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目的 :以60 Coγ射线为参比射线 ,研究裂变中子诱导小鼠胸腺细胞凋亡的时间效应 ,并初步探讨其分子机制。方法 :用形态学观察、DNA电泳、流式细胞仪 (FCM )分析及二苯胺 (DPA)法检测细胞凋亡 ;用斑点杂交方法检测 p53与bcl 2的基因表达。 结果 :小鼠经裂变中子 2 .5Gy照射后 2～ 2 4h ,在各时间点上均检测到胸腺细胞DNA梯状条带 ,利用光镜与电镜观察到具有典型凋亡特征的胸腺细胞 ;以DPA法、FCM分析与形态观察计数测定的结果 ,绘制时间效应曲线 ,发现胸腺细胞凋亡比例随着照射时间的延长而增加 ,在 6h前上升较快 ,8～ 10h达到峰值 ,12h后开始下降 ,2 4h较 4～ 12h明显降低 (P <0 .0 5) ,但略高于正常水平。γ射线 5.0Gy照射后 ,胸腺细胞凋亡比例随时间的变化规律与之相似 ,但在 6h之前增加较缓 ;此外 ,在照后 2 4h检测不到DNA梯状条带。裂变中子 2 .5Gy照射后 ,小鼠胸腺细胞 p53基因表达水平在 2、4、12、2 4h均比未照射组有显著增加 (P <0 .0 5或P <0 .0 1) ;bcl 2基因表达水平均比未照射组明显降低 (P <0 .0 1)。结论 :裂变中子 2 .5Gy照射小鼠可诱导其胸腺细胞凋亡 ,且与γ射线 5.0Gy照射有类似的时相性 ,但裂变中子诱导的胸腺细胞凋亡比γ射线增加快、持续时间长 ,表明裂变中子对小鼠免疫系统损伤较
OBJECTIVE: To study the time effect of fission neutrons on the apoptosis of thymocytes induced by fission neutrons in mice by using 60 Coγ-ray as reference radiation and to explore the molecular mechanism. Methods: Apoptosis was detected by morphological observation, DNA electrophoresis, flow cytometry (FCM) and diphenylamine (DPA). The gene expression of p53 and bcl2 was detected by dot blot hybridization. Results: Thymocytes DNA ladder bands were detected at 2 ~ 24 hours after irradiation with 2.5G fission neutrons in mice. Thymocytes with typical apoptotic features were observed by light and electron microscopy. The results of DPA method, FCM analysis and morphometric observation were used to draw the time-effect curve. It was found that the proportion of apoptotic thymocytes increased with the increase of irradiation time, rising rapidly 6h before reaching the peak at 8-10h and beginning at 12h Decreased 24 h compared with 4 ~ 12 h (P <0.05), but slightly higher than the normal level. The proportion of apoptotic cells in thymocytes changed with time after γ-rays irradiation at 5.0Gy, but increased slowly before 6h. In addition, no DNA ladder was detected at 24 hours after irradiation. After irradiated with 2.5G fission neutrons, the expression of p53 gene in mouse thymocytes increased significantly at 2, 4, 12, and 24 hours (P <0.05 or P <0.01). bcl 2 gene expression levels were significantly lower than the non-irradiated group (P <0.01). CONCLUSION: Mice exposed to 2.5G fission neutrons can induce apoptosis of thymocytes and have a similar phase-contrast to γ-rays irradiated at 5.0Gy, but the apoptosis of thymocytes induced by fission neutron is faster than that of γ-rays, and the duration Long, indicating that fission neutrons on the immune system damage in mice