大鼠耳蜗胶质细胞的分离培养和纯化  

In vitro Methods for Isolation, Culture and Purification of Cochlear Glial Cells from Rats

作　　者：孙菲 周柯 田克勇 常会敏[3] 王洁[4] 邱建华 查定军 Sun Fei;Zhou Ke;Tian Keyong;Chang Huimin;Wang Jie;Qiu Jianhua;Zha Dingjun(Department of Otolaryngology Head and Neck Surgery,Xijing Hospital,Air Force Military Medical University,Xi'an,710032,China;不详)

机构地区：[1]空军军医大学西京医院耳鼻咽喉头颈外科,西安710032 [2]空军军医大学西京医院检验科全军临床检验医学研究所,西安710032 [3]西安医学院附属医院耳鼻咽喉头颈外科 [4]西安市儿童医院耳鼻咽喉头颈外科

出　　处：《听力学及言语疾病杂志》2020年第5期535-539,共5页Journal of Audiology and Speech Pathology

基　　金：国家重点基础研究发展计划(973计划,2014CB541700);国家自然科学基金(81870732、81470695);陕西省创新能力支撑计划(2018PT-01);陕西省重点研发计划(2017ZDXM-SF-061);空军军医大学西京医院学科助推项目(XJZT18ML54)资助。

摘　　要：目的探讨大鼠耳蜗胶质细胞的体外分离培养和纯化的方法,为进一步研究耳蜗胶质细胞功能提供实验基础。方法分离出生后3天的新生大鼠(18只,36耳)耳蜗轴组织,通过组织酶消化及阿糖胞苷补充法、组织酶消化及免疫磁珠分选法、组织块培养及细胞冷喷注法三种方法(各2只,4耳,均重复三次)进行耳蜗胶质细胞分离、培养和纯化,通过细胞计数、CCK-8检测以及胶质细胞标记物免疫荧光染色计算细胞的产量、活性及纯度。结果组织块培养及细胞冷喷注法获得的耳蜗胶质细胞的产量尽管最低,但其细胞活性最高并优于其它两种方法,且细胞纯度较高,接近组织酶消化及免疫磁珠分选法。结论蜗轴组织块培养及细胞冷喷注法获得的耳蜗胶质细胞活性良好且细胞纯度较高,操作简便且成本低,可作为一种便捷的耳蜗胶质细胞体外培养和纯化方法。Objective To evaluate the advantages of in vitro methods for isolation,culture and purification of cochlear glial cells of rats,and provide experimental basis for further study of their specific functions.Methods The neonatal rats(12 ears from 6 rats)cochlear modiolus tissues containing spiral ganglion were dissected,and the isolated cochlear glial cells were cultured and purified by combined use of tissue enzymatic digestion and cytarabine supplement,or immunomagnetic bead sorting,or combined use of modiolus explants culture and a cold jet technique.The cell yield,viability and purity of cochlear glial cells obtained by each method were calculated by direct cytometry,cell counting-kit 8(CCK-8)assay,and immunofluorescence staining of glial cell marker.Results The combined use of modiolus explants culture and cold jet method obtained the lowest yield of cochlear glial cells,but the resulting cell viability was the highest and superior than the other two methods,and resulting high cell purity was similar as that of tissue enzymatic digestion combined with an immunomagnetic purification procedure.Conclusion The combined use of modiolus explants culture and cold jet method is simple to operate and cost-effective,and can obtain highly purified cochlear glial cells with good viability,thus can be used as a convenient method for in vitro culture and purification of cochlear glial cells.

关 键 词：耳蜗 胶质细胞 螺旋神经节 培养 纯化 

分 类 号：R764.35[医药卫生—耳鼻咽喉科]