黄芪总黄酮对正常细胞和肝癌细胞的辐射防护差异性研究
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摘要
目的：本实验以川产膜夹黄芪(Astragalus)为原料,以乙酸乙酯改良萃取技术分离提取黄芪总黄酮(Total Flavonoids of Astragalus TFA),主要研究TFA对放射损伤人正常骨髓间充质细胞(human mesenchymal stem cells hMSCs)和人肝癌细胞HepG-2的不同放射防护效应；从DNA和蛋白水平上深入探讨TFA对正常细胞和肿瘤细胞放射防护作用差异性的分子机理。
方法：以肝癌细胞HepG-2和人体正常细胞hMSCs为实验对象,设置空白对照组(不照射,不给药),单纯照射组(只照射,不给药)TFA用药照射组(TFA处理+照射)三个实验组；当细胞生长至对数生长期时,给予照射剂量为6Gy的60Coγ射线一次性照射。MTT法检测不同浓度TFA处理组与未处理组hMSCs和HepG-2经60Coγ射线照射后细胞的存活率；流式细胞技术分析照射后6h、24h、48h各实验组细胞的凋亡率；琼脂糖电泳技术分析照射后24h和72h各实验组细胞DNALadder的形成；Western blot方法分析照射后24h和72h各实验组HepG-2细胞凋亡相关蛋白Fas, Bcl-2, Bax的表达。
结果：MTT检测结果显示在照射后24h,经0.05mg/ml、0.10mg/ml、0.15mg/ml、0.20mg/ml不同浓度TFA预处理的hMSCs,其细胞存活率分别是单纯照射组的1.15、1.38、1.80、1.95倍；与此相反,经相同浓度TFA预处理的肝癌细胞HepG-2,其细胞存活率分别只有单纯照射组的0.53、0.51、0.41、0.23倍；说明TFA对人体正常细胞具有良好的放射防护作用,对肿瘤细胞具有显著的放射增敏效应,并呈现出良好的剂量依赖性。琼脂糖电泳结果显示,TFA对正常细胞和肿瘤细胞核DNALadder形成具有不同的影响。TFA用药照射组hMSCs核DNA形成的DNA Ladder明显低于单纯照射组；而TFA用药照射组HepG-2核DNA形成的DNA Ladder明显高于单纯照射组。流式细胞分析显示,经60Coγ射线照射6h、24h、48h后,单纯照射组hMSCs的凋亡率分别为29.3%、24.9%、13.6%,TFA用药照射组hMSCs的凋亡率分别为23.3%、11.2%、2.9%；单纯照射组HepG-2的凋亡率分别为6.9%、9.3%、15.8%；TFA用药照射组HepG-2的凋亡率分别为11.6%、17.3%、20.1%。结果提示,TFA预处理能够降低60Coγ射线对正常细胞的凋亡诱导,促进60Coγ射线对肿瘤细胞的凋亡诱导,说明TFA对细胞凋亡具有双重调节作用。Western blot结果显示,在肝癌细胞HepG-2中,单纯照射组Fas蛋白表达较弱与空白对照组Fas表达量相似；TFA用药照射组Fas表达量明显高于单纯照射组和空白对照组。各组细胞Bax的表达与Fas表达基本一致,Bcl-2的表达刚好与Bax和Fas的表达相反。
结论：TFA对人正常骨髓间充质细胞具有明显的放射防护作用,对肝癌细胞不仅没有放射防护作用反而具有促凋亡作用,TFA通过调控凋亡相关蛋白表达从而增强γ射线对肝癌细胞的杀伤作用。
Objective:In this study, we use Astragalus Membranaceus from Sichuan as raw material and make use of the improved ethyl acetate extraction technology to separate the total Flavonoids of Astragalus(TFA) from Astragalus. Then, we focus our research priorities on the different radioprotective effects of TFA on normal human mesenchymal stem cells (hMSCs) and hepatoma cells injured by irradiation and explore the molecule mechanism of the different radioprotective effects of TFA on normal cells and tumor cells in DNA and protein level.
Methods:The HepG-2 hepatocellular carcinoma cells and hMSCs were used as experimental subject. The experiment was set up three experimental groups:blank control group (no irradiation, no TFA), direct irradiation group (radiation only, no TFA), TFA treated irradiation group (TFA treatment+radiation). The experiment cells were irradiated by 60Coγ-ray with a irradiation dose of 6Gy one time when it reached logarithmic growth phase. MTT assay was used to detect the survival rates of hMSCs and HepG-2 cells which were pretreated with different concentrations of TFA before irradiation, and flow cytometer was used to detect the cell apoptosis rates of all the groups in 6h、24h、48h time points. Agarose gel electrophoresis was utilized to examine the DNA ladders of HepG-2 and hMSCs cells which were irradiated by 60Co y-ray before 24 hour or 72 hour. Western blot technique was applied to detect the expression of apoptotic protein on HepG-2 cells such as Fas, Bcl-2 and Bax in all the experiment groups.
