990 resultados para G2


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本实验旨在研究电离辐射对不同肿瘤细胞细胞周期进程的影响,为肿瘤的放射治疗提供科学依据。以3Gyγ射线照射指数生长期的肿瘤细胞,用流式细胞术测定细胞DNA含量并确定细胞周期各时相的细胞百分比。实验结果显示,γ射线照射后,SMMC-7721,hepG2和HO-8910细胞的G2/M期发生明显阻滞,且都在辐射后12h累积达到最大值,但S期只发生短暂的延迟,即辐射后6h明显累积,之后下降至接近对照水平或低于对照;Hela细胞的G2/M期和S期均发生显著阻滞。实验结果说明,电离辐射后,不同肿瘤细胞的G2/M期和S期检查点的激活和维持是有差异的。

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利用早熟染色体凝集技术预测研究了γ射线对肝癌细胞SMMC7721的辐射效应。结果表明,G1和G2期细胞内的染色单体和等点染色单体断裂数与照射剂量之间存在线性相关性,染色单体断裂总数与细胞存活率之间存在良好的线性相关性。说明辐射诱导的染色单体断裂可以作为预测SMMC7721细胞内在辐射敏感性的指标,也可为临床诊断和治疗肝癌提供依据。

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Human tumor cell lines of SMMC-7721 (liver cancer), hepG2 (liver cancer), HO-8910 (ovary cancer), and Hela (cervix cancer) were irradiated to 3Gy by Co γ rays and different cell cycle responses were found. The re- 60 sults showed that the SMMC-7721, hepG2 and HO-8910 cells displayed G2 / M phase arrest and S phase tempo- rally delayed after the irradiation. The Hela cells had an increased number of cells in both G2 / M and S phase, which indicated that the G2 / M and S checkpoints were both activated.

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以人肝癌细胞 hepG2 为研究对象,用流式细胞术测定并分析了低剂量 γ 射线(5cGy)预照射对高剂量照射(3Gy)诱导的细胞周期阻滞的影响。结果显示:(1)低剂量照射可引起 hepG2 细胞在 G2/M 期短暂累积(至照射后 4h);(2)低剂量照射促进 hepG2 细胞的生长;(3)高剂量照射后,hepG2 细胞的 G2期发生阻滞,而 S 期只发生短暂延迟;(4)与单纯高剂量照射相比,低剂量照射预处理后 4h 给予高剂量照射可进一步促进hepG2 细胞在 G2/M 期累积,但预处理后 8h 给予高剂量可促进细胞通过 G2/M 期。实验结果表明,低剂量照射预处理对高剂量照射诱导的细胞周期阻滞的影响,取决于两次照射的时间间隔。

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实验研究了低剂量γ射线预照射对人肝癌细胞系hepG2和人宫颈癌细胞系HeLa的细胞周期进程的影响.结果显示(1)低剂量(5cGy)辐射后,两种细胞的G2/M期细胞短暂累积;(2)低剂量辐射促进肿瘤细胞的生长;(3)高剂量(3Gy)辐射后,hepG2细胞发生G2期阻滞,HeLa细胞发生S期和G2期阻滞;(4)与单纯高剂量照射相比,低剂量辐射预处理后4h,再给予高剂量辐射,可进一步促进hepG2细胞在G2/M期累积,但是预照射对HeLa细胞的周期进程没有明显影响.因此,低剂量辐射预处理对高剂量诱导的细胞周期阻滞的影响依赖于肿瘤细胞的类型.

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This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/ m), and were then stimulated to obtain dividing cells. PBLs were treated with 100nMcalyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy−1. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.

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In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u(12)C(6+) ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to C-12(6+) ions.

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The purpose of this paper is to prepare for an easy and reliable biodosimeter protocol for radiation accidents involving high-linear energy transfer (LET) exposure. Human peripheral blood lymphocytes were irradiated using carbon ions (LET: 34.6 keV mu m(-1)), and the chromosome aberrations induced were analyzed using both a conventional colcemid block method and a calyculin A induced premature chromosome condensation (PCC) method. At a lower dose range (0-4 Gy), the measured dicentric (dics) and centric ring chromosomes (cRings) provided reasonable dose information. At higher doses (8 Gy), however, the frequency of dics and cRings was not suitable for dose estimation. Instead, we found that the number of Giemsa-stained drug-induced G2 prematurely condensed chromosomes (G2-PCC) can be used for dose estimation, since the total chromosome number (including fragments) was linearly correlated with radiation dose (r = 0.99). The ratio of the longest and the shortest chromosome length of the drug-induced G2-PCCs increased with radiation dose in a linear-quadratic manner (r = 0.96), which indicates that this ratio can also be used to estimate radiation doses. Obviously, it is easier to establish the dose response curve using the PCC technique than using the conventional metaphase chromosome method. It is assumed that combining the ratio of the longest and the shortest chromosome length with analysis of the total chromosome number might be a valuable tool for rapid and precise dose estimation for victims of radiation accidents.

