60Co γ射线照射致小鼠肝脏的miRNAs表达改变

目的 应用miRNA芯片筛选4 Gy60Co γ射线照射后小鼠肝脏中差异表达的miRNAs,生物信息学方法探索差异表达miRNAs调控的主要功能.方法 SPF级C57BL/6J小鼠接受4 Gy60Co γ射线单次全身照射后,进行外周血白细胞计数和骨髓嗜多染红细胞微核计数.应用miRNA芯片筛选照射后小鼠肝脏中差异表达的miRNAs,用miRNA特异引物对部分差异表达miRNA进行实时定量PCR(real time PCR)验证.运用生物信息学方法,对差异miRNAs靶基因及调控功能进行预测.结果 4 Gyγ射线照射后,外周血白细胞总数与对照组相比显著减少(t=2.87,P＜0.05),而骨髓嗜多染红细胞微核率与对照组相比显著增加(t=-2.91,P＜0.05).miRNA芯片结果显示,照射组与对照组差异表达的miRNAs共17个,其中9个表达上调,8个表达下调.miR-124和miR-34a的实时荧光定量RT-PCR验证结果与芯片结果一致.GO分析发现,与黏附、细胞周期相关的通路被抑制,一些免疫相关通路被激活.结论 miR-34a和miR-194参与了急性辐射损伤的调控,起主要调控作用的miRNAs还有miR-124、miR-382和miR-92a*.

Abstract：

Objective To investigate the differential expression profiles of microRNAs in the liver of 60Co γ-ray irradiated mice using microRNA microarray and to explore their main functions by bioinformatic analysis.Methods After SPF C57BL/6J mice expose to 4 Gy-single whole body radiation,total number of peripheral WBC and the fMNPCE were measured at 3 d.The differentially expressed miRNAs in mouse liver were detected with miRNA microarray,miRNA-124 and miR-34a were confirmed by real time RT-PCR assay.Bioinformatic analysis was applied to explore target genes and the main functions of the differential expressed miRNAs.Results Compared with control group,the total number of peripheral WBC decreased( t = 2.87,P ＜ 0.05 ) ,while the fMNPCE in bone marrow increased ( t =-2.91,P ＜0.05) after 4 Gy γ-ray irradiation.miRNA microarray revealed that 17 miRNAs were differentially expressed,in which 9 up-regulated,8 down-regulated.The expression levels of miR-124 and miR-34a were coincident with the result of real time RT-PCR.GO analysis showed that some pathways including adherens junction and cell cycle were suppressed,while some immune-related pathways were activated.Conclusions miR-34a and miR-194 were involved in the regulation of acute radiation damage,some other miRNAs including miR-124、miR-382 and miR-92a* also played important roles in radiation process.     

黄芪总黄酮对人体正常细胞和肿瘤细胞辐射防护作用的差异性研究

目的 研究黄芪总黄酮(total flayonoids of astragalus,TFA)对60°Co γ射线辐射损伤的人体正常骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)和肝癌细胞HepG-2辐射防护作用的差异性.方法 MTT法检测TFA处理组与单纯照射组hMSCs和HepG-2的细胞活性;HepG-2细胞克隆形成实验检测细胞的辐射敏感性;流式细胞技术分析细胞凋亡率;Western blot技术分析凋亡相关蛋白Fas,Bcl-2,Bax的表达.结果 MTT检测结果显示,当给予6 Gy γ射线一次性照射后,TFA预处理组hMSCs细胞活性分别比单纯照射组提高了1.15～1.95倍;经相同浓度TFA预处理的肝癌细胞HepG-2的细胞活性仅为单纯照射组的53%～23%;TFA的给药浓度与细胞存活率(survival rate)之间显现出良好的量效关系.细胞克隆形成实验结果显示:TFA+照射组能够明显抑制HepG-2细胞增殖,作用强于单纯TFA给药组和单纯照射组.流式细胞分析表明,经6 Gy γ射线一次性照射6、24和48 h后,TFA预处理组hMSCs的细胞凋亡率分别为23.3%,11.2%和2.9%.单纯照射组hMSCs的细胞凋亡率相应为29.3%,24.9%和13.6%;TFA预处理组肝癌细胞HepG-2的细胞凋亡率分别为11.6%,17.3%和20.1%,单纯照射组HepG-2的细胞凋亡率分别为6.9%、9.3%和15.8%.Western blot分析显示,在肝癌细胞HepG-2中,TFA预处理照射组促凋亡蛋白Fas和Bax 的表达量显著高于单纯照射组和对照组(t=11.17～-2.8,-12.35～3.4,P＜0.05);凋亡抑制蛋白Bel-2的表达量,TFA预处理照射组明显低于单纯照射组和对照组(f<6.36～17.61.P<0.05).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凋亡促进作用;TFA对肝癌细胞的促凋亡作用,主要通过上调促凋亡蛋白Fas和Bax的表达与下调凋亡抑制蛋白Bcl-2的表达,从而大大增强了60 Co γ射线对肝癌细胞的凋亡诱导作用.

Abstract：

Objective To investigate the different radioprotective effects of total flavonoids of Astragalus (TFA) on human normal mesenchymal stem cells(hMSCs) and hepatoma cells injured by 60 Coγ-ray radiation.Methods hMSCs and HepG-2 cells were cultured and randomly divided into TFA-treated and untreated groups.The cells of different groups were irradiated with 60 Co γ-rays at the dose of 6 Gy.MTT method was utilized to detect the survival rates of the hMSCs and HepG-2 cells pretreated or untreated with TFA before irradiation.Cell clone formation test was used to measure the cellular radiosensitivity.The apoptosis rates of different groups were determined by flow cytometer assay.The expression rates of the apoptosis-promoting proteins Fas and Bax and the apoptosis-inhibiting protein Bcl-2 were analyzed by Western blotting.Results MTT showed that the survival rates of hMSCs pretreated by TFA were 1.15-1.95 times higher than that of the pure irradiation group.On the contrary,the survival rates of the TFA pretreated HepG-2 cells were only 0.53-0.23 times that of the pure irradiation group.There was a good dose-effect relationship between the cell survival rate and the TFA concentration.Cell clone formation rate indicated that combined treatment of TFA and radiation inhibited the cell proliferation more effectively than single TFA or pure radiation.Flow cytometry showed that 6,24 and,48 h post-irradiation to 6 Gy,the apoptosis rates of the hMSCs were 23.3% ,11.2% ,and 2.9% ,respectively in the TFA pretreated group and were 29.3% ,24.9% ,and 13.6% in the pure radiation group.However,the apoptosis rates of the HepG-2 cells at 6,24,and 48 h post-irradiation to 6 Gy were 11.6% ,17.3% ,and 20.1% ,respectively in the TFA pretreated group and were 6.9% ,9.3% ,and 15.8% ,respectively in the direct radiation group.Western blotting showed that the expression levels of Fas and Bax proteins in the HepG-2 cells were significantly higher in the TFA pretreated group than in the pure radiation group.On the contrary,the expression level of the apoptosis inhibiting protein Bcl-2 was significantly lower in the TFA pretreated group than in the pure radiation group.Conclusions TFA has obvious effects of radiological protection on human hMSCs and has no effects of radiological protection but effects of apoptosis enhancement on hepatoma cells.The promotion of apoptosis of TFA on hepatoma cells is primarily through increasing the expression of apoptotic proteins such as Fas and Bax and reducing the expression of anti-apoptotic protein Bcl-2.     

X射线对人肺癌A549细胞CCR7表达影响的研究

目的 研究不同剂量X射线照射及照射后不同时间点对人肺癌A549细胞CC-趋化因子受体7(CCR7)表达的影响.方法 体外培养A549细胞,实验组采用直线加速器X射线一次性照射,细胞吸收剂量分别为2、4、6和8 Gy(源皮距100 cm;剂量率442.89 cGy/min),照射后4、12、24、48和72 h分别采用实时荧光定量PCR技术及Western blot方法分别进行CCR7 mRNA及蛋白质表达水平检测;对照组A549细胞除不接受x射线照射外,余处理同实验组.结果 A549细胞经2、4、6和8 Gy的X射线照射后,CCR7 mRNA及蛋白质在照射4 h后开始表达升高,达到高峰后相继出现下降;72 h后6和8 Gy组mRNA表达量仍高于对照组水平(t=6.75～7.26,P＜0.01),2和6 Gy组蛋白质表达量高于对照组(t=11.13～14.17,P＜0.01),而4和8 Gy组蛋白质表达量在48和72 h已降至对照组水平.结论 2、4、6和8 Gy的X射线照射A549细胞后,A549细胞CCR7mRNA及蛋白质的表达量明显增加,可能与一定剂量X射线辐射促进A549细胞增殖和转移有关.

Abstract：

Objective To study the effects of X-ray radiation on CC-chemokine receptor 7(CCR7) expression in human non-small cell lung cancer (NSCLC) cells.Methods Humanadenocarcinoma cells of the line A549 were cultured and irradiated by X-ray at the absorbed doses of 2,4,6,and 8 Gy respectively by linear accelerator (with the source skin distance of 100 cm and dose rate of 442.89 cGy/min).The relative levels of CCR7 mRNA and protein expression in the A549 cells were respectively detected by real time-PCR and Western blotting 4,12,24,48,and 72 h after radiation.Untreated A549 cells were used as control group.Results The expression levels of CCR7 mRNA and protein in the A549 cells began to increase since 4 h after radiation and then decreased gradually after they reached the peak.The CCR7 mRNA expression levels 72 h after radiation of the 6 and 8 Gy groups were still significantly higher than those of the control group (t = 6.75-7.26,both P < 0.01),and the CCR7 protein expression levels of the 2 and 6 Gy group were still significantly higher than those of the control group(t=11.13-14.17,both P <0.01).Then the CCR7 protein expression levels of the 4 and 8 Gy groups decreased to the control group level 48 and 72 h after radiation respectively.Conclusions The CCR7 mRNA and protein expression levels in the NSCLC cells increase after X-ray irradiation,which may be correlated with the promotion of proliferation and metastasis of NSCLC cells by X-ray irradiation at a certain dose.     

辐射诱导人外周血淋巴细胞DNA损伤反应相关基因表达变化的实验研究

目的 采用实时定量PCR技术,检测人外周血淋巴细胞DNA损伤反应相关基因表达对X射线全身照射的反应,为探索新型辐射生物标志物奠定基础.方法 以吸收剂量为0、1、2、3、4、5 Gy X射线照射正常人外周血,在照射后4和24 h,应用实时定量PCR法,对淋巴细胞细胞周期素依赖性蛋白激酶抑制物蛋白1a(Cdknla)、生长阻滞和DNA损伤基因45a(Gadd45α)基因的表达变化进行检测.应用胞质分裂阻滞微核法(CB微核法),检测淋巴细胞微核率变化.结果 Cdknla基因在人外周血淋巴细胞受到1～5 Gy照射后4和24 h,其相对表达量均较对照组显著性升高,至4 Gy达到峰值,5 Gy后不再继续增加.Cdknla基因表达与照射剂量呈线性相关(r=0.946、0.975,P<0.05).Gadd45ct基因在1～5 Gy照后4和24 h,其相对表达量均呈剂量依赖性升高,且照射后4 h的表达高于24 h(r=0.936、0.797,P<0.05).CB微核法中,在1～5 Gy X射线照射后4和24 h,各剂量组淋巴细胞微核率均显著增多,呈现良好的线性关系(r=0.990、0.984,P<0.05).结论 辐射使Cdknla基因和Gadd45α基因表达上调,表现出较好的剂量线性关系,有可能成为研制新型辐射生物剂量计的候选基因.

Abstract：

Objective To detect the expression of DNA damage response genes induced by radiation in human peripheral blood lymphocyte,and to explore the new biomarkers of radiation.Methods The human peripheral blood cells were irradiated to X-rays at different doses of 0,1,2,3,4,and 5 Gy.The quantitative real.time qPCR wag used to detect the expressions of cyclin-dependent kinase inhibitor l a gene(Cdknl a)and growth arrest and DNA damage inducible gene(Gadd45a)in lymphoeytes at 4 and 24 h post-irradiation,respectively.The method of CB mieronucleus was used to determine the change of micronucleus ratio.Results The expression of Cdknl a in peripheral blood lymphocytes wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy.reached the peak at 4 Gy and began to decrease at 5 Gy,which showed a dose-dependent manner(r=0.946,0.975,P<0.05).Similarly,the expression of Gadd45α in human peripheral blood lymphocytes was also increased significantly at 4 and 24 h post-irradiation to 0-5 Gy in a dose-dependent manner,while the expression of Gadd45a at 4 h wag higher than that at 24 h(r=0.936,0.797,P<0.05).The ratio of micronuclei wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy(r=0.990,0.984,P<0.05).Conciusions Cdknl a and Gadd45α expression could be increaged significandy at 4 and 24 h post-irradiation to 0-5 Gy,showing a good linear relationship.which might be candidate for radiation biological dosimeter.     

青蒿琥酯对人宫颈癌HeLa细胞的放射增敏作用

Objective To investigate the radiosensitizing effects of artesunate on human HeLa cells of cervical cancer in vitro.Methods Hela cells were irradiated with 60Co γ-rays.The dose rate was 0.635 Gy/min and the radiation dose was 0,1,2,4,6 Gy,respectively.The anti-proliferation activities of artesunate on HeLa cells were evaluated with MTT assay,to determine the most appropriate drug concentration.The effect of radiosensitivity was observed by using clonogenic assay.The single-hit multitarget model was used to plot the HeLa cell's dose-survival curve,to calculate mean lethal dose,quasithreshold dose and sensitization enhancement rate,and to evaluate its radiosensitization effect.The apoptosis was analyzed with flow cytometry (FCM) to further test the radiation senseitization of artesunate on HeLa cells.Results The inhibition of artesunate on HeLa cells increased with concentration.In radiation group,the cell cloning efficiency were 91.67% ,82.02% ,58.60% ,25.01%,respectively,and in artesunate (2.0 μ mol/L) + radiation group,the cell cloning efficiency were 74.93% ,60.53% ,22.38% ,5.05%.In radiation group and artesunate (2.0 μmol/L) + radiation group,the mean lethal dose(D0) was 2.95 and 2.07 Gy,respectively,while the qusai-threshold dose (Dq) were 2.01 and 1.24 Gy,respectively,and SER was 1.43.Compared with 2 and 6 Gy radiation group,the apoptosis rate of drug + radiation group increased from 12.26% ,40.08% to 22.71% ,59.92%.Conclusions The inhibiting effect of artesunate on HeLa cells is concentration-dependent.Artesunate has radiosensitizing effect on HeLa cells in vitro.     

γ射线诱导人淋巴细胞损伤及磷酸化组蛋白H2AX和ATM表达

目的 探讨人外周血经不同剂量60co γ射线照射后淋巴细胞损伤情况,以及与磷酸化的H2AX、ATM表达的关系.方法 永生化淋巴细胞及人新鲜外周血淋巴细胞经0～8 Gy 60 Coγ射线照射后0.5 h,分别采用Western blot和流式细胞术检测磷酸化ATM和γ-H2AX的表达情况,并通过CB微核法检测受照射后人外周血淋巴细胞微核验证细胞损伤程度.结果 流式细胞检测发现人外周血淋巴细胞照射后0.5 h,γ-H2AX表达的剂量效应关系呈线性平方模式,其拟合曲线为:Y=3.96+11.29D-0.45D2,而相同方法检测磷酸化ATM蛋白的表达未见明显变化.Western blot检测结果表明,照射后0.5 h,各受照射组磷酸化ATM表达在0.1～8 Gy范围内具有呈剂量依赖性增高的趋势.人外周血淋巴细胞微核结果显示,γ射线照射后DNA损伤情况明显加重,随吸收剂量的增加,微核细胞率明显增多.结论 60Co γ射线可诱导DNA双链断裂,随着吸收剂量的增加,微核率明显增加,磷酸化的H2AX、ATM表达水平亦明显增高,本研究为辐射损伤及辐射事故生物剂量估算领域的研究提供了理论依据.

Abstract：

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.     

X射线对人肺腺癌A549细胞Pokemon基因表达的影响

目的 研究不同剂量X射线照射及照射后不同时间点对人肺腺癌A549细胞Pokemon基因表达的影响.方法 用吸收剂量分别为2、4、6和8 Gy的X射线照射体外堵养的人肺腺癌A549细胞,2、4、8、12、24、48和72 ha,用实时定量PCR技术检测其中的Pokemon mRNA表达水平,以未照射组为对照.结果 在2、4、6、8 Gy X射线照射后的早期(除2 Gy照射后的2和4 h外)Pokemon mRNA的表达降低,但在晚期(48 h以后)呈升高趋势,在大部分时间点实验组与对照组的差异有统计学意义(t=3.40～154.76,P<0.05).结论 较大剂量的X射线在早期可下调A549细胞Pokemon基因mRNA的表达,诱导肿瘤细胞凋亡;但在晚期又可诱导A549细胞高表达PokemonmRNA,这可能与辐射所致A549细胞的DNA损伤修复和细胞周期调控有关.

Abstract：

Objective To study the dose-and time-effects of X-ray irradiation on the expression of Pokemon gene in A549 cells of human lung adenocarcinoma.Methods A549 cells were cultured in vitro and exposed to X-rays with the doses of 2,4,6 and 8 Gy,respectively.Untreated A549 cells were used as control group.The relative levels of Pokemon mRNA expