Ⅱ型胶原酶体外分离并培养成年小鼠血管纹边缘细胞  

Detachment of Adult Mouse Stria Vascularis Marginal Cell with CollagenaseⅡ and Cells Culture in Vitro

作　　者：王燕[1,2] 褚汉启[1] 周良强[1] 陈金[1] 李志勇[1] 刘云[1] 张平[1] 王春芳[1] 黄孝文[1] 崔永华[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院耳鼻咽喉头颈外科,武汉430030 [2]武汉市妇幼儿童保健中心(武汉市儿童医院)耳鼻咽喉头颈外科

出　　处：《听力学及言语疾病杂志》2011年第5期451-454,共4页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金资助项目(30672307)

摘　　要：目的探讨单纯Ⅱ型胶原酶消化法体外分离并培养成年小鼠耳蜗血管纹边缘细胞(marginal cell,MC)的方法。方法选取6只出生75±3 d的健康C57BL/6J小鼠,显微分离其耳蜗血管纹组织,以Ⅱ型胶原酶原代消化培养边缘细胞。倒置显微镜观察细胞形态,噻唑蓝(MTT)法测细胞生长曲线,透射电镜观察细胞的超微结构,免疫荧光法检测上皮细胞标志物——中间丝角蛋白18(CK18)和耳蜗血管纹的独特标志物KCNQ1的表达,RT-PCR法检测CK18和KCNQ1mRNA的表达。结果接种24～48 h后可见细胞贴壁增殖,形成大小不等的细胞岛,一周后细胞迅速生长,相互融合,紧密连接,形成单层极性上皮层,呈典型的"铺路石"样外观。透射电镜观察可见多角形细胞表面有较多微绒毛,胞浆内线粒体、内质网等细胞器丰富。免疫荧光检测显示CK18和KCNQ1蛋白表达阳性,RT-PCR结果示CK18和KCNQ1mRNA均有表达。结论采用原代消化培养技术可成功建立成年小鼠耳蜗血管纹MC的体外原代培养,为进一步研究成年期MC的功能和某些内耳疾病的发病机制提供了实验基础。Objective To investigate the method of isolation and culturing marginal cells of adult mouse by solo collagenase in vitro. Methods The marginal cell were isolated from the stria vascularis of adult mice by collagenase Ⅱ for culturing, The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The ultrastructure of the cultured cells was observed by transmission electron microscopy. The expression of CKI8 and KCNQ1 were detected by immunofluorescence and reverse transcription-- polymerase chain reaction （RT--PCR）. Results The primary cultured cells attached within 24-48 h of seeding and proliferated in an island like monolayer. After one week, more and more epithelial--like cells had a tendency to become confluent and became tightly packed, growing into a monocellular layer, ＂cobblestone--like appearance. Transmission electron microscope showed typical epithelia with microvilli on the cells surface, massive mitochondria and rough endoplasmic reticulurn in the cells. Expression of CK18 and KCNQ1 in MC was confirmed by immunofluorescence staining, and expression of CK18--mRNA and KCNQ1- mRNA was also detected by RT --PCR. Conclusion The cell culture system of marginal cells of adult mouse has been successfully established by enzyme digestion which can provide a stable source of MC for investigation of the ongoing function of the marginal cell.

关 键 词：成年小鼠 边缘细胞 酶消化法 细胞培养 

分 类 号：R339.16[医药卫生—人体生理学]