Results:The MTT results showed that the hMSCs survival rates of TFA treated irradiation group were respectively as 1.15,1.38,1.80 and 1.95 times as the direct irradiation group when the treated concentration of TFA were 0.05mg/ml, 0.10mg/ml,0.15mg/ml and 0.20mg/ml. Inversely, under the same pretreatment, the HepG-2 survival rates of TFA treated irradiation group were respectively as 0.53,0.51,0.41 and 0.23 folds as the direct irradiation group. It indicated that TFA had a strong radioprotection on human normal cells and a significant radiosensitization on tumor cells as well as an evident dose dependent. Agarose gel electrophoresis showed that TFA had different effects on the DNA Ladder of normal cells and tumor cells. Compared with the direct radiation group, the apoptotic DNA ladders of hepatoma cells in TFA treated irradiation group were apparently higher, on the other hand the apoptotic DNA ladders of hMSCs in TFA treated irradiation group were obviously lower. Flow cytometry showed that after being irradiated 6h、24h、48h by 60Coγray the apoptosis rates of hMSCs in the direct radiation group were 29.3%、24.9%、13.6% and the apoptosis rates of hMSCs in the TFA treated irradiation group were 23.3%、11.2%、2.9%; however, the apoptosis rates of HepG-2 in the direct radiation group were 6.9%、9.3%、15.8% and the apoptosis rates of HepG-2 in TFA treated irradiation group were 11.6%、17.3%、20.1%. These results indicate that TFA can decrease the apoptosis in normal cells and promote the apoptosis in tumor cells induced by 60Coγray. It indicated that TFA had a dual role on the regulation of the apoptosis of the cells. The results of Western blot demonstrated that the expression of apoptotic protein Fas on hepatoma cells both in blank control group and in direct irradiation group were poor and similar. However, the expression of Fas in TFA treated irradiation group was higher than that of the direct radiation group and the blank control group significantly. The expressions of Bax in all the groups were the same as Fas. As for the expression of Bcl-2, it was just the opposite expression of Bax and Fas.
Conclusion:TFA has obvious effects of radiological protection on human hMSCs, and has not effects of radiological protection and also has effects of apoptosis enhancement on hepatoma cells. TFA can strengthen the lethal effect of 60Coγray on hepatoma carcinoma cell through the expression regulation of apoptosis associated protein.
方法：以肝癌细胞HepG-2和人体正常细胞hMSCs为实验对象,设置空白对照组(不照射,不给药),单纯照射组(只照射,不给药)TFA用药照射组(TFA处理+照射)三个实验组；当细胞生长至对数生长期时,给予照射剂量为6Gy的60Coγ射线一次性照射。MTT法检测不同浓度TFA处理组与未处理组hMSCs和HepG-2经60Coγ射线照射后细胞的存活率；流式细胞技术分析照射后6h、24h、48h各实验组细胞的凋亡率；琼脂糖电泳技术分析照射后24h和72h各实验组细胞DNALadder的形成；Western blot方法分析照射后24h和72h各实验组HepG-2细胞凋亡相关蛋白Fas, Bcl-2, Bax的表达。
结果：MTT检测结果显示在照射后24h,经0.05mg/ml、0.10mg/ml、0.15mg/ml、0.20mg/ml不同浓度TFA预处理的hMSCs,其细胞存活率分别是单纯照射组的1.15、1.38、1.80、1.95倍；与此相反,经相同浓度TFA预处理的肝癌细胞HepG-2,其细胞存活率分别只有单纯照射组的0.53、0.51、0.41、0.23倍；说明TFA对人体正常细胞具有良好的放射防护作用,对肿瘤细胞具有显著的放射增敏效应,并呈现出良好的剂量依赖性。琼脂糖电泳结果显示,TFA对正常细胞和肿瘤细胞核DNALadder形成具有不同的影响。TFA用药照射组hMSCs核DNA形成的DNA Ladder明显低于单纯照射组；而TFA用药照射组HepG-2核DNA形成的DNA Ladder明显高于单纯照射组。流式细胞分析显示,经60Coγ射线照射6h、24h、48h后,单纯照射组hMSCs的凋亡率分别为29.3%、24.9%、13.6%,TFA用药照射组hMSCs的凋亡率分别为23.3%、11.2%、2.9%；单纯照射组HepG-2的凋亡率分别为6.9%、9.3%、15.8%；TFA用药照射组HepG-2的凋亡率分别为11.6%、17.3%、20.1%。结果提示,TFA预处理能够降低60Coγ射线对正常细胞的凋亡诱导,促进60Coγ射线对肿瘤细胞的凋亡诱导,说明TFA对细胞凋亡具有双重调节作用。Western blot结果显示,在肝癌细胞HepG-2中,单纯照射组Fas蛋白表达较弱与空白对照组Fas表达量相似；TFA用药照射组Fas表达量明显高于单纯照射组和空白对照组。各组细胞Bax的表达与Fas表达基本一致,Bcl-2的表达刚好与Bax和Fas的表达相反。
结论：TFA对人正常骨髓间充质细胞具有明显的放射防护作用,对肝癌细胞不仅没有放射防护作用反而具有促凋亡作用,TFA通过调控凋亡相关蛋白表达从而增强γ射线对肝癌细胞的杀伤作用。
Objective:In this study, we use Astragalus Membranaceus from Sichuan as raw material and make use of the improved ethyl acetate extraction technology to separate the total Flavonoids of Astragalus(TFA) from Astragalus. Then, we focus our research priorities on the different radioprotective effects of TFA on normal human mesenchymal stem cells (hMSCs) and hepatoma cells injured by irradiation and explore the molecule mechanism of the different radioprotective effects of TFA on normal cells and tumor cells in DNA and protein level.
