膜联蛋白V与碘化丙啶用于细胞周期特异性凋亡的检测

Analysis of cell cycle specific apoptosis by FTTC-Annexin V/PI

目的 建立一种新的细胞周期特异性凋亡检测方法。方法 将急性早幼粒细胞白血病细胞株 (HL 60 )分别经喜树碱 (CAM)、紫外线诱导 ,及喜树碱和N 甲苯磺酰基 L 苯基丙氨酸氯甲基酮(TPCK)共同诱导后 ,用异硫氰酸荧光素标记的膜联蛋白V(AnnexinV)进行标记 ,经 1 %不含甲醇的甲醛溶液冰浴固定 ,再用含洋地黄皂苷的碘化丙啶染色液染色后 ,流式细胞术分析凋亡细胞的周期特异性和细胞凋亡率 ,并与DNA链缺口标记法 (TdT)和常规AnnexinV法检测结果进行比较。结果 细胞凋亡的周期特异性分析 :膜联蛋白V/碘化丙啶法 (PA法 )和TdT法结果一致 ,CAM和紫外线诱导的细胞凋亡分别发生在S期和G1期 ;CAM和TPCK共同诱导试验显示 ,PA法对早期凋亡具有较高的检测灵敏度 ,TdT法则不能有效检测 ;而常规AnnexinV法无法进行凋亡细胞的周期特异性分析。细胞凋亡率的检测 :HL 60经CAM、紫外线、CAM与TPCK三种方法诱导后 ,PA法检测凋亡率的结果 (1 7 3 %、34 .7%、35 .9% )与常规AnnexinV法的检测结果 (1 8.1 %、35 .6 %、37.0 % )一致 (P >0 .0 5) ,而TdT法(1 0 .7%、1 9.5 %、- )则明显偏低 (P <0 .0 1 )。结论 PA法可以用于凋亡细胞周期特异性检测 ,其稳定、可靠。

Objective The current study was designed to establish a new method for the analysis of cell cycle specific apoptosis. Methods Cells of the human promyelocytic HL 60 line treated with camptothecin (CAM), ultraviolet (UV), CAM and N tosyl L phenylalanine chloromethyl ketone (TPCK) combined respectively were labeled with FITC conjugated Annexin V, prefixed with 1% methanol free formaldehyde on ice, stained with propidium iodide solution containing digitonin, then analyzed the cell cycle specificity of apoptosis and apoptosis rate by flow cytometry. The results were compared respectively with those analyzed by TdT assay and common Annexin V assay. Results The cell cycle specificity of apoptotic cells analyzed by new method(PA assay)were the same with those analyzed by TdT method, the apoptotic cells induced by CAM and UV occurred in S phase and G1 phase respectively. CAM and TPCK study showed that new method could detect early apoptosis sensitively and TdT assay failed. Common Annexin V assay failed to analyze the cell cycle specificity of apoptotic cells in those studies. The apoptosis rate of cells induced respectively by CAM, UV, CAM and TPCK detected by PA assay (17 3%?34.7%?35.9%) were the same with that detected by common Annexin V assay (18.1%?35.6%?37 0%; t test, P >0.05), but the results detected by TdT assay (10.7%?19.5%?-) were obviously low ( t test, P <0.01). Conclusion The new method for apoptosis detection is stable, reliable, sensitive, and can be used to analyze the cell cycle specificity of apoptotic cells.