嗅鞘细胞的分离纯化及上清液中神经营养因子的测定  

Purification of olfactory ensheathing cells and determination of neurotrophic factors in the supernatant

作　　者：尹丽慧[1] 叶爱芳[1] 陈迟琪[1] 吴建波[1] 倪文飞[2] 

机构地区：[1]温州医学院附属第一医院医科所 [2]温州医学院附属第二医院骨科,温州325000

出　　处：《神经解剖学杂志》2011年第4期433-437,共5页Chinese Journal of Neuroanatomy

基　　金：浙江省自然科学基金(Y2111114;Y2110402)

摘　　要：目的：创建理想的纯化嗅鞘细胞（OECs）的方法,并观察不同时间段OECs上清液中神经营养因子的含量,为选择OECs移植的最佳时机提供理论依据。方法：采用差速贴壁法结合免疫亲和吸附法纯化培养OECs,采用荧光染色法鉴定OECs并进行纯度测定;采用ELISA法检测培养上清液中神经生长因子（NGF）,脑源性神经营养因子（BDNF）和胶质源性神经营养因子（GDNF）的水平。结果：通过上述方法,可以得到纯度85%以上的OECs,光镜下OECs突起细长,以典型的双极或三极细胞为主,背景有少量扁平的、折光性差的细胞存在;NGF、BDNF和GDNF在上清液中均有存在,其中NGF含量最高,NGF和BDNF含量随着培养时间延长逐渐升高,11 d达到高峰,GDNF仅在培养第11 d后检测到。结论：采用差速贴壁法结合免疫亲和吸附法可以获得较高纯度的OECs,培养1114 d,OECs数量多且分泌旺盛,适合用于细胞移植。Objective： The present study tried to establish a perfect culture method for olfactory ensheathing cells（OECs） with better viability and purification and to determine the concentration of neurotrophic factors in olfactory ensheathing cells conditioned media at different culturing time so as to provide the theoretic support for OECs transplantation with optimal time.Methods： Olfactory ensheathing cells were purified with a method based on the differing rates of attachment of the various harvested cell types combined with the method of immune affinity adsorption.The purity of OECs was identified with immunofluorescence staining.The nerve growth factor（NGF）,brain-derived neurotrophic factor（BDNF） and glia cell line-derived neurotrophic factor（GDNF） were detected by the method of ELISA.Results： Immunostaining demonstrated that OECs with purity of over 85% were obtained.The OECs exhibited dipolar or tripolar appearance with long thin processes.Few flat cells with weak refraction on background were also observed.NGF,BDNF and GDNF were detected in OECs supernatant,and the content of NGF was highest.Concentration of NGF and BDNF was increased gradually with extending culture time,and peak value appeared at the 11th day of cell culture.GDNF was detected just after the 11th day of cell culture.Conclusion： OECs with high purity can be obtained by the method of differential adherence combined with immune affinity adsorption.From the 11th to the 14th day,the amount of OECs was largest and their secretion was strongest.Therefore the best time for cell transplantion is between the 11th and 14th days after cell culture.

关 键 词：嗅鞘细胞 原代培养 神经营养因子 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]