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Aim: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines. Methods: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique. Results: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to gamma-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r=0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant. Conclusion: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

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To investigate the protective effects of melatonin against high-LET ionizing radiation, V79 Chinese hamster cells were irradiated with 100 keV/mu m carbon beam. Parallel experiments were performed with 200 kV X-rays. To avoid the impact from extra solvents, melatonin was dissolved directly in culture medium. Cells were cultured in melatonin medium for 1 hr before irradiation. Cell inactivation was measured with conventional colony forming assay, medium containing 6-thioguanine was used for the selection of mutants at hprt locus, and the cell cycle was monitored by flow cytometry. Both carbon beam and X-rays induced cell inactivation, hprt gene mutation and cell cycle G2 block dose-dependently. But carbon beam showed stronger effects as indicated by all three endpoints and the relative biological effectiveness (RBE) was 3.5 for cell killing (at 10% survival level) and 2.9 for mutation induction (at 5 x 10(-5) mutants/ cell level). Melatonin showed protective effects against ionizing radiation in a dose-dependent manner. In terms of cell killing, melatonin only increased the survival level of those samples exposed to 8Gy or larger of X-rays or 6 Gy or larger of carbon beam. In the induction of hprt mutation and G2 block, melatonin reduced such effects induced by carbon beam but not by X-rays. The results suggest that melatonin reduces the direct interaction of particles with cells rather than an indirect interaction. Further studies are required to disclose the underlying mechanisms.

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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. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

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探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.

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The influence of survivin expression on the radiosensitivity of tumor cells to high linear energy transfer (LET) radiation is investigated. Survivin-specific short-interfering RNA (siRNA) oligonucleotides were synthesized based on the survivin sequence provided by GenBank. Human hepatoma HepG2 cells were transfected with survivin-specific siRNA to inhibit its expressions. It was found that the transfection with surviving-specific siRNA increased the levels of G2/M arrest and the apoptotic rates induced by radiation in HepG2 cells. After exposure to high-LET carbon ions, a reduced clonogenic survival effect was observed in the cells treated with siRNA. These results show that survivin plays a key role in mediating the radioresistance of cells to high-LET radiation.

