放射诱导HL-60细胞凋亡及其与克隆存活率的关系

RADIATION-INDUCED HL-60 APOPTOSIS AND RELATIONSHIP BETWEEN APOPTOSIS AND LOSS OF CLONOGENICITY

目的研究放射诱导的ＨＬ－６０细胞凋亡及其与放射后细胞克隆存活率的关系。方法应用电子显微镜、琼脂糖电泳、流式细胞术和ＴｄＴ介导的缺口和末端标记法（ＴＵＮＥＬ）等方法分析放射诱导ＨＬ－６０细胞凋亡，应用有限稀释法测定放射后细胞克隆形成率。结果放射后ＨＬ－６０细胞表现出细胞体积缩小、染色质固缩致密化、边缘化、染色体断裂、出现凋亡小体、细胞膜保持完整和电泳时ＤＮＡ“梯谱”等典型的细胞凋亡特征。放射诱导ＨＬ－６０细胞凋亡有明显的时间、剂量效应，照射后４ｈ细胞凋亡已比较明显，放射诱导ＨＬ－６０细胞凋亡随着照射剂量增加而增加。但是，放射引起ＨＬ－６０细胞克隆存活率的减少比放射诱导ＨＬ－６０细胞凋亡更敏感，如８Ｇｙ照射后克隆存活率减少到０．００１６，而此剂量时的细胞凋亡水平几乎与对照组水平相同。结论放射能诱导ＨＬ－６０细胞凋亡，并有明显的时间、剂量效应关系。但是，放射诱导ＨＬ－６０细胞凋亡并不能解释ＨＬ－６０细胞克隆存活率的减少。

URPOSE To detect radiation-induced HL-60 apoptosis and explore the relationship between apoptosis and loss of clonogenicity.METHODS Light microscope, electron microscope, agarose gel electrophoresis and flow cytometry (FCM) were used to detect both qualitatively and quantitatively radiation-induced HL-60 apoptosis. Limiting dilution assay was performed to determine the HL-60 cell colony forming efficiency (CPE).RESULTS The irradiated HL-60 cell exhibited condensation of the cytoplasm, chromatin condensation, chromosal clumping and margination, apoptotic body and the nuclear DNA degradation with characteric “DNA ladder” subjected to electrophoresis. Radiation-induced HL-60 apoptosis showed a time-dose response curve with most apparent apoptosis at 4h after irradiation. The induced apoptotic cell number increaed with dose. However, HL-60 cell were much more sensitive to radiation-induced loss of clonogenicity than to induction of apoptosis at 6h, e.g. 8Gy radiation reduced the surviving fraction to 0.0016 in a clonogenic assay, wheras the irradiation induced apoptotic cell number was nearly the same as the control.CONCLUSIONS Our data show irradiation can induce HL-60 apoptosis, but the apoptotic cell number is not able to account for its radiation-induced loss of clonogenicity.