两种染色方法在细胞核DNA含量检测中应用及比较.docVIP

两种染色方法在细胞核DNA含量检测中应用及比较 　 作者：焦红丽， 冶亚平 夏潮涌 【摘要】 目的 探讨改良、快速两种Feulgen染色方法达到最佳染色效果的水解时间段，为科研和临床的应用提供方法学指导。方法 选取5只成年健康雄性SD大鼠的肝组织，制成肝细胞涂片，分别在室温和60℃温度下水解不同时间，应用改良和快速两种方法染色，TIGER图像分析仪检测和分析单个肝细胞核的DNA含量。结果 （1） 不同大鼠肝细胞涂片在同一水解温度、时间作用下，相同DNA倍体含量肝细胞的DNA含量大致相同，CV值均小于10%；（2） 二、四、八倍体肝细胞核的DNA含量比值均接近2或4；（3） 同一大鼠相同水解温度不同水解时间，同一倍体的肝细胞核DNA含量存在差异：60℃水解温度下，IOD(5～7 min)gt;IOD(9～15 min)gt;IOD(1 min，20 min)，室温水解温度下，IOD(50 min)gt;IOD(20～30 min，70～90 min)gt;IOD(5～1 min，100 min)。结论 HCL水解肝细胞涂片时间过长或过短均不能理想的染色，快速法和改良法Feulgen染色达较佳染色效果的时间段分别为：快速法5～7 min，改良法20～90 min。 【关键词】 DNA分析； 染色与标记方法； Feulgen染色； 肝细胞涂片 Application and comparison of two kinds of staining methods for Abstract: Objective To find out the suitable hydrolysis time for two staining methods of Feulgen and to provide guidline on research and clinic. Methods Smears of liver cell suspension were obtained from five healthy rats. All the hepatocytes are, respectively, hydrolyzed in different time at room temperature and 60℃, and then stained with modified Feulgen stain and quick one. DNA content and ploid of intact hepatocyte nuclei was analyzed by TIGER cell image analysis system. Results (1) the nuclear DNA contents are almost identical from same DNA content ploidy of different rats under the same temperature and time of hydrolysis, and the CV of IOD from three types of DNA content ploidy was less than 10 percent. (2) The IOD ratio among diploid, tetraploid and octaploid was close to 2 and 4. (3) The DNA contents of hepatocyte nuclei from same DNA content ploidy of different rats were distinct under the same temperature and different hydrolysis time: hydrolysis at 60℃, IOD(5～7 min)gt;IOD(9～15 min)gt;IOD(1 min，20 min); hydrolysis at room temperature, IOD(50 min)gt;IOD(20～30 min，70～90 min)gt;IOD(5～10 min，100 min). Conclusion nuclei DNA cant be stained better for extra long or short hydrolysis time, the better hydrolysis time of quick Feulgen stain and modified one are, respectively, 5～7 min and 20～90 min. Key words: DNA analysis; stainin