低剂量率γ射线杀伤肿瘤细胞机制的实验研究

Study on the anti-cancer mechanisms of low dose-rate γ irradiation

用60Co源以1Gy/min剂量率照射Hela细胞,剂量分别为1、2、5、10、15Gy。用AnnexinV和PI双染法观察凋亡细胞形态;DNA梯形条带证实凋亡存在;克隆形成分析细胞增殖能力。结果显示:(1)Hela细胞凋亡率随照射剂量和时间的增加呈上升趋势,照射后168h组各剂量点凋亡率高于其他各时间组。2Gy以下时凋亡率改变不大,达5Gy时凋亡率显著增加,且达峰值(72.57±2.04)%(P<0.001)。(2)早期凋亡细胞,PS外翻,胞膜呈绿色荧光圈。凋亡晚期出现AnnexinV-FITC及PI染色均阳性的外绿内红的细胞图像。坏死细胞则为红色。(3)凋亡细胞碎片呈“梯状”条带。(4)1Gy/min的剂量率照射,剂量为2—15Gy,克隆形成率由(58.95±0.36)%降至(1.67±0.35)%(P<0.001)。表明低剂量率γ射线照射可诱导Hela细胞凋亡,其凋亡率与照射剂量相关,在5Gy时凋亡率最高。

To investigate appropriate dose range in low-dose-rate γ irradiation and to establish a useful method for the research of radioactive biological effect produced by low does-rate γ irradiation, we have studied apoptosis of Hela carcinoma cells induced by different low dose rates of y irradiation in vitro. The cultured cells were exposed to defined radiation doses from a ^60Co therapy source. The radiation source delivered a dose rate of 1Gy/min and gave total dose of 1, 2, 5, 10 and 15 Gy respectively. Using Annexin V labeled by fluorescein and PI, followed by flow cytometry and DNA ladder analysis and confocal microscope, apoptotic cells were detected and quantified. The results are as follows： （1） Determination of apoptosis by flow cytometry indicated that percentage of apoptofic ceils was increasing with dose and time. When cells were cultured for 168 h after irradiation, the highest percentage of apoptosis was yielded in all groups. When the cells were irradiated with 1-2 Gy, no significant increase in the number of apoptotic ceils could be seen. After a total dose of 5 Gy, however, a significant increase in the ratio of apoptotic/nonapoptotic cells was observed, and apoptosis reached a peak level of （72.57±2.04）% （P 〈0.001）. （2） The typical appearance of Hela cells stained by annexin V-Fluos for detection of apoptosis exhibited a plasma mererahe-localized fluorescence that is an early sign of apoptosis under laser confocal microscope. PI entered into the cells slowly while in later apoptosis, with special laser which were red in center and green around, however, necrotic cell only emitted red laser. （3）The presence of apoptotic cells were confirmed by the DNA ladder formation assay, with typical formation of apoptotic fragments. DNA laddering reached the greatest after a single radiation dose of 5 Gy. （4） Over the dose interval from 2 to 15 Gy with a dose rate of 1 Gy/min, the survival of the cells decreased from （58.95±0.36）% to （1.67±9.35）% （P 〈0.001） by clonogenic assay. It was concluded that the low-dose, low-dose-rate γ irradiation delivered by ^60Co sources to Hela cells can induce a significant apoptosis rate, and a maximum level of apoptosis as high as （72.57±2.04）% for an irradiation dose of 5Gy can be reached.