碳离子辐射对人肝癌细胞SMMC-7721细胞周期和P53、MDM2及P21表达的影响

研究^12C^6＋离子辐照对体外培养人肝癌细胞SMMC-7721细胞周期和P53、MDM2及P21表达的影响。采用0、1．0、2．0、4．0、6．0Gy^12C^6＋离子束辐照细胞,用克隆形成法观察细胞存活情况;同时在辐照24h后用流式细胞仪检测细胞周期的变化,Western-blot检测细胞中P53、MDM2及P21蛋白表达情况。结果发现,重离子辐照后细胞存活率显著下降;1．0Gy、4．0Gy和6．0Gy照射组发生G0／G1期阻滞,而2．0Gy照射组出现G2／M期阻滞;Western-blot结果显示细胞辐照后MDM2的57kD蛋白表达水平无明显变化,而76kD蛋白表达水平随辐照剂量逐渐上升;P53和P21蛋白表达水平随辐照剂量增高。以上结果提示不同剂量的^12C^6＋离子束照射可激活SMMC-7721细胞不同的细胞周期检测点,其中G0／G1期阻滞与P53和P21蛋白以及MDM2截短体76kD蛋白的表达水平升高有关。     

Potential mechanisms involved in resistant phenotype of MCF-7 breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage, carbon ion irradiation resulted in an approximately 1.9-2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might, at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.     

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation.     

X射线低剂量多次辐射对小鼠外周血淋巴细胞的影响

为了评估低剂量多次辐射对健康机体产生的生物学风险,检测了X射线多次照射对BALB/c小鼠免疫系统的影响.采用X射线全身多次照射,第1天照射0.07 Gy,之后每天照射0.08 Gy共12 d,剂量率0.2 Gy/min,累积剂量为0、0.07、0.23、0.39、0.55、0.71、0.87和1.03 Gy时取样.照射24 h后取血,用流式细胞仪检测外周血中免疫细胞周期和死亡的变化.结果表明,外周血淋巴细胞的周期在0.39和1.03 Gy时,被阻滞在G2/M期,在0.07、0.23、0.71和0.87Gy时,被阻滞在G0/G1期;细胞的死亡随着累积剂量的增加而增加,在0.71Gy时达到峰值.经过X射线多次全身照射小鼠后,可引起外周血淋巴细胞周期和死亡比例发生变化,符合线性平方模型,造成其免疫系统一定的损伤.     

The role of autophagy in cell survival from heavy ion irradiation in the plateau region

To study cytotoxic effect of heavy ion irradiation in the plateau region, and investigate whether autophagy in- duced by heavy ion irradiation is cytoprotective, HeLa cells were irradiated with 350 MeV/u carbon ions beams, and the clonogenic survival was analyzed. The results showed that cell survival decreased with increasing doses. It was also found that G2/M-phase cells increased, and the autophagy-related activity was significantly higher than the control. When autophagy was blocked by 3-methyladenine in carbon-ion irradiated cells, G2/M phase arrest and the percentage of apoptosis cells were further elevated, and cell survival decreased significantly, in- dicating the induction of cytoprotective autophagy by carbon-ion irradiation. Our results demonstrated that autophagy induced by carbon ion irradiation provided a self-protective mechanism in HeLa ceils, short-time inhibition of autophagy before carbon-ion irradiation could enhance radiation cytotoxicity in HeLa cells.     

重离子诱导人舌鳞癌细胞凋亡和Bax/Bcl-2蛋白表达的变化

探讨重离子辐照对人舌鳞癌Tb细胞的凋亡及Bax/Bcl-2蛋白表达的影响.采用0、0.5、1.0、2.0、4.0 Gy重离子束辐照人舌鳞癌Tb细胞,应用MTT法检测细胞存活,流式细胞技术检测细胞周期变化,Hoechst 33258/PI复染法观察Tb细胞凋亡形态,并采用Western-blot法检测Bax/Bcl-2蛋白表达情况.结果发现,Tb细胞经12C6+离子束辐照后存活率显著下降,呈剂量依赖性的生长抑制;Tb细胞呈现蓝色荧光浓集成团的凋亡形态,且凋亡比例随辐照剂量增加;G2/M期细胞百分数随照射剂量增加而增加(P＜0.05).Western-blot结果显示Bax蛋白表达水平随辐照剂量逐渐上升,但在4 Gy组其表达不再增高,Bcl-2蛋白在1.0、2.0、4.0 cy组随剂量增大呈下降趋势.以上结果提示重离子束辐照对Tb细胞有抑制作用,Bax/Bcl-2蛋白表达是重离子治癌的机制之一.     