Methods:The HepG-2 hepatocellular carcinoma cells and hMSCs were used as experimental subject. The experiment was set up three experimental groups:blank control group (no irradiation, no TFA), direct irradiation group (radiation only, no TFA), TFA treated irradiation group (TFA treatment+radiation). The experiment cells were irradiated by 60Coγ-ray with a irradiation dose of 6Gy one time when it reached logarithmic growth phase. MTT assay was used to detect the survival rates of hMSCs and HepG-2 cells which were pretreated with different concentrations of TFA before irradiation, and flow cytometer was used to detect the cell apoptosis rates of all the groups in 6h、24h、48h time points. Agarose gel electrophoresis was utilized to examine the DNA ladders of HepG-2 and hMSCs cells which were irradiated by 60Co y-ray before 24 hour or 72 hour. Western blot technique was applied to detect the expression of apoptotic protein on HepG-2 cells such as Fas, Bcl-2 and Bax in all the experiment groups.
Results:The MTT results showed that the hMSCs survival rates of TFA treated irradiation group were respectively as 1.15,1.38,1.80 and 1.95 times as the direct irradiation group when the treated concentration of TFA were 0.05mg/ml, 0.10mg/ml,0.15mg/ml and 0.20mg/ml. Inversely, under the same pretreatment, the HepG-2 survival rates of TFA treated irradiation group were respectively as 0.53,0.51,0.41 and 0.23 folds as the direct irradiation group. It indicated that TFA had a strong radioprotection on human normal cells and a significant radiosensitization on tumor cells as well as an evident dose dependent. Agarose gel electrophoresis showed that TFA had different effects on the DNA Ladder of normal cells and tumor cells. Compared with the direct radiation group, the apoptotic DNA ladders of hepatoma cells in TFA treated irradiation group were apparently higher, on the other hand the apoptotic DNA ladders of hMSCs in TFA treated irradiation group were obviously lower. Flow cytometry showed that after being irradiated 6h、24h、48h by 60Coγray the apoptosis rates of hMSCs in the direct radiation group were 29.3%、24.9%、13.6% and the apoptosis rates of hMSCs in the TFA treated irradiation group were 23.3%、11.2%、2.9%; however, the apoptosis rates of HepG-2 in the direct radiation group were 6.9%、9.3%、15.8% and the apoptosis rates of HepG-2 in TFA treated irradiation group were 11.6%、17.3%、20.1%. These results indicate that TFA can decrease the apoptosis in normal cells and promote the apoptosis in tumor cells induced by 60Coγray. It indicated that TFA had a dual role on the regulation of the apoptosis of the cells. The results of Western blot demonstrated that the expression of apoptotic protein Fas on hepatoma cells both in blank control group and in direct irradiation group were poor and similar. However, the expression of Fas in TFA treated irradiation group was higher than that of the direct radiation group and the blank control group significantly. The expressions of Bax in all the groups were the same as Fas. As for the expression of Bcl-2, it was just the opposite expression of Bax and Fas.
Conclusion:TFA has obvious effects of radiological protection on human hMSCs, and has not effects of radiological protection and also has effects of apoptosis enhancement on hepatoma cells. TFA can strengthen the lethal effect of 60Coγray on hepatoma carcinoma cell through the expression regulation of apoptosis associated protein.
引文
[1]Kang KA, Zhang R, Chae S, et al.Phloroglucinol (1,3,5-trihydroxybenzene) protects against ionizing radition-induced cell damage through inhibition of oxidative stress in vitro and in vivo[J].Chem Biol Interact,2010, Feb 25(Epub ahead of print)
[2]Kokotkiewicz A, Jaremicz Z, Luczkiewicz M. Aronia Plants:A Review of Traditional Use, Biological Activities, and Perspectives for Modern Medicine[J].J Med Food, 2010, Feb 19:(Epub ahead of print).
[3]Qian L, Cao F, Cui J, et al. Radioprotective.effect of hydrogen in cultured cells and mice[J].Free Radic Res,2010,44 (3):275-82.
[4]Assayed ME. Radioprotective effects of black seed (Nigella sativa) oil against hemopoietic damage and immunosuppression in gamma-irradiated rats[J].Immunopharmacol Immunotoxicol,2010, Jan 27 (epub ahead of print)
[5]Muramoto GG, Russell JL, Safi R, et al. Inhibition of Aldehyde Dehydrogenase Expands Hematopoietic Stem Cells with Radioprotective Capacity[J].Stem Cells, 2010, Jan 6 (epub ahead of print)
[6]Winczura P, Jassem J. Combined treatment with cytoprotective agents and radiotherapy[J].Cancer Treat Rev,2009, Dec 29 (Epub ahead of print)
[7]Beaven AW, Shea TC. The effect of palifermin on chemotherapyand radiation therapy-induced mucositis:a review of the current literature[J].Support Cancer Ther,4 (4):188-97.
[8]Muller AC, Pigorsch S, Beyer C, et al. Radioprotective effects of amifostine in vitro and in vivo measured with the comet assay [J]. Strahlenther Onkol,2004,180 (8):517-25.
[9]Andreassen CN, Grau C, Lindegaard JC, et al. Chemical radioprotection:a critical review of amifostine as a cytoprotector in radiotherapy[J]. Semin Radiat Oncol, 2003,13 (1):62-72.
