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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 presence of ATP in the cultured rat neonatal rat stria vascularistem cells, that is, whether ATP vesicles exist in the cells and whether the released ATP can be detected in vitro. Methods: Sprague-Dawley rats aged 1 to 3 days were used to culture, purify and identify vascular lineage cells in vitro. The DNA of rabbits was stained with ATP-labeled ATPase and the vesicles in the marginal cells were observed under a fluorescence microscope. The bioluminescence method was used to detect the concentration of ATP released by extracellular fluid of marginal cells. Results: The purity of the keratin and vimentin in epithelial cells was detected by flow cytometry. The cells obtained from the culture were found to be marginal cells. After the quinoline was stained by fluorescence microscopy, there were a large number of green star spotted cells in the cytoplasm of border cells. The bioluminescence method was used to detect the concentration of ATP in the extracellular fluid of marginal cells. The concentration of ATP was calculated by the fluorescence value of the cells. Conclusion: There are ATP vesicles in the vascular borderline cells of neonatal rat cochlea and can secrete ATP.