12C^6＋离子束诱导HeLa细胞DNA损伤效应

本文研究了HeLa细胞经过12C^6＋离子柬辐照之后的DNA损伤效应,及辐照后p53激活的分子机制。运用中性单细胞电泳技术,检测了HeLa细胞经过4Gy12C6＋离子束辐照间隔0、3、6和12h之后DNA的损伤情况,及0.5、1、2和4Gy12C^6＋离子束辐照后即时的DNA损伤情况。同时运用细胞生长实时监测仪监测了HeLa细胞在经过0、0．5和1Gy也C6＋离子束辐照之后的生长变化,并运用AO／EB双染检测了辐照细胞24h后的凋亡情况。另外,利用8mmol／L的咖啡因[抑制ATM（ataxia-telangiectasia,mutated）和ATR（ATMand Rad3．relatedkinase）1和20μmol／L的wortmannin[抑制ATM和DNA—PK（DNA-dependent protein kinase）】处理HeLa细胞后再进行1Gy12C^6＋离子束辐照,通过westernblot检测p53的表达。结果显示,12C^6＋离子束辐照可造成HeLa细胞的DNA损伤,损伤随剂量升高而升高但随测定间隔时间降低,诱导HeLa细胞发生凋亡;而且辐照后p53表达升高。结果证明12C^6＋离子束辐照可造成HeLa细胞的DNA损伤并诱导损伤修复及凋亡等效应,损伤效应相关因子p53被激活,并且激活依赖于ATM。     

硫酸镁对人脐静脉血管内皮细胞的辐射防护作用

对数生长期的人脐静脉血管内皮细胞(Human umbilical vein endothelial cells,HUVECs)分3组进行不同处理后采用流式细胞仪检测细胞周期分布;Annexin V/PI双染法测定细胞凋亡率;Western blot检测Bcl-2蛋白的表达情况;RT-PCR法检测Bcl-2 mRNA的表达情况。结果显示,硫酸镁可以在X-射线照射24 h后改善HUVECs细胞G2/M期阻滞;在辐照后24、48、72 h可以提高细胞对X-射线的辐射敏感性,并增加细胞凋亡率(t=4.24、7.19、3.20,p0.05);在辐照后48、72 h能明显抑制抗凋亡蛋白Bcl-2蛋白的表达;照后24、48、72 h的Bcl-2 mRNA表达量减少(t=3.034、6.182、3.957,p0.05)。结果提示,硫酸镁可以缓解X-射线照射后细胞G2/M期阻滞,增加照射后HUVECs的细胞凋亡率,降低X-射线诱导的抗凋亡蛋白Bcl-2的表达。     

电离辐射诱导EL-4淋巴瘤细胞G_1期阻滞及相关蛋白的表达

探讨X射线诱导EL 1细胞G1期阻滞及相关蛋白的表达。采用PI荧光标记 ,流式细胞术检测细胞周期的变化。用单克隆抗体免疫荧光标记 ,流式细胞术检测蛋白表达的变化。结果表明 ,2 0Gy和 4 0Gy照射后 12 72h ,G1期EL 4细胞百分数显著高于假照射组 ( p <0 0 5- p <0 0 0 1)。 4 0Gy照射后EL 4细胞 p53蛋白表达从照射后 2h开始明显增高 ,持续至照射后2 4h ( p <0 0 5- p <0 0 0 1) ;p2 1蛋白表达在照射后 2h开始明显增高 ,持续至照射后 4 8h ( p <0 0 5- p <0 0 0 1) ;GADD4 5蛋白表达在照射后 2h开始明显增高 ,持续至照射后 4 8h ( p <0 0 5- p <0 0 0 1) ;MDM 2蛋白表达在照射后 4h开始明显增高 ,持续至照射后 2 4h ( p <0 0 5- p <0 0 0 1)。结果提示 ,中等剂量X射线照射可诱导EL 4细胞G1期阻滞。p53、p2 1和GADD4 5蛋白表达在电离辐射诱导EL 4细胞G1期阻滞中起重要作用     

