TUNEL检测心肌细胞凋亡的影响因素探讨

目的：探讨避免TUNEL法检测心肌细胞凋亡出现的假阳性和消除非特异性反应的方法以及选择合适的蛋白酶K浓度。方法：4%多聚甲醛固定心肌组织,制作石蜡切片。①将试剂盒说明常规步骤加以改良,将0.3%H2O2放在反应液标记DNA片段之后,先后用小牛血清或羊血清两次封闭非特异性反应;②按蛋白酶K不同剂量分为10、20μg/mL蛋白酶K和不加蛋白酶K组（均n=5）,再按照改良的TUNEL法检测心肌细胞凋亡。结果：①改良TUNEL法,避免了假阳性,背景清晰;②未加蛋白酶K组较10或20μg/mL蛋白酶K组心肌细胞凋亡率显著减少（均P〈0.05）,两组蛋白酶K组心肌细胞凋亡率差异未见有统计学意义（P〉0.05）。结论：①改良后的TUNEL方法适用于心肌细胞凋亡的检测;②TUNEL法检测心肌细胞凋亡选择10、20μg/mL蛋白酶K是适当的。

STUDY ON THE INFLUENTIAL FACTORS OF CARDIOMYOCYTE APOPTOSIS DETECTED BY TUNEL METHOD

Objective：To explore a way of avoiding false positive reactivity and clearing non-specific reactivity in the TUNEL apoptotic detection in situ and how to select appropriate concentrations of proteinase K.Methods：The paraffin sections of myocardium tissues were produced which were fixed by 4% paraformaldehyde.①The inhibition of endogenous peroxidase with 0.3%H2O2 was performed after marking the DNA fragments with there activity fluid. Bovine serum and normal sheep serum were used to inhibit the non-specific reactivity for two times.②Rats were divided into 10,20μg/ml proteinase K groups and non-proteinase K group.Result：①The improved method avoided false positive reactivity,and a clear background was observed. ②Comparing to 10,20 μg/mL proteinase K groups,the cardiomyocyte apoptosis rate was significally decreased in the non-proteinase K group（all P〈0.05）.There was no statistical difference between 10,20 μg/mL proteinase K groups（P〉0.05）.Conclusion：①The improved TUNEL staining method can be used to detect the apoptotic cardiomyocytes. ②10,20 μg/mL proteinase K is appropriate selection in the cardiomyocyte TUNEL apoptotic detection.