量子点联合Cy5双重荧光标记人舌鳞状细胞癌细胞蛋白质的应用研究

Double labeling and comparison of fluorescence photostability between quantumdots and Cy5 in human tongue squamous cell carcinoma

目的比较以量子点(quantum dots,QDs)与Cy5荧光染料对人舌鳞癌细胞株Tca8113细胞中热休克蛋白70(heat shock protein 70,HSP70)和survivin蛋白质进行双重免疫荧光标记时蛋白质的分布及荧光的稳定性。方法以QDs和Cy5荧光染料对Tca8113细胞内HSP70和survivin蛋白质进行双重免疫荧光标记,在激光共聚焦显微镜下同时观察Tca8113细胞内的HSP70和survivin蛋白质表达及分布。激光连续照射2 420 391 ms,用激光共聚焦显微镜自带软件Leica Confocal Software测量QDs和Cy5的荧光信号强度变化。结果激光共聚焦显微镜下可见Tca8113细胞内HSP70和survivin蛋白质均有明显表达,分别表现为绿色和红色荧光,其中两种蛋白质重叠处呈黄色荧光。激光连续照射2 420 391 ms,QDs标记的绿色荧光未见明显衰减,而Cy5荧光染料标记的红色荧光基本淬灭,随着Cy5荧光的淬灭,绿红黄三色通道下所见的红绿黄3种颜色的荧光也逐渐消退直至变成单色的绿色荧光。结论 QDs对光漂白的抵抗能力强,比Cy5荧光染料具有更高的光稳定性,更加适用于细胞内蛋白质的长时间动态监测的研究。

Objective To evaluate the application of quantum dots （QDs） and the Cy5 labeling technique in Tca8113 cells, and to compare their fuorescence photostability. Methods Using two-colour confocal microscopy, QDs and Cy5 were applied to tag HSP70 and survivin respectively with indirect immunofluorescence. The expression of HSPT0 and survivin in TcaS113 cells was observed in confocal laser microscopy, and the fluorescence photostability between QDs and Cy5 was compared. Results Survivin and HSPT0 expression was clearly detected in the TcaSll3 cells. After 40 rain of continuous laser exposure, no significant decline in the QD fluorescent markers was observed, whereas the Cy5 fluorescent signals faded very quickly and became undetectable. Conclusion QDs have better fluorescence photostability than Cy5, and are more appropriate for observation of protein changes in cells dynamicly.