流式细胞术检测胱氡肽酶3原位激活与两种检测细胞凋亡方法的比较性研究

Comparison of Caspase 3 activity, subG1 and Annexin V/PI in detecting apoptosis by flow cytometry

目的 用一种新的荧光染色技术 (FLICA/PI)检测放射诱导T淋巴细胞白血病细胞(MOLT 4 )的细胞凋亡过程中胱氡肽酶 3(Caspase 3)原位激活及探讨其活性变化特点 ,并与亚G1峰和膜联蛋白V/碘化丙啶 (AnnexinV/PI)检测细胞凋亡方法的灵敏度进行比较性评价。方法 以放射诱导的MOLT 4细胞凋亡为模型 ,用荧光标记的Caspase 3抑制剂 (FLICA)标记原位激活的Caspase 3,然后 ,用流式细胞仪定量检测并分析其活性变化 ;同时 ,用亚G1峰法和AnnexinV/PI检测细胞凋亡。结果 FLICA/PI检测到的Caspase 3激活 ,在照射后 2h开始升高 (5 84 % ) ,而AnnexinV/PI在 4h时检测到的凋亡率为 5 35 % ,FLICA/PI比AnnexinV/PI可提前 2h检测到细胞凋亡 ,Caspase 3活性的出现早于细胞膜反转。FLICA阳性率在照射后 4h暴发性增高至 12 39% ,显示有时间效应。两种检测方法均比亚G1峰法敏感 ,但AnnexinV/PI检测的凋亡率低于FLICA/PI法。结论 X 射线能诱导MOLT 4细胞凋亡 ,并有明显的时间效应关系 ;Caspase 3激活有阈值效应 ,是照射后细胞发生凋亡过程中早于形态学变化的检测指标。用Caspase 3活性检测细胞凋亡更直接 ,更敏感。

Objective Investigate apoptosis by a new flow cytometric method of FLICA/PI and compared with subG1 and Annexin V/PI Methods Cells of human lymphocytic leukemia cell line MOLT-4 irradiated by X-ray were subjected to flow cytometric analysis of apoptosis And a new fluorescence labeled inhibitor of Caspase (FLICA) was used to detect Caspase activity in situ in individual cells and apoptosis index were compared with that of subG1, Annexin V/PI analysisResults Caspase 3 was activated 2 h after X-ray detected by FLICA/PI method And the apoptosis index increased on the function of time It was 2 hours earlier for FLICA/PI than Annexin V/PI to identify cell apoptosis Apoptosis index detected by subG1 was lower than both of FLICA/PI and Annexin V/PI Conclusion MOLT-4 cells undergo apoptosis after irradiated by X-ray And apoptosis index increased on the function of time Caspase 3 activation is earlier than cell membrane reverse and DNA fragmentation FLICA/PI method for detecting early apoptosis is more sensitive and directly than others