[10]Ma C, Xie J, Jiang Z, et al.Does amifostine have radioprotective effects on salivary glands in high-dose radioactive iodine-treated differentiated thyroid cancer[J].Eur J Nucl Med Mol Imaging,2010, Feb 4 (Epub ahead of print)
[11]Dzieqielewski J, Goetz W, Murtey JS, et al.Amifostine metabolite WR-1065 disrupts homologous recombination in mammalian cells[J].Radiat Res,2010,173 (2): 175-83.
[12]周丽君,方允中,章扬培,等.绿茶多酚等五种抗辐射药物对质粒pBR322DNA辐射损伤防护效应的研究[J].中华医学与防护杂志,1995,6(18)：416-419.
[13]梁长玲.国内抗辐射天然药物简介[J].中草药,2000,31(4)：315-317.
[14]王春燕,陈德清.刺五加的抗辐射诱发染色体畸变作用[J].中华医学与防护杂志,1996,16(2)：封四.
[15]郭雪峰,岳永德.黄酮类化合物的提取分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007,35(26)：8083-8086.
[16]徐莲英,陶建生,冯怡,等.中药制剂发展的回顾[J].中成药,2000,22(1)：6-21.
[17]张福海,马丽娟.黄芪乙酸乙酯层有效成分的分离及鉴定[J].中医药信息,2007,24(3)：45-46.
[18]周光明,李文建,王菊芳,等.γ射线诱导的肝癌细胞DNA双链断裂[J].核技术,2000,23(11)：776-779.
[19]陈建业,陈俊杰,李瑞祥,等.护生宝口服液对H22肝癌抑癌作用的实验研究[J].中草药,2002,33(10)：929-931.
[20]陈建业,曾益新,陈小君,等.护生宝口服液对辐射的防护作用研究[J].中华与防护杂志,2000,20(4)：273-275.
[21]李宗山,张迪,邱世翠,等.黄芪抗γ射线辐射作用的研究[J].时针国医国药,2003,14(12)：733-734.
[22]刘遵峰,刘雪萍,梁晓勇,等.黄芪总黄酮和总甙的提取和分离[J].南开大学学报,2003,36(3)：22-25.
[23]周萍,汝海龙,蒋惠娣.大孔吸附树脂对花生壳总黄酮的纯化研究[J].大理学院 学报,2007,6(2)：10-12.
[24]侯世祥,徐莲英.中药制药工艺技术解析[M].北京；人民卫生出版社,2003,67-94.
[25]房树标,李艳燕,刘必旺,等.紫外分光光度发测定蒙古黄芪中总黄酮的含量[J].中国药物与临床,2007,7(12)899-901.
[26]YuQT, QiLW, LiP, et al.Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi (Radix astragali) by HPLC-DAD-ELSD[J].J Sep Sci,2007,30 (9):1292-9
[27]肖卫华,韩鲁佳.黄芪黄酮乙醇回流提取工艺的研究[J].食品工业科技,2008,29(1)：233-235.
[28]王宗伟,黄兆胜,罗霄云,等.中华芦荟多糖对非瘤细胞辐射后细胞凋亡和凋亡蛋白表达的影响[J].中国药理学通报,2004,20(5)：531-7.
[29]钱素娟,周令望,迟月明,等.Bcl-2蛋白的表达与胃癌细胞凋亡的关系.哈尔滨医科大学学报,1998,32：6-7.
[30]Oltval ZN, Milliman CL, Korsmeyer SJ.Bcl-2 heterodimerizes in vivo with a conserved hemology, Bax, that accelerates programmed cell death[J].Cell,1993, 74:609.
[31]刘海峰,刘为纹,房殿春,等.胃癌组织中促凋亡基因Bax的表达及意义.第三军医大学学报,1998,20：121-123.
[32]Yin XM, Oltval ZN, Korsmeyer SJ.BH1 and BH2 domains of bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax[J].Nature,1994,369: 321-323.
[33]步宏,石毓君,吴杨.分子病理学进展[M].四川大学出版社.2006,11(1)：133-143.
[34]O'Reilly LA, Tail L, Lee L, et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[J]. Nature,2009,461 (7234):659-63.
[35]Verbrugge I, Maas C, Heijkoop M, et al. Radiation and anticancer drugs can facilitate mitochondrial bypass by CD95/Fas via c-FLIP downregulation[J]. Cell Death Differ,2009, Oct (2):141.
[36]Lin CH, Shih WL, Lin FL, et al. Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway[J]. Apoptosis,2009,14 (7):864-77.
[37]Lu Y, Wu LQ, Dong Q, et al. Eperimental study on the effect of Kang-Lai-Te induced apoptosis of human hepatoma carcinoma cell HepG2[J]. Hepatobiliary pancreat Dis Int,2009,8 (3):267-72.
[38]梁素华,朱洵良,冯碧珍.电离辐射诱导小白鼠染色体畸变的比较研究[J].川北医学院学报,1991,6(2)：13-16.
[39]张冬青,汪德清.应用蛋白组学方法研究黄芪总黄酮对人肝癌细胞的影响[J].感染、炎症、修复,2009,10(1)：26-28.
[40]汪德清,李煜,田亚萍,等.黄芪总黄酮对BEL7402细胞的体外抑制作用[J].军医进修学报,2005(5)：321-323.
[41]Bensidhoum M, Gobin S, Chapel A, et al. Therapeutic effect of human mesenchymal stem cells in skin after radiation damage[J]. J Soc Biol,2005,199 (4):337-41.