电离辐射对小鼠骨髓造血细胞周期进程的影响

采用流式细胞术（ＦＣＭ）观察了Ｘ射线全身照射后小鼠骨髓造血细胞周期进程的时程变化和剂量效应关系,为探讨电离辐射对小鼠骨髓细胞周期进程的影响。结果显示,２ＧｙＸ射线全身照射后１２ｈ,骨髓细胞出现Ｇ１期细胞百分数升高（ｐ〈０．０５）,４ｈＳ期细胞百分数下降（ｐ〈０．０５）,Ｇ２＋Ｍ期细胞百分数于后４ｈ和１２ｈ升高（ｐ〈０．０５）。量效研究显示,０．５～６Ｇｙ照射后１２ｈＧ１期细胞百分数高于对照组,其中     

电离辐射对HelaS3细胞凋亡与细胞周期的影响

研究了电离辐射对HelaS3细胞凋亡和细胞周期的影响,进一步探讨细胞凋亡与细胞周期的关系,为肿瘤放化疗方案的制定提供基础资料。采用PI、Hoechst33342双梁和PI单梁,用流式细胞术检测细胞凋亡和细胞周期。结果表明,给予0.5-4.0Gy X射线照射后8h细胞凋亡开始增加,12h达最大值,12－24h有下降趋势,但24h开始又逐渐增加,到72h时又接近12h水平。S期和G2／M期细胞均明显增加,并呈剂理依赖性,Go/G1期细胞则呈剂量依赖性减少,且这种变化在受照射后24h最明显。说明一定剂量电离辐射可以诱导明显的G2/M阻滞和S期阻滞,并可诱导HelaS3细胞凋亡的产生,两者之间有一定的关系。     

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of “Tail DNA (%)” (TD) and “Olive tail moment” (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radiosensitivity.     

80MeV/u 12C6+离子辐照小鼠头部对骨髓细胞周期分布的影响

研究重离子辐照小鼠头部对骨髓细胞周期分布的影响,为重离子放射治疗癌症和太空防护提供基础数据.80MeV/u能量的12C6 离子对BALB/c小鼠头部给以0、0.5、1、2、4、10Gy的照射,用流式细胞仪测骨髓细胞周期分布.随着重离子辐照剂量的增加,G1/G0期细胞出现明显阻滞(P＜0.05),而G2/M期细胞出现显著减少(P＜0.05).说明重离子辐照小鼠头部对小鼠骨髓细胞周期分布有明显影响,也同时表明电离辐射对骨髓细胞周期分布的影响也有一种间接作用.     

硝普钠对人A172胶质瘤细胞X射线诱导细胞周期改变的影响

Nitric oxide (NO) has been implicated both in regression and progression of tumors due to its production by both tumor cells and infiltrating leukocytes. Ionizing radiation causes the regression of tumors, and can augment the production of NO by macrophages in vitro. The authors investigated the effect of X-ray irradiation on cell cycle of A172 human glioma cells pretreated with nitroprusside sodium (NPS), the NO donor.Human glioma A172 cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactive fetal calf serum in a 5 % CO2 incubator at 37 ℃. The experiment was performed in exponentially growing cultures.The cells in cell culture flasks treated with or without 1 mM NPS for 4 h. were irradiated to a dose of 4 Gy X-rays using 8MeV CL 2100C electron linear accelerator (Varian, USA) in General Hospital of Lanzhou Military Area.After the irradiation, the cells were collected by trypsinization, washed with phosphate buffer solution, f1xed in 70 % ethanol, treated with RNase and the nuclei were stained with propidium iodide. The DNA content was evaluated using Becton Dickinson FACSCalibur and analyzed with UMCM cylchred cell cycle analyzer software.The cell culture medium was replaced after irradiation and cells were harvested 24h later, labeled with propidium iodide and analyzed by flow cytometry. After the 4 Gy X-ray irradiation, the cell cycle distribution of A172 glioma cells changed markedly, with increased percentage of G2/M cells and decreased of G0/G1 and S cells compared to control cells(Fig. 1 a, lb). The cells pretreated with 1 mM NPS 4 h displayed similar cell cycle distributions to those of the control (Fig. lc). But X-ray irradiated cells preincubated with 1 mM NPS showed different cell cycle distributions from those of unpretreated cells (Fig. 1 d).It was reported that NO or NO release agents were as effective as oxygen to radiosensitize hypoxic cells in vitro. It was also revealed that NO involved in the resistance to ionizing radiation by by-stander effects. The results in the present research demonstrated that NO donor inhibit A 172 glioma cell undergo cell cycle arrest following X-ray irradiation. Several studies have shown that p53 protein accumulates in the nuclei of cells exposed to ionizing radiation. Whether impairing p53 function involved in prevention of NO to cell cycle arrest induced by X-ray remained further investigation.     

