新生大鼠耳蜗血管纹缘细胞中ATP存在的证据  

Evidence for the existence of ATP in the marginal cells of the neonatal rat cochlea

作　　者：陈洁[1] 何珊[2] 杨军[3] 

机构地区：[1]上海交通大学医学院附属儿童医学中心耳鼻咽喉科,上海200127 [2]上海浦东新区人民医院耳鼻咽喉科 [3]上海交通大学医学院附属新华医院耳鼻咽喉-头颈外科

出　　处：《临床耳鼻咽喉头颈外科杂志》2010年第10期462-465,共4页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：国家自然科学基金资助项目(No:30772396)

摘　　要：目的:研究体外培养的新生大鼠耳蜗血管纹缘细胞中存在ATP的证据,即细胞中是否存在ATP囊泡,体外培养液中能否检测到所释放的ATP。方法:采用出生1～3 d的Sprague-Dawley大鼠,进行体外血管纹缘细胞培养、纯化、鉴定。特异性标记ATP囊泡的喹丫因染色后在荧光显微镜下观察缘细胞中的ATP囊泡。采用生物发光法检测缘细胞细胞外液中所释放的ATP的浓度。结果:体外培养的新生大鼠耳蜗血管纹缘细胞,经流式细胞法检测上皮细胞标志性的角蛋白和波形蛋白的纯度,证实培养所获得的细胞为缘细胞。经喹丫因染色后在荧光显微镜下可见缘细胞细胞质中存在大量的绿色星点状染色。采用生物发光法检测缘细胞细胞外液中ATP的浓度,通过细胞荧光值可计算出ATP的浓度。结论:新生大鼠耳蜗血管纹缘细胞中存在ATP囊泡,并能分泌ATP。Objective：To study the evidence of adenosine triphosphate（ATP） in the marginal cells of the stria vascularis in the neonatal rat cochlea,namely,whether ATP vesicles could be detected in the marginal cells and ATP release could be detected in the culture solution in vitro.Method：The marginal cells of 1-3 day old Sprague-Dawley rats were cultivated,purified and verified.ATP vesicles were observed in the marginal cells under fluorescence microscope using quinacrine staining technique.The concentration of ATP released from the cells in the extracellular solution was determined through bioluminescence assay.Result：The marginal cells were verified in vitro by detecting antibodies of cytokeratin and vimentin using flow cytometry.A large number of asterion-like staining could be observed in cultured marginal cells that were incubated with quinacrine.The concentration of ATP released from the cells in the culture solution could be determined,and the concentration of ATP could be calculated by fluorescent intensity.Conclusion：The ATP vesicles exist in the marginal cells and can release ATP.

关 键 词：三磷酸腺苷 耳蜗 血管纹 缘细胞 细胞培养 生物发光法 

分 类 号：R764[医药卫生—耳鼻咽喉科]