离子辐射通过caspase途径而非p53途径诱导Jurkat细胞凋亡

目的:研究离子辐射诱导Jurkat细胞凋亡的动态趋势及其机制。方法:分别以5、10、20Gy的辐射量照射Jurkat细胞,其中一份样本在照射前加入zVAD.fmk,在照射后培养6、12、24、36和48h收获细胞,应用AnVPI双染和呈现subG1峰技术,在流式细胞仪上定量测定凋亡细胞。采用WesternBlot技术检测p53蛋白的表达。结果:细胞受照射后,凋亡细胞数随培养时间的延长和辐射量的加大而增加。在6h,只有20Gy才诱导细胞显著凋亡(P<0.005),5Gy的照射要到24h才出现明显的凋亡(P<0.05)。在48h,20Gy照射诱导的凋亡细胞数是所有时间点和剂量强度中最高的(P<0.001)。加有zVAD.fmk的细胞,尽管都是10Gy照射,比未加zVAD.fmk的细胞表现出凋亡细胞明显减少(P<0.005),但与未加zVAD.fmk、照射量是5Gy的细胞相比,凋亡细胞数仍显著增加(P<0.005)。Jurkat细胞在照射前后都没有p53表达。结论:离子辐射诱导细胞凋亡呈现出时间和剂量效应关系;capase抑制剂zVAD.fmk部分抑制离子辐射诱导细胞凋亡;离子辐射诱导细胞凋亡的机制独立于肿瘤抑制基因p53,caspase可能在其中起重要作用。

p53-independent, caspase-involved apoptosis by ionizing radiation in Jurkat cells

Objective To investigate the apoptosis pattern induced by ionizing radiation and its possible mechanisms. Method Jurkat cells were irradiated by ionizing radiation with doses of 5,10,20 Gy, of which one sample was pretreated with zVAD.fmk prior to irradiation. As soon as the cells were harvested at 6,12,24,36,48 hours, apoptosis was quantified with AnV/PI staining and presence of sub-G1 on flow cytometry. p53 expression was detected with Western blot. Results Induction of apoptosis presented increasd along with the culture time prolonging and the dose of radiation increasing. At 6 hours, only dose of 20 Gy significantly induced apoptosis (P<0.005), while dose of 5 Gy did not develop significant induction of apoptosis until 24 hours (P<0.05). At 48 hours, apoptosis induced by dose of 20 Gy was the highest among all time points and intensities (P<0.001). Comparison of the cells with zVAD.fmk and cells without zVAD.fmk under the same dose of 10 Gy showed at 12,24,36,and 48 hours demonstrated that zVAD.fmk significantly inhibited induction of apoptosis (P<0.005), but induction of apoptosis in cells with zVAD.fmk by dose of 10 Gy was significantly higher (P<0.005) than that in cells without zVAD.fmk by dose of 5 Gy at 24,36 and 48 hours. Absence of p53 expression was found in Jurkat cells before and after irradiation. Conclusion Ionizing radiation induces apoptosis in a time- and dose-dependent fashion. zVAD.fmk partly inhibits induction of apoptosis by irradiation. The mechanism,independent of the tumor suppressor gene p53,involves of caspase activation.