双向差速法制备高纯度雪旺细胞的实验研究  

Novel method of Schwann cell purification by differential velocity of cell attachment and detachment

作　　者：刘炎[1,2] 张莉[1] 宫旭[2] 陈继凤[1] 王玉[1] 赵斌[1] 任志午[1] 詹胜峰[1] 许文静[1] 彭江[1] 卢世璧[1] 

机构地区：[1]解放军总医院骨科研究所,北京100853 [2]吉林大学白求恩第一医院,长春130021

出　　处：《中国矫形外科杂志》2011年第16期1373-1376,共4页Orthopedic Journal of China

基　　金：国家863计划(编号:2009AA03Z312);全军十一五专项项目(编号:06Z057);国家支撑计划(编号:2009BAI87B02)

摘　　要：[目的]利用乳鼠的雪旺细胞与成纤维细胞贴壁及复合酶消化分离速度不同的特点,建立简单而快速提取和纯化雪旺细胞的方法。[方法]取3 d SD大鼠双侧坐骨神经,在解剖镜下剥离去除神经外膜,剪碎后用0.2%复合胶原酶(Collagenase NB4)消化,细胞悬液接种于预先用多聚赖氨酸包被的培养瓶差速贴壁30 min,然后将细胞悬液移入一新的培养瓶培养48 h;用0.05%复合胶原酶37℃消化30 min,振荡分离雪旺细胞与成纤维细胞,培养48 h后在相差显微镜下观察细胞形态;计数、纯度测定;免疫细胞化学鉴定和流式细胞仪检测所得的雪旺细胞的纯度。[结果]经过2次纯化后,每25 cm2培养瓶可获取约(117.2±3.4)×104个细胞;第一轮纯化后雪旺细胞纯度达86.99%±1.53%,经2次纯化后纯度为98.32%±0.12%,两轮纯化之间有显著性差异,P<0.001;P75NTR免疫细胞化学鉴定细胞为阳性,流式细胞仪检测雪旺细胞纯度达97.9%。[结论]双向差速复合胶原酶消化法是一种高效、快捷的雪旺细胞纯化方法。[Objective]To establish a novel efficient method of Schwann cell purification by the characteristic that there has been a different velocity of attachment and detachment between Schwann cells and fibroblast.[Method]Postnatal SD rat＇ s（3 day old） sciatic nerve segments were harvested and epinuims were striped off aseptically under a dissecting microscope.The nerve segments were digested by Collagenase NB4 at a concentratin of 0.2% with a rotator for 15 min.After enzymatic and mechanical dissociation,the cell digest was allowed to settle on polylysine-coated flasks for 30 min with intermittent shaking,and then the suspended cells were transferred to new flasks.After 48h primary culture,culture media was replaced with the 0.05% collagenase.After incubation at 37 ℃ for 30 min,the flasks were shaked horizontally to release rounded up or detaching cells after another 48h culture.The final purified cells were identified with immunocytochemistry and flow cytometric analysis.[Result]After two rounds of attach and detach purification the number of Schwann cells reached about（117.2±3.4）×104 per 25 cm2 and purity was achieved 97.9%.[Conclusion]A large number of purified Schwann cells can be obtained by our simple,fast and safe method within only 72h,and might be valuable for studies related to basic science of peripheral nerve injury and regeneration.

关 键 词：雪旺细胞 纯化 差速贴壁法 差速分离法 复合胶原酶 周围神经再生 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]