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肿瘤是严重威胁人类生命健康的常见病、多发病,不仅病因复杂、发生发展异常迅速,而且到目前为止,发病机理不完全清楚,尚无适应范围广和有特异疗效的治疗方法。因此,肿瘤治疗方法的探索依然是医学、生物学及其相关学科研究的热点。肿瘤的重离子治疗和基因治疗是近年来发展起来的新的肿瘤治疗方法。但它们同样或多或少存在一些不足。在肿瘤治疗方法的探索中,将两种或两种以上理化特性或生物学作用原理不尽相同的现有治疗方法有机结合,充分利用各自优势,取长补短,使治疗效果叠加,对肿瘤发挥协同或相加抑制作用。本研究将重离子辐射与p53腺病毒重组体(AdCMV-p53)转染有机结合,探讨了重离子辐射联合p53基因转导对肿瘤细胞的生物学作用及其可能机理。在低剂量γ辐射联合AdCMV-p53/GFP转染HT-29和PC-3细胞研究基础上,我们用不同剂量的AdCMV-p53/GFP转染经0.5 Gy、1.0 Gy、2.0 Gy 12C6+束/γ射线预辐射处理的人非小细胞肺癌(H1299细胞系,nullp53),人肝癌细胞(HepG2细胞系,wtp53)和人宫颈癌细胞(Hela细胞系,wtp53,wtp53低水平表达)。用流式细胞分析法检测肿瘤细胞绿色荧光蛋白(GFP)、p53蛋白表达水平和细胞周期。DAPI染色后用荧光显微镜检测细胞凋亡。用RT-PCR检测外源性p53转录。用Western Blot检测外源性p53、MDM2和p21蛋白表达。用克隆形成法测定肿瘤细胞存活。通过与γ辐射联合腺病毒重组体转染组比较,观察了12C6+ 辐射联合腺病毒重组体转染对肿瘤细胞外源性p53蛋白表达、细胞周期阻滞、细胞凋亡和细胞增殖的影响。结果显示,12C6+ 辐射对AdCMV-GFP转染H1299、HepG2和Hela细胞的诱导作用明显强于γ辐射(p<0.05)。与γ辐射诱导AdCMV-GFP转染组相比,0.5 Gy 12C6+束辐射联合20 MOI AdCMV-p53转染组H1299细胞GFP阳性率增加约50% (其GFP阳性率提高到约90%)。0.5 Gy、1.0 Gy 12C6+辐射联合40 MOI AdCMV-p53转染组HepG2细胞GFP阳性率增加约44%(其阳性率分别达56.6%和76.4%)。0.5 Gy、1.0 Gy 12C6+ 束辐射联合40 MOI AdCMV-p53转染组Hela细胞GFP阳性率分别增加37.8%和50%(其阳性率分别达43.4%和59.8%)。12C6+ 辐射对AdCMV-p53转染H1299、HepG2和Hela细胞外源性p53蛋白表达的增强作用明显强于γ辐射(p<0.05)。12C6+ 辐射联合AdCMV-p53转染组各种细胞p53阳性率明显高于其它处理组同种细胞p53阳性率(p<0.05)。转染后第5天,γ辐射联合AdCMV-p53转染组3种细胞p53阳性率均降至对照水平。转染后第13天,12C6+ 辐射联合AdCMV-p53转染组3种细胞p53阳性率仍高达6-44%。12C6+ 辐射联合AdCMV-p53转染H1299细胞G0/G1、G2/M期细胞所占比例明显高于其它处理组G0/G1、G2/M期细胞所占比例(p<0.05)。与γ辐射联合AdCMV-p53转染组相比,12C6+ 辐射联合AdCMV-p53转染组G0/G1期细胞增加6-36%,G2/M期细胞增加了13-86%。12C6+ 辐射联合AdCMV-p53转染HepG2细胞G0/G1期细胞所占比例明显高于其它处理组G0/G1期细胞所占比例(p<0.05);转染后第5天,1.0、2.0 Gy 12C6+ 辐射联合AdCMV-p53转染组G2/M期细胞所占比例明显高于γ辐射联合AdCMV-p53转染组G2/M期细胞所占比例(p<0.05)。各12C6+束辐射联合AdCMV-p53转染Hela细胞G0/G1和G2/M期细胞所占比例均明显高于单纯12C6+ 辐射组和γ射线辐射联合AdCMV-p53转染组G0/G1和G2/M期细胞所占比例(p<0.05)。各12C6+ 辐射联合AdCMV-p53转染H1299、HepG2和Hela细胞凋亡率明显高于等剂量12C6+ 单纯辐射和等剂量γ辐射联合AdCMV-p53转染组细胞凋亡率(p<0.05)。与等剂量单纯12C6+辐射和等剂量γ辐射联合AdCMV-p53转染组相比,12C6+ 辐射联合AdCMV-p53转染H1299细胞凋亡率分别增加8.0-66.0%和9.3-63.5%;12C6+束辐射联合AdCMV-p53转染HepG2细胞凋亡率分别增加0.8-32.7%和4.5-27.1%; 12C6+束辐射联合AdCMV-p53转染Hela细胞凋亡率分别增加4.8-30.7%和3.1-22.7%。低剂量12C6+ 辐射联合AdCMV-p53转染细胞存活率明显低于其它处理组同种细胞存活率(p<0.05)。结果提示,低剂量碳离子辐射对腺病毒重组体转染肿瘤细胞和靶细胞内外源p53蛋白表达的促进作用明显强于低剂量γ辐射。碳离子辐射联合AdCMV-p53转染通过促进外源性p53转导、靶细胞外源性p53蛋白表达、细胞周期阻滞和细胞凋亡等增强对肿瘤细胞的抑制。碳离子辐射联合AdCMV-p53转染对肿瘤细胞生物学作用与肿瘤细胞内在p53基因状态有关。总之,我们的研究表明,低剂量碳离子辐射联合AdCMV-p53转染,可通过促进腺病毒重组体对肿瘤细胞的转染、增强靶细胞外源性p53蛋白稳定表达及其由此而诱发的细胞周期阻滞与细胞凋亡等有效抑制肿瘤细胞。在临床上,碳离子辐射联合AdCMV-p53转染有望在提高肿瘤治疗效果的基础上,进一步降低碳离子辐射与AdCMV-p53转染的各自临床用量,减少碳离子辐射的毒副作用,降低AdCMV-p53转染的潜在生物危险性