[42]Francois S, Bensidhoum M, Mouiseddine M, et al. Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs:a study of their quantitative distribution after irradiation damage[J]. Stem Cells,2006,24 (4):1020-9.
[43]Tomuleasa CI, F oris V, Soritau O, et al. Effects of 60Co gamma-rays on human osteoprogenitor cells[J].Rom J Morphol Embryol,2009,50 (3):349-55.
[44]Serakinci N, Christensen R, Graakjaer J, et al. Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart[J].Exp Cell Res,2007,313 (5):1056-67.
[45]Chen MF, Lin CT, Chen WC, et al. The sensitivity of human mesenchymal stem cells to ionizing radiation[J]. Int J Radiat Oncol Biol Phys,2006,66 (1):244-53.
[46]Graakjaer J, Christensen R, Kolvraa S, et al. Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation[J].BMC Mol Biol,2007, 13 (8):49.
[47]Christensen R, Alsner J, Brandt Sorensen F, et al. Transformation of human mesenchymal stem cells in radiation carcinogenesis:long-term effect of ionizing radiation[J].Reqen Med,2008,3 (6):849-61.
[48]Soule BP, Simone NL, DeqraffWG, et al. Loratadine dysregulates cell cycle progression and enchances the effect of radiation in human tumor cell lines[J].Radiat Oncol,2010,5 (1):8.
[49]Schneideman MH, Hofer KG, Schneideman GS. Association between the division delay and DNA late in the cell cycle[J].Radiat Res,1990,22 (3):337-4.
[50]Shi W, Feng Z, Zhang J, et al. The Role of RPA2 phosphorylation in Homologous Recombination in Response to Replication Arrest[J].Carcinogenesis,2010,3 (epub ahead of print)
[51]汪德清,沈文梅,田亚平,等.黄芪总黄酮对DNA损伤防护作用的研究[J].生物化学与生物物理进展,1996,4,346-348.
[1]金华,王德文,彭瑞云.电磁脉冲辐射后小鼠免疫器官的病理研究[J].军事医学科学院院刊,2004,28(6)：537-547.
[2]谢漪,党秉荣,张红,等.低剂量X射线辐射对BALB/C小鼠免疫系统的影响[J].辐射研究与辐射工艺学报,2006,(3)：60-62.
[3]罗灿,苏旭,刘建香,等.低剂量X射线全身照射对小鼠脾细胞转铁蛋白受体表达的影响[J].辐射研究与辐射工艺学报,1999,2(6)：97-100.
[4]韩京.辐射损伤对巨核细胞增殖与分化的影响及相关机制的研究[A].中国学位论文全文数据库,2005,11.
[5]陈光珍.60Co Y射线后小鼠早期骨髓造血机制细胞的增殖指数及细胞凋亡的变化[J].第三军医大学报,1999,7(30)：22-25.
[6]侯殿俊,乔建维,李洁清,等.低剂量电离辐射对人体健康状况的影响[J].职业与健康,2003,35(5)：5-7.
[7]刘永彪,赵杰,刘颖,等.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究[J].辐射研究与辐射工艺学学报,2004,05(9)：42--47.
[8]许浪,张碧清,孙远昌,等.黄芪对辐射损伤大鼠脾脏促进再生作用[J].彬州医学高等专科学校学报,2002,4(1)：9-10.
[9]焦艳,闻杰,张德山.膜荚黄芪茎叶总黄酮对小鼠免疫功能的影响[J].中医药信息,1997,(5)：44.
[10]焦艳,闻杰,于晓红,等.膜荚黄芪茎叶总黄酮对小鼠细胞免疫功能的影响[J].中国中西医结合杂志,1999,19,(6)：356-368.
[11]沈敬华,杨丽敏,吕炳申,等.五种中药提取物对正常小鼠细胞免疫的影响[J].中国实验方剂学杂志,2006,12(2)：57-59.
[12]王胜春,蒋永培,胡永武,等.黄芪对60Co辐射小鼠外周血细胞和骨髓细胞的影响[J].中国中西医结合杂志,1997,1：98.
[13]林善文,周乃康,丁保国,等.黄芪总黄酮在术中放疗时对机体免疫力影响的实验观察[J].军医进修学院学报,1995,16(1)：5-7.
[14]汪德清,房征宇,沈文梅,等.黄芪总黄酮抗紫外线损伤作用的实验研究[J].劳动医学,1998,15(4)：200—202.
[15]李宗山,邱正翠,等.黄芪对X射线辐射损伤小鼠造血功能的影响[J].中国中医中药,2003,10(2)：99.
[16]李宗山,张迪,邱正翠,等.黄芪抗γ射线辐射作用的研究[J].时珍国医国药,2003,14(12)：733-734.
[17]全宏勋,李海生.黄芪对辐射小鼠红系造血功能的影响[J].中国公共卫生,2001,17(5)：393.
[18]陆天池,秦志丰,文海英.黄芪注射液穴位注射足三里治疗化疗白细胞减少的疗效观察[J].时珍国医国药,2006,17(11)：2262-2265.
[19]吕艳,冯雪梅,祝彼得,等.黄芪注射液对贫血小鼠骨髓有核细胞表达抗凋亡蛋白的影响[J].中药药理与临床,2005,21(5)：30-32.
