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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 investigate the correlation between the trend of hydrolysis products and the dyeing effect and the hydrolysis time to achieve the best dyeing effect by two different dyeing methods, which will provide valuable reference for the scientific research and clinical application. Methods Five adult healthy male Sprague-Dawley rats were selected, and some of them were made into a constant number of cells. After the 5N HCL was hydrolyzed, the maximum absorption peak wavelength of the product was scanned by UV spectrophotometer, and the OD of the product The other part of liver tissue was made into hepatocyte smear, which was hydrolyzed at room temperature and 60 ℃ for different time respectively. Two kinds of modified and fast Feulgen method were used for staining. The DNA content and ploidy of single nucleus were detected and analyzed by image analyzer. Results (1) Hydrolyzate of hepatocyte slides showed the maximum absorption peak at 260 nm. (2) The amount of hydrolysates measured by UV spectrophotometer at room temperature and hydrolysis temperature of 60 ℃ increased gradually. ( 3) In the same rat, the content of hepatic nuclear DNA in the same diploid was different at the same hydrolysis temperature at different hydrolysis time: IOD (5 ~ 7 min)> IOD (9 ~ 15 min)> IOD (1 min, 20 min), IOD (50 min)> IOD (20-30 min, 70-90 min)> IOD (5-10 min, 100 min) at room temperature hydrolysis temperature. Conclusions Hydrolysis of HCL causes cleavage of glycosidic bonds in DNA double-stranded chains, exposure of aldehyde groups and cleavage of bases to HCL. The HCL hydrolysis time is not proportional to the staining effect, and the two Feulgen staining methods are more specific The best staining time was 5 ~ 7 min for rapid method and 20 ~ 90 min for modified method.