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研究目的: 1.研究碳离子辐射对小鼠不同组织抗氧化酶活性及细胞周期进程的影响。 2.研究低剂量碳离子预辐射对离体培养的黑色素瘤B16细胞及正常小鼠诱导的适应性反应。 3. 研究褪黑素(MLT)对碳离子的辐射损伤防护效应。 4. 研究碳离子层叠法辐照对H22荷瘤小鼠的治疗效果。研究方法: 1.采用不同剂量的碳离子辐照小鼠,用黄嘌呤氧化酶法检测外周血、肝脏及脑组织中抗氧化酶活性变化,流式细胞仪检测细胞周期阻滞情况。 2.采用低剂量碳离子预辐射离体培养的黑色素瘤B16细胞及正常小鼠,间隔4h后再以攻击剂量辐照,常规组织切片染色观察各组织器官病理变化,流式细胞仪检测细胞周期阻滞情况,黄嘌呤氧化酶法检测小鼠胸腺、脾脏及B16细胞中抗氧化酶活性,Western-blot法检测胸腺细胞及B16细胞中P53及P21蛋白表达情况,RT-PCR法检测CHK2及CDC25mRNA的表达水平。 3.在碳离子辐照小鼠1h前腹腔注射褪黑素,单细胞电泳方法检测胸腺、脾脏细胞的DNA损伤情况,微核法表征外周血的染色体损伤,黄嘌呤氧化酶法测定胸腺、脾脏细胞的抗氧化酶活性。 4.以碳离子层叠法辐照H22荷瘤小鼠,统计不同剂量照射后的肿瘤体积变化、肿瘤抑制率、肿瘤生长延迟天数及治愈率。结果: 1、小鼠血清和肝脏组织在辐射剂量较低(≤0.75Gy)时SOD活性高于对照组,随着辐射剂量的增高,SOD活性趋于降低;MDA含量在辐射剂量较低(≤0.3Gy)时低于对照,随着辐射剂量的增高其含量趋于升高;脑组织GSH浓度在照射剂量较低(≤0.5Gy)时大于对照组,随着照射剂量的升高其浓度趋于降低;低剂量辐射小鼠引起胸腺G2期细胞比例增加,脾脏G1期细胞比例增加。 2、小鼠肝脏、脾脏、肺脏及脑组织在攻击剂量辐射后,出现明显的病理变化,低剂量预辐射处理后病理变化减轻;低剂量预辐射增加胸腺G2期细胞及脾脏G1期细胞比例;相对于单纯攻击剂量辐射组,低剂量预辐射组胸腺组织P53及P21蛋白表达升高;CHK2 mRNA水平升高,CDC25 mRNA水平降低;脾脏及胸腺组织中SOD活性降低程度减弱,MDA含量升高趋势减弱。B16细胞经低剂量预辐射处理后上述指标均未发生明显变化。 3、与辐照处理组相比,褪黑素处理后小鼠的脾脏胸腺细胞DNA损伤拖尾率及彗尾长度明显降低;SOD活性升高,MDA含量降低,外周血微核率降低。 4、在不同剂量的碳离子辐照处理后观察的12天内,各组肿瘤生长速度减慢,生长延迟,肿瘤抑制率随时间而增加。15Gy照射组肿瘤生长速度最慢,肿瘤抑制率最大,肿瘤生长延迟最为明显,而且肿瘤治愈率达到30%。结论: 1. 低剂量12C6+离子全身辐照小鼠应激激活机体抗氧化系统,随辐射剂量的增加,机体抗氧化能力明显降低,导致脂质过氧化发生;低剂量的碳离子辐射导致小鼠胸腺细胞G2期阻滞,脾脏细胞发生G1期阻滞。 2. 低剂量12C6+离子预辐射引发小鼠正常机体产生适应性反应,减轻随后的大剂量辐射造成的损伤;低剂量12C6+离子预辐射对小鼠黑色素瘤B16细胞未引发适应性反应。 3. 15mg/kg的MLT可以对小鼠的重离子辐射损伤产生明显的防护效果。 4. 12C6+离子适形治疗小鼠移植性肿瘤H22,荷瘤鼠的存活时间、肿瘤体积变化、肿瘤的控制率、治愈率等结果显示,15Gy为最佳治疗剂量