光敏剂亚细胞定位研究方法的应用比较

Comparison of techniques to determine the subcellular localization of photosensitizers

目的探讨不同光敏剂亚细胞定位方法的应用特点。方法应用电感耦合器材(CCD)荧光显微成像系统,选择细胞器荧光探针BODIPY标记细胞内高尔基体。分别采用直接观察法、伪彩色融合法、波形比较法、相关系数法及细胞器-细胞荧光强度比值法,对光敏剂血卟啉单甲醚(HMME)进行细胞内分布的定性与定量研究。结果采用直接观察法比较光敏剂和细胞器探针荧光图像,发现HMME和BODIPY的荧光分布模式有相似之处,采用伪彩色融合法得到的二者融合图像,显示存在黄色的空间重叠区域。应用波形比较法发现,直线路径上所有像素在HMME荧光图像与BODIPY荧光图像中的灰度值相对空间坐标的曲线波形相似。应用相关系数法得出,各像素的HMME荧光灰度值与细胞器探针荧光灰度值之间的相关系数值为0.6024。采用细胞器-细胞荧光强度比值法发现,随着参数m的增大,即细胞器聚集程度的降低,高尔基体聚集区域的平均荧光强度比值(J1/J2)呈下降趋势,二者具有明显的相关性(P<0.05)。结论通过直接观察、伪彩色融合及波形比较的方法,基本可以判定HMME在高尔基体聚集区域有分布;采用相关系数法,尤其是细胞器-细胞荧光强度比值法能得到更为精确的量化结果,以弥补定性方法的不足。

Objective To compare and evaluate Organelle-cell fluorescence intensity ratio analysis established in this study with other techniques to determine the subcellular localization of photosensitizers. Methods CCD fluorescence microscopy imaging system was applied and a kind of special organelle probe BODIPY was selected to label Golgi body. Directly observing, pseudo-color fusing, wave-shape comparing, correlation coefficient calculating and Organelle-cell fluorescence intensity ratio analysis were adopted, respectively, to study the intracellular distribution of domestic photosensitizer Hematoporphyrin monomethyl ether （ HMME）. Results Fluorescence distributing modes of HMME and BODIPY were similar with each other. There ware yellow space-overlap areas in the fusion image. Wave body of changing curve of gray scale value of all pixels in the straight line in two images corresponding to space coordinate was similar with each other. Correlation coefficient in each pixel between fluorescence intensity of HMME and that of BODIPY was 0. 602 4. With increasing of parameter m, namely degree of organelles congregated reducing, the average fluorescence intensity ratio （J1/J2） of Golgi complex was decreasing, the two parameters possessed obvious relativity. P 〈0.05 was got by One way ANOVA test. Conclusion By Directly observing, pseudocolor fusing, wave-shape comparing, HMME distributing in Golgi complex clustered areas is presumedly determined; correlation coefficient calculating and Organelle-cell fluorescence intensity ratio analysis can gain much more accurate quantitative results, which can make up qualitative techniques.