摘要
目的探讨水解产物变化趋势与染色效果的相关性以及两种不同染色方法达到最佳染色效果的水解时间,为科研和临床的应用提供有价值的参考。方法选取5只成年健康雄性SD大鼠的肝组织,一部分制成恒定细胞数的肝细胞滴片,5N HCL水解后,紫外分光光度计扫描产物的最大吸收峰波长,并检测该产物OD值的变化; 另一部分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Feulgen法染色,图像分析仪检测和分析单个细胞核的DNA含量与倍体。结果（1）肝细胞滴片水解产物扫描在260 nm处有最大吸收波峰; （2）紫外分光光度计在室温和60℃水解温度下测得水解产物的量随着时间的延长逐渐增加; （3）同一大鼠在相同水解温度下,经过不同时间水解,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）〉IOD（9-15 min）〉IOD（1 min,20 min）,室温水解温度下,IOD（50 min）〉IOD（20-30 min,70-90 min）〉IOD（5-10min,100 min）。结论HCL对细胞核的水解作用使DNA双链中糖苷键断裂,醛基暴露,碱基脱至HCL中,且量逐渐增加; HCL水解时间与染色的效果不成正比,两种Feulgen染色法存在各自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。
Objective To explore the correlation between the trend of changes of nuclear hydrolysis products and staining results, to determine the optimal hydrolysis time for two Feulgen staining procedures, and to provide a valuable reference for research and clinical applications. Methods Liver cell suspension was prepared from five healthy SD rats. One portion was used to make smears with constant cell number, then these hepatocytes were hydrolyzed by 5N HCL. The wavelength of the maximum absorption peak of the products was scanned detected. The other portion room temperature and 60℃ staining, respectively. DNA by an UV spectrophotometer, and the quantity of the hydrolysis products was was used to make ordinary smears, and the hepatocytes were hydrolyzed at , respectively, and stained with modified Feulgen staining and fast Feulgen content and ploid of intact hepatocyte nuclei were analyzed by a TIGER cell image analysis system. Results （ 1 ） The wavelength of the maximum absorption peak of the hydrolytic products of hepatocytes on smears was 260 nm. （2） The quantity of hydrolytic products was increasing with the prolongation of hydrolysis time. （3） For the same rat, the DNA contents of hepatocyte nuclei measured from the same DNA content ploidy were different under the same temperature and different hydrolysis time： hydrolysis at 60℃, IOD（5 -7 min） 〉 IOD（9 - 15 min） 〉 IOD（1 min, 20 min） ; hydrolysis at room temperature, IOD（50 min） 〉 IOD（20 -30 min, 70 -90 min） 〉 IOD（5 -10 min, 100 min）. Conclusion Because of HCL hydrolysis, the glucosidic bond is broken, free aldehyde groups are exposed from the DNA backbone structure, and the quantity of aldehyde groups is increased gradually. Nuclei DNA can not be stained better for an extra long or short hydrolysis time, the better hydrolysis time of fast Feulgen staining and modified one are 5 -7 min and 20 -90 min, respectively.
出处
《中国体视学与图像分析》
2009年第4期423-428,共6页
Chinese Journal of Stereology and Image Analysis
关键词
HCL水解
OD值
DNA含量
肝细胞涂片、滴片
DNA analysis
DNA hydrolysate
Optical density
Hepatocytes, smear
Feulgen staining