80MeV/u 12C6+离子辐照小鼠头部对脾脏细胞周期分布的影响

研究重离子辐照小鼠头部对脾脏细胞周期分布的影响，为重离子放射治疗癌症和太空防护提供基础数据。80MeV／u能量的^12C^6＋对BALB／c小鼠头部给以0、0．5、1、2、4、10Gy的照射，用流式细胞仪测脾脏细胞周期分布。重离子辐照后36h，小鼠脾脏细胞S期细胞随着辐照剂量的增加显著减少（p〈0．05）；0．5Gy组、4Gy组和10Gy组出现G0／G0期阻滞明显阻滞（p〈0．05），1Gy组和2Gy组无显著变化（p〉0．05）；0．5Gy组G2／M期细胞显著减少（p〈0．01），其它剂量组明显阻滞（p〈0．05）。重离子辐照小鼠头部对小鼠脾脏细胞周期分布有明显影响。     

不同LET辐射对HepG2肝癌细胞的辐射敏感性与周期阻滞的影响

本工作研究不同LET射线辐照对HepG2肝癌细胞辐射敏感性、周期进程和凋亡的影响,为重离子治疗癌症的临床应用积累基础数据.以0、0.5、1、2、4、8Gy剂量的12C6 离子及X射线分别照射处于指数生长期的HepG2细胞,用克隆形成率测定细胞辐射敏感性,通过流式细胞术测定细胞DNA含量以确定各时相细胞的比例及细胞凋亡情况.实验结果显示,12C6 离子辐照所致的HepG2细胞存活率明显低于X射线.随着吸收剂量的增加和修复时间的延长,12C6 离子能导致更显著的细胞S期阻滞、G2/M期阻滞延迟和细胞凋亡.说明与X射线相比,12C6 离子辐照能更有效地杀伤HepG2肝癌细胞并诱导其凋亡.     

MG132联合X射线对非小细胞肺癌生长转移和凋亡的影响

探讨蛋白酶体抑制剂MG132联合X射线对非小细胞肺癌H1299细胞生长、转移侵袭和细胞凋亡的影响及机制。采用MTT法检测不同MG132浓度不同时间处理后肺癌H1299细胞的增殖;Transwell小室实验测定肺癌细胞的迁移和侵袭能力;流式细胞术测定肺癌细胞的凋亡;Western—blot法测定蛋白表达水平。结果表明,MG132能明显抑制H1299细胞的生长,并呈现剂量-效应和处理时间-效应关系;MG132在无毒性剂量下联合X射线可显著抑制H1299细胞的迁移及侵袭能力,并明显诱导细胞凋亡;MG132能明显降低H1299细胞的基质金属蛋白酶-2、-9的表达水平,降低抗凋亡蛋白Bcl-2的表达水平,同时增加凋亡蛋白Bax的表达水平。提示MG132可以显著抑制人非小细胞肺癌H1299细胞的生长,且在无毒性剂量下联合X射线可以明显降低癌细胞转移和侵袭能力,能显著加强X射线诱导的细胞凋亡作用。     

电离辐射联合顺铂对人肺癌细胞中BTG3基因表达的影响

加速器产生的6-MV X-射线一次性照射体外培养的人肺腺癌细胞株A549和SPCA-1,细胞的吸收剂量分别设置为0、1、3、5和8 Gy,细胞照射后24 h及单次接受5 Gy照射后6、12和24 h后取样;X-射线联合顺铂对BTG3表达的影响研究设立单纯照射组(5 Gy)、顺铂处理组(20μmol·L-1顺铂)和联合处理组(5 Gy+20μmol·L-1顺铂),上述实验均以未给予任何处理的细胞为对照组;采用Western blot及RT-PCR法检测受到不同剂量电离辐射及照射后不同时间肿瘤细胞中BTG3表达水平。Western blot与PCR检测结果均显示,A549和SPCA-1细胞在受到不同剂量的X-射线照射后24 h,其BTG3蛋白及mRNA表达量均明显增加,并随电离辐射剂量的增加而增加,与对照组比较有统计学差异(t=5.25-15.75,p0.05);照射后不同时间检测结果显示,细胞在X-射线照射4 h后BTG3蛋白及mRNA的表达量即开始上升,并持续到照射后24 h,与照射前相比有统计学差异(t=7.52-11.18,p0.05);联合处理组BTG3的表达量均明显高于单纯照射组、顺铂处理组和对照组(t=7.02-15.86,p0.05)。电离辐射联合顺铂可以上调肺癌细胞株中BTG3的表达,BTG3基因有可能作为肺癌放化疗的靶基因。