[20]汪德清,沈文梅, 田亚平,等.黄芪总黄酮对羟自由基所致V79细胞DNA链断裂损伤的防护作用[J].中国药理学通报,1995,11(4)：311-313.
[21]汪德清,沈文梅,田亚平,等.黄芪总黄酮对DNA损伤防护作用的研究[J].生物化学与生物物理进展,1996,4：346-348.
[22]汪德清,田亚平,末淑珍,等.黄芪总黄酮抗突变作用实验研究[J].中国中药杂志,2003,28(12)：164-116.
[23]汪德清,田亚平,向兰.黄芪总黄酮生物学活性作用的化学成分基础研究[J].军医进修学院学报,2006,27(1)：13-15.
[24]杨映雪,陈建业,王亚平.黄芪总黄酮的抗氧化作用[J].川北医学院学报,2007,22(6)：606-608.
[25]郑敏,杨宏杰,张丹,等.黄芪提取液清除自由基的实验研究[J].山东中医杂志,2004,23(12)：743-745.
[26]欧阳雁玲,赵富玺.黄芪活性成分对膜损伤的防护作用[J].山西医药杂志,2002,31(3)：209-210.
[27]汪德清,沈文梅.黄芪总黄酮对犬模拟肺癌术中放疗抗辐射损伤作用的研究[J]. 中华医学与防护杂志,1996,16(6)：399-401.
[28]丁利君,冼建毅.黄芪中黄酮类化合物提取及其对羟自由基清除作用[J].食品与机械,2002,(3)：20-2.
[29]沈文梅,王成彬.X射线诱导小鼠脑组织一氧化氮合酶活性的变化[J].中国应用生理学杂志,1997,13(4)：298.
[2]Kokotkiewicz A, Jaremicz Z, Luczkiewicz M. Aronia Plants:A Review of Traditional Use, Biological Activities, and Perspectives for Modern Medicine[J].J Med Food, 2010, Feb 19:(Epub ahead of print).
[3]Qian L, Cao F, Cui J, et al. Radioprotective.effect of hydrogen in cultured cells and mice[J].Free Radic Res,2010,44 (3):275-82.
[4]Assayed ME. Radioprotective effects of black seed (Nigella sativa) oil against hemopoietic damage and immunosuppression in gamma-irradiated rats[J].Immunopharmacol Immunotoxicol,2010, Jan 27 (epub ahead of print)
[5]Muramoto GG, Russell JL, Safi R, et al. Inhibition of Aldehyde Dehydrogenase Expands Hematopoietic Stem Cells with Radioprotective Capacity[J].Stem Cells, 2010, Jan 6 (epub ahead of print)
[6]Winczura P, Jassem J. Combined treatment with cytoprotective agents and radiotherapy[J].Cancer Treat Rev,2009, Dec 29 (Epub ahead of print)
[7]Beaven AW, Shea TC. The effect of palifermin on chemotherapyand radiation therapy-induced mucositis:a review of the current literature[J].Support Cancer Ther,4 (4):188-97.
[8]Muller AC, Pigorsch S, Beyer C, et al. Radioprotective effects of amifostine in vitro and in vivo measured with the comet assay [J]. Strahlenther Onkol,2004,180 (8):517-25.
[9]Andreassen CN, Grau C, Lindegaard JC, et al. Chemical radioprotection:a critical review of amifostine as a cytoprotector in radiotherapy[J]. Semin Radiat Oncol, 2003,13 (1):62-72.
[10]Ma C, Xie J, Jiang Z, et al.Does amifostine have radioprotective effects on salivary glands in high-dose radioactive iodine-treated differentiated thyroid cancer[J].Eur J Nucl Med Mol Imaging,2010, Feb 4 (Epub ahead of print)
[11]Dzieqielewski J, Goetz W, Murtey JS, et al.Amifostine metabolite WR-1065 disrupts homologous recombination in mammalian cells[J].Radiat Res,2010,173 (2): 175-83.
[12]周丽君,方允中,章扬培,等.绿茶多酚等五种抗辐射药物对质粒pBR322DNA辐射损伤防护效应的研究[J].中华医学与防护杂志,1995,6(18)：416-419.
[13]梁长玲.国内抗辐射天然药物简介[J].中草药,2000,31(4)：315-317.
[14]王春燕,陈德清.刺五加的抗辐射诱发染色体畸变作用[J].中华医学与防护杂志,1996,16(2)：封四.
[15]郭雪峰,岳永德.黄酮类化合物的提取分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007,35(26)：8083-8086.
[16]徐莲英,陶建生,冯怡,等.中药制剂发展的回顾[J].中成药,2000,22(1)：6-21.
[17]张福海,马丽娟.黄芪乙酸乙酯层有效成分的分离及鉴定[J].中医药信息,2007,24(3)：45-46.
[18]周光明,李文建,王菊芳,等.γ射线诱导的肝癌细胞DNA双链断裂[J].核技术,2000,23(11)：776-779.
[19]陈建业,陈俊杰,李瑞祥,等.护生宝口服液对H22肝癌抑癌作用的实验研究[J].中草药,2002,33(10)：929-931.
[20]陈建业,曾益新,陈小君,等.护生宝口服液对辐射的防护作用研究[J].中华与防护杂志,2000,20(4)：273-275.
[21]李宗山,张迪,邱世翠,等.黄芪抗γ射线辐射作用的研究[J].时针国医国药,2003,14(12)：733-734.
[22]刘遵峰,刘雪萍,梁晓勇,等.黄芪总黄酮和总甙的提取和分离[J].南开大学学报,2003,36(3)：22-25.
[23]周萍,汝海龙,蒋惠娣.大孔吸附树脂对花生壳总黄酮的纯化研究[J].大理学院 学报,2007,6(2)：10-12.
[24]侯世祥,徐莲英.中药制药工艺技术解析[M].北京；人民卫生出版社,2003,67-94.
[25]房树标,李艳燕,刘必旺,等.紫外分光光度发测定蒙古黄芪中总黄酮的含量[J].中国药物与临床,2007,7(12)899-901.
[26]YuQT, QiLW, LiP, et al.Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi (Radix astragali) by HPLC-DAD-ELSD[J].J Sep Sci,2007,30 (9):1292-9
[27]肖卫华,韩鲁佳.黄芪黄酮乙醇回流提取工艺的研究[J].食品工业科技,2008,29(1)：233-235.
[28]王宗伟,黄兆胜,罗霄云,等.中华芦荟多糖对非瘤细胞辐射后细胞凋亡和凋亡蛋白表达的影响[J].中国药理学通报,2004,20(5)：531-7.
[29]钱素娟,周令望,迟月明,等.Bcl-2蛋白的表达与胃癌细胞凋亡的关系.哈尔滨医科大学学报,1998,32：6-7.
[30]Oltval ZN, Milliman CL, Korsmeyer SJ.Bcl-2 heterodimerizes in vivo with a conserved hemology, Bax, that accelerates programmed cell death[J].Cell,1993, 74:609.
[31]刘海峰,刘为纹,房殿春,等.胃癌组织中促凋亡基因Bax的表达及意义.第三军医大学学报,1998,20：121-123.
[32]Yin XM, Oltval ZN, Korsmeyer SJ.BH1 and BH2 domains of bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax[J].Nature,1994,369: 321-323.
[33]步宏,石毓君,吴杨.分子病理学进展[M].四川大学出版社.2006,11(1)：133-143.
[34]O'Reilly LA, Tail L, Lee L, et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis[J]. Nature,2009,461 (7234):659-63.
[35]Verbrugge I, Maas C, Heijkoop M, et al. Radiation and anticancer drugs can facilitate mitochondrial bypass by CD95/Fas via c-FLIP downregulation[J]. Cell Death Differ,2009, Oct (2):141.
[36]Lin CH, Shih WL, Lin FL, et al. Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway[J]. Apoptosis,2009,14 (7):864-77.
[37]Lu Y, Wu LQ, Dong Q, et al. Eperimental study on the effect of Kang-Lai-Te induced apoptosis of human hepatoma carcinoma cell HepG2[J]. Hepatobiliary pancreat Dis Int,2009,8 (3):267-72.
[38]梁素华,朱洵良,冯碧珍.电离辐射诱导小白鼠染色体畸变的比较研究[J].川北医学院学报,1991,6(2)：13-16.
[39]张冬青,汪德清.应用蛋白组学方法研究黄芪总黄酮对人肝癌细胞的影响[J].感染、炎症、修复,2009,10(1)：26-28.
[40]汪德清,李煜,田亚萍,等.黄芪总黄酮对BEL7402细胞的体外抑制作用[J].军医进修学报,2005(5)：321-323.
[41]Bensidhoum M, Gobin S, Chapel A, et al. Therapeutic effect of human mesenchymal stem cells in skin after radiation damage[J]. J Soc Biol,2005,199 (4):337-41.
[42]Francois S, Bensidhoum M, Mouiseddine M, et al. Local irradiation not only induces homing of human mesenchymal stem cells at exposed sites but promotes their widespread engraftment to multiple organs:a study of their quantitative distribution after irradiation damage[J]. Stem Cells,2006,24 (4):1020-9.
[43]Tomuleasa CI, F oris V, Soritau O, et al. Effects of 60Co gamma-rays on human osteoprogenitor cells[J].Rom J Morphol Embryol,2009,50 (3):349-55.
[44]Serakinci N, Christensen R, Graakjaer J, et al. Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart[J].Exp Cell Res,2007,313 (5):1056-67.
[45]Chen MF, Lin CT, Chen WC, et al. The sensitivity of human mesenchymal stem cells to ionizing radiation[J]. Int J Radiat Oncol Biol Phys,2006,66 (1):244-53.
[46]Graakjaer J, Christensen R, Kolvraa S, et al. Mesenchymal stem cells with high telomerase expression do not actively restore their chromosome arm specific telomere length pattern after exposure to ionizing radiation[J].BMC Mol Biol,2007, 13 (8):49.
[47]Christensen R, Alsner J, Brandt Sorensen F, et al. Transformation of human mesenchymal stem cells in radiation carcinogenesis:long-term effect of ionizing radiation[J].Reqen Med,2008,3 (6):849-61.
[48]Soule BP, Simone NL, DeqraffWG, et al. Loratadine dysregulates cell cycle progression and enchances the effect of radiation in human tumor cell lines[J].Radiat Oncol,2010,5 (1):8.
[49]Schneideman MH, Hofer KG, Schneideman GS. Association between the division delay and DNA late in the cell cycle[J].Radiat Res,1990,22 (3):337-4.
[50]Shi W, Feng Z, Zhang J, et al. The Role of RPA2 phosphorylation in Homologous Recombination in Response to Replication Arrest[J].Carcinogenesis,2010,3 (epub ahead of print)
[51]汪德清,沈文梅,田亚平,等.黄芪总黄酮对DNA损伤防护作用的研究[J].生物化学与生物物理进展,1996,4,346-348.
[1]金华,王德文,彭瑞云.电磁脉冲辐射后小鼠免疫器官的病理研究[J].军事医学科学院院刊,2004,28(6)：537-547.
[2]谢漪,党秉荣,张红,等.低剂量X射线辐射对BALB/C小鼠免疫系统的影响[J].辐射研究与辐射工艺学报,2006,(3)：60-62.
[3]罗灿,苏旭,刘建香,等.低剂量X射线全身照射对小鼠脾细胞转铁蛋白受体表达的影响[J].辐射研究与辐射工艺学报,1999,2(6)：97-100.
[4]韩京.辐射损伤对巨核细胞增殖与分化的影响及相关机制的研究[A].中国学位论文全文数据库,2005,11.
[5]陈光珍.60Co Y射线后小鼠早期骨髓造血机制细胞的增殖指数及细胞凋亡的变化[J].第三军医大学报,1999,7(30)：22-25.
[6]侯殿俊,乔建维,李洁清,等.低剂量电离辐射对人体健康状况的影响[J].职业与健康,2003,35(5)：5-7.
[7]刘永彪,赵杰,刘颖,等.SOD对肿瘤细胞凋亡影响和抗氧化损伤的研究[J].辐射研究与辐射工艺学学报,2004,05(9)：42--47.
[8]许浪,张碧清,孙远昌,等.黄芪对辐射损伤大鼠脾脏促进再生作用[J].彬州医学高等专科学校学报,2002,4(1)：9-10.
[9]焦艳,闻杰,张德山.膜荚黄芪茎叶总黄酮对小鼠免疫功能的影响[J].中医药信息,1997,(5)：44.
[10]焦艳,闻杰,于晓红,等.膜荚黄芪茎叶总黄酮对小鼠细胞免疫功能的影响[J].中国中西医结合杂志,1999,19,(6)：356-368.
[11]沈敬华,杨丽敏,吕炳申,等.五种中药提取物对正常小鼠细胞免疫的影响[J].中国实验方剂学杂志,2006,12(2)：57-59.
[12]王胜春,蒋永培,胡永武,等.黄芪对60Co辐射小鼠外周血细胞和骨髓细胞的影响[J].中国中西医结合杂志,1997,1：98.
[13]林善文,周乃康,丁保国,等.黄芪总黄酮在术中放疗时对机体免疫力影响的实验观察[J].军医进修学院学报,1995,16(1)：5-7.
[14]汪德清,房征宇,沈文梅,等.黄芪总黄酮抗紫外线损伤作用的实验研究[J].劳动医学,1998,15(4)：200—202.
[15]李宗山,邱正翠,等.黄芪对X射线辐射损伤小鼠造血功能的影响[J].中国中医中药,2003,10(2)：99.
[16]李宗山,张迪,邱正翠,等.黄芪抗γ射线辐射作用的研究[J].时珍国医国药,2003,14(12)：733-734.
[17]全宏勋,李海生.黄芪对辐射小鼠红系造血功能的影响[J].中国公共卫生,2001,17(5)：393.
[18]陆天池,秦志丰,文海英.黄芪注射液穴位注射足三里治疗化疗白细胞减少的疗效观察[J].时珍国医国药,2006,17(11)：2262-2265.
[19]吕艳,冯雪梅,祝彼得,等.黄芪注射液对贫血小鼠骨髓有核细胞表达抗凋亡蛋白的影响[J].中药药理与临床,2005,21(5)：30-32.
[20]汪德清,沈文梅, 田亚平,等.黄芪总黄酮对羟自由基所致V79细胞DNA链断裂损伤的防护作用[J].中国药理学通报,1995,11(4)：311-313.
[21]汪德清,沈文梅,田亚平,等.黄芪总黄酮对DNA损伤防护作用的研究[J].生物化学与生物物理进展,1996,4：346-348.
[22]汪德清,田亚平,末淑珍,等.黄芪总黄酮抗突变作用实验研究[J].中国中药杂志,2003,28(12)：164-116.
[23]汪德清,田亚平,向兰.黄芪总黄酮生物学活性作用的化学成分基础研究[J].军医进修学院学报,2006,27(1)：13-15.
[24]杨映雪,陈建业,王亚平.黄芪总黄酮的抗氧化作用[J].川北医学院学报,2007,22(6)：606-608.
[25]郑敏,杨宏杰,张丹,等.黄芪提取液清除自由基的实验研究[J].山东中医杂志,2004,23(12)：743-745.
[26]欧阳雁玲,赵富玺.黄芪活性成分对膜损伤的防护作用[J].山西医药杂志,2002,31(3)：209-210.
[27]汪德清,沈文梅.黄芪总黄酮对犬模拟肺癌术中放疗抗辐射损伤作用的研究[J]. 中华医学与防护杂志,1996,16(6)：399-401.
[28]丁利君,冼建毅.黄芪中黄酮类化合物提取及其对羟自由基清除作用[J].食品与机械,2002,(3)：20-2.
[29]沈文梅,王成彬.X射线诱导小鼠脑组织一氧化氮合酶活性的变化[J].中国应用生理学杂志,1997,13(4)：298.