成年大鼠嗅球嗅鞘细胞的纯化实验(英文)  

Purifying olfactory ensheathing cells from the olfactory bulb of adult rats

作　　者：朱仲庚[1] 吴小涛[2] 蒋赞利[2] 

机构地区：[1]东南大学临床医学院 [2]东南大学临床医学院附属中大医院骨科,江苏省南京市210009

出　　处：《中国组织工程研究与临床康复》2007年第15期2971-2975,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:嗅鞘细胞的纯化方法以及嗅鞘细胞纯度的不同被认为与嗅鞘细胞移植后的效果有关。因此,开发高效、便于统一的纯化方法对嗅鞘细胞移植研究的标准化非常重要。目的:为嗅鞘细胞移植研究的标准化提供一个高效、便于统一的纯化方法。设计:随机对照实验。单位:东南大学临床医学院附属中大医院骨科,东南大学临床医学院中心实验室,东南大学临床医学院实验动物中心。材料:实验于2006-02/08在东南大学临床医学院中心实验室完成。选用28只成年雌性SD大鼠,体质量200￣250g;DMEM/F-12(GIBCO);2.5g/L胰蛋白酶(GIBCO);左旋多聚赖氨酸(SIGMA);牛垂体萃取液(SIGMA);胎牛血清(杭州四季青生物试剂公司);兔抗p75抗体(SIGMA);生物素化羊抗兔IgG(武汉博士德生物技术公司);MTT试剂盒(SIGMA)。方法:将SD大鼠嗅球中分离出嗅鞘细胞的原代培养物,体外培养8d,将培养物分为4组:差速贴壁组、免疫吸附组、改良方法组、对照组。①改良方法组细胞悬液接种到未经包被的培养瓶在37℃、体积分数为0.05CO2的环境中孵育1h,将上清液接种到培养瓶中(底面用1mg/L的兔抗p75抗体润湿后在37℃下烘干,再用DMEM/F-12洗涤1次)。上清液在这种已包被兔抗p75抗体的培养瓶中37℃、体积分数为0.05CO2下孵育45min,DMEM/F-12洗涤5遍以清除未贴壁的细胞,用细胞刮刀收获贴壁细胞、离心、重新悬浮在含20mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中;Nash差速贴壁组细胞按Nash的方法进行操作;抗p75抗体免疫吸附组细胞按照Ramo’n-Cueto的方法进行操作;上述纯化方法获得的3组细胞悬液分别接种到包被左旋多聚赖氨酸的24孔细胞培养板中,在37℃、体积分数为0.05CO2的环境中培养14d。对照组未经纯化的细胞悬液同样重新悬浮在含20mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中并接种到包被左旋多聚赖氨酸的24孔细胞培养板中,�BACKGROUND：The diversity of purification procedures resulting in various pudties of olfactory ensheathing cells （OECs） used for grafting is considered to be relevant in the effectiveness of OECs transplant. It is important to develop a well-defined method which produces OECs of great purity and is easy to unify for the future standardization of research involving OECs. OBJECTIVE： To establish a method being easy to unify for purifying OECs to acquire highly and uniformly enriched population of OECs for standardized studies on cell transplantation. DESIGN： Randomized and controlled experiment SETTING： Department of Orthopaedics, Affiliated Zhongda Hospital of Southeast University School of Clinical Medicine; Central Laboratory of Southeast University School of Clinical Medicine; Experimental Animal Center of Southeast University School of Clinical Medicine. MATERIALS： This experiment was carded out in the Central Laboratory of Southeast University School of Clinical Medicine from February to August 2006. Twenty-eight adult female SD rats weighing 200-250 g were selected in this study. The main reagents were detailed as follows： DMEM/F-12 （GIBCO）; 2.5 g/L trypsin （GIBCO）; poly-L-lysine （SIGMA）; bovine pituitary extract （BPE, SIGMA）; fetal bovine serum （FBS, Sijiqing Biological Agent Co., Ltd., Hangzhou）; rabbit anti-low-affinity nerve growth factor receptor （anti-P^75, SIGMA）; biotinylated goat anti-rabbit IgG （Boster Bioengineering Co., Ltd,, Wuhan）; methyl thiazolyl tetrazolium （MTT） kit （SIGMA）. METHODS： Pdmary cultures of OECs were separated from adult SD rats olfactory bulbs. At day 8 in vitro, the primary cultures were divided randomly into 4 groups, namely differential adhesion method group, immunoadsorption method group, the modified method group, and control group. ① The cell suspension in the modified method group was seeded into uncoated flasks and incubated at 37 ℃ in 0.05 volume fraction of CO2 for 1 hours. The supernatants were seeded

关 键 词：嗅球 嗅鞘细胞 纯化 纯度 细胞活力 

分 类 号：R339.12[医药卫生—人体生理学]