对嗅鞘细胞培养条件的报道各不相同,个别方法重复性较差,不利于实际应用。目的:观察差速贴壁+化学药物+胰酶限时消化法纯化大鼠嗅鞘细胞的效果,并与化学药物法、差速贴壁法培养结果进行比较。设计、时间及地点:细胞学体外对照观察,于2007-06/2008-06在福建医科大学人体解剖与组织胚胎学系实验室完成。材料:新生2d的SD大鼠8只,由福建医科大学试验动物中心提供。方法:无菌条件下完整取出大鼠双侧嗅球,剥除嗅球表面的软脑膜及毛细血管网,剪成0.5mm3的小块进行胰蛋白酶消化,过筛后按4.0×108L-1密度种植于包被多聚左旋赖氨酸的培养瓶中进行原代培养。设立4组:①未经纯化的常规对照组。②化学药物组加入5μmol/L阿糖胞苷抑制成纤维细胞分裂。③差速贴壁组采用Nash差速贴壁法纯化嗅鞘细胞。④差速贴壁+化学药物+胰酶限时消化组首先采用Nash差速贴壁法去除大部分成纤维细胞,而后对于少量残留的成纤维细胞加入阿糖胞苷处理,培养6d贴壁细胞大部分融合后,加入1.25g/L胰蛋白酶消化1min,显微镜下见突起回缩、细胞变圆、少量细胞浮起时终止消化。主要观察指标:嗅鞘细胞的形态,NGFRp75免疫细胞荧光染色结果。结果:体外培养的新生大鼠嗅球嗅鞘细胞主要为双极或三极细胞,其突起细长;未经纯化的常规对照组培养7d时视野内成纤维细胞已达60%以上,至14d成纤维细胞完全长满;经过纯化处理的另外3组始终以嗅鞘细胞居多,嗅鞘细胞的形态与原代基本相同。存活的双极和3极嗅鞘细胞呈NGFRp75阳性反应,但化学药物组、差速贴壁组嗅鞘细胞纯度偏低,仅为75%;差速贴壁+化学药物+胰酶限时消化组嗅鞘细胞纯化效率明显增高,达85%以上。结论:实验结果证实了差速贴壁+化学药物+胰酶限时消化法的三合一操作技术,是一种高效率的纯化培养嗅球嗅鞘细胞的方法。

BACKGROUND：Reports of culture condition of olfactory ensheathing cells （OECs） vary.And some methods have bad reproducibility,not appropriate for actual application.OBJECTIVE：To observe the effect of differential attachment,chemicals in combination with trypsin digestion to purify rat OECs in vitro and to compare the effect with differential attachment or chemicals alone.DESIGN,TIME AND SETTING：In vitro controlled observation of cytology was performed at the Laboratory of Department of Human Anatomy Histology and Embryology,Fujian Medical University from June 2007 to June 2008.MATERIALS：A total of 8 SD rats,2 days old,were provided by the Laboratory Animal Center of Fujian Medical University.METHODS：The OECs were dissociated from the postnatal rat olfactory bulbs under sterile condition,and seeded in poly-L-lysine-coated culture flask at a density of 4.0×10^8 /L for primary culture.The cells were divided into 4 groups：control group （not purified）;chemicals group （5 μmol/L arabinose）;differential attachement group （Nash differential attachement）;combination group （Nash differential attachement to eliminate most of the fibroblasts,followed by arabinose;when the cells were confluent at 6 days,the cells were digested with 1.25 g/L trypsin for 1 minute until the processes were shrank,cells became round,with some cells floating）.MAIN OUTCOME MEASURES：Morphological changes of the cultured OECs and NGFRp75 immunocytochemistry were observed.RESULTS：The OECs displayed a very characteristic morphological appearance.Most of OECs were bipolar or tripolar with long and slim processes.In the unpurified control group the rapidly proliferating fibroblasts were in the majority （60%） after 7 days in culture,and confluent at day 14.The OECs occupied the most in the other groups,and their appearance remained unchanged.The surviving bipolar or tripolar OECs were positive for NGFRp75.The purifity by chemicals and differential attacehment methods was low （75%）,while the combination group was high （85%）.CONCLUSION：The method of purifing OECs through a combination of differential attacehment,chemicals and trypsinization is effective.