中文摘要：

目的 观察体外培养的新生大鼠耳蜗K（o）lliker器支持细胞是否存在并释放ATP,初步探讨其释放机制.方法 选取出生后ld的Sprague-Dawley大鼠,分离耳蜗膜迷路,采用机械分离与酶消化相结合的方法获得单离的K（o）lliker器支持细胞.观察膜迷路和K（o）lliker器支持细胞的喹丫因染色情况.采用生物发光法,通过影响K（o）lliker器支持细胞ATP代谢、改变细胞内外Ca2浓度、抑制细胞内磷脂酶信号通路及添加缝隙连接半通道阻断剂,观察K（o）lliker器支持细胞释放ATP浓度的变化.结果 用喹丫因染色体外培养的K（o）lliker器支持细胞,发现胞质中存在大量绿色星点状染色.采用生物发光法检测的ATP标准曲线呈明显的对数线性关系.随着巴佛洛霉素A1浓度增加,K（o）lliker 器支持细胞培养液中ATP浓度逐渐降低,而随着己二酸二癸酯浓度的增加,培养液中ATP浓度逐渐升高;在一定浓度范围内,随着细胞外Ca2＋浓度增加,K（o）lliker器支持细胞ATP的释放减少,而随着细胞内游离Ca2浓度增加,K（o）lliker器支持细胞释放ATP量增加;培养液中加入甘珀酸钠或乌热酸抑制半通道后可以显著的降低ATP释放.此外,抑制细胞内磷脂酶信号通路也可以减少ATP的释放.结论 体外培养的新生大鼠耳蜗K（o）lliker器支持细胞存在并释放ATP,细胞内、外液中Ca2＋浓度的变化可能通过调节半通道的开放而影响其ATP的释放.

英文摘要：

Objective The specific mechanism underlying in the Adenosine triphosphate （ATP） release from the K（o）lliker＇s organ is still unknown.The present study was designed to investigate whether the supporting cells in the K（o）lliker organ in vitro release ATP and to explore the mechanism of ATP releasing from these cells.Methods Supporting cells in the K（o）lliker organ from P1 rats were isolated,purified and cultured with a combinatorial approach of enzymatic digestion and mechanical separation.Quinacrine staining was used to observe the cochlear membranous labyrinth and supporting cells.the bioluminescence assay was chosen to explore the release ATP from supporting cells in the K（o）lliker organ,when the ATP metabolism of the cells was influenced,the intracellular or extracellular Ca2 ＋ concentration changed,the hemichannels blocked,and the phospholipase signaling pathways inhibited.Results There were intensely numerous starlike green spots of quinacrine staining in the cytoplasm of supporting cells.There was a strong log-linear relationship in the ATP standard curve generated by the bioluminescence assay.With increasing concentrations of bafilomycin Al,the ATP concentration in the culture medium of the supporting cells in the K（o）lliker organ decreased,while with adipic acid didecyl,it increased.In a certain concentration range,with increasing extracellular Ca2＋ concentration,the supporting cells in the K（o）lliker organ releasing ATP decreased,while the intracellular Ca2＋ concentration increased,the results showed the elevation of the amount of ATP release.Adding chelerythrine chloride or aristolochid acid into the culture medium of the supporting cells in the K（o）lliker organ could decrease the ATP release significantly via inhibiting the hemichannels.In addition,by reducing intracellular Ca2＋ concentration,inhibition of intracellular signaling pathways phospholipase also decreased ATP release.Conclusions This study demonstrated the presence and release of ATP from the support