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

王燕;褚汉启;周良强;陈金;李志勇;刘云;张平;王春芳;黄孝文;崔永华;

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

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

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。
关键词
成年小鼠;边缘细胞;酶消化法;细胞培养
基金项目(Foundation):
国家自然科学基金资助项目(30672307)
作者
王燕;褚汉启;周良强;陈金;李志勇;刘云;张平;王春芳;黄孝文;崔永华;
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