摘要
本文以地塞米松（25mg／kg）腹腔注射10h后诱导的BALB／c鼠（n=5x）胸腺细胞作为凋亡阳性细胞，比较了碘化丙啶（PI，propidiumiodium）染色DNA含量分析和TUNEL（TdT—mediatedX-dUTPnickendlabeling）标记DNA断裂点分析两种方法在检测凋亡中的差异。流式细胞仪分析结果表明，PI检测凋亡的阳性率29．3±8．1％（x±s）明显低于TUNEL检测的阳性率（40．2±8．7％），P<0．01（配对t检验）。进一步用PI和TUNEL双参数分析显示，在PI检测阳性的颗粒中有4．7±2．2％是TUNEL检测阴性的，即没有DNA断裂点的增加，这些颗粒很可能是细胞碎片，它们导致了PI检测的假阳性。另外TUNEL阳性的细胞中有42．8±10．6％处在S期和G2／M期，因而用PI检测为阴性，造成PI方法的漏检，即假阴性。我们的资料表明，PI检测凋亡与TUNEL相比有很高的漏检率和一定的假阳性，因此PI只适用于凋亡初步分析。要进行准确的定量分析应该用TUNEL法。
Eukaryotic cell apoptosis is usually assessed by flow cytometry on reduction of DNA c0ntent or formation of DNAbreaks. We compared the character of the two methods in detecting apoptosis. The apoptosis of mice (n= 5) thymocytes were in-duced by abdominal cavity injection of dexamethasone (25mg/kg), and after 10hr the cells were separated. Propidium iodium(Pl)staining and TdT enzyme mediated deoxynucleotidy nick end labeling (TUNEL)kits were used to measure the DNA content and theDNA strand breaks, respectively. The positive rates of apoptosis detected with the methods of Pl and TUNEL were 29. 3±8. 1 %(x±s) and 40. 2±8. 7%, respectively, P<0. 01 (matched t-test). Analysing with double parameters of Pl and TUNEL, in thepositive part of Pl staining, there were 4. 7±2. 2% of particles which had n0 increased DNA breaks (TUNEL negative). Theseparticles, we think, may be the debris of cells and contribute to the false positive of Pl detection. On the other hand, about 42. 8±10. 6% of TUNEL positive cells had no reduction of DNA content, which resulted in the false negative of Pl measunement. Our re-sults indicated that the method of DNA breaks labeling with TUNEL is superior to the methods of DNA content assay by Pl stain-ing to detect apoptosis.
出处
《上海医学》
CAS
CSCD
北大核心
1997年第2期65-67,共3页
Shanghai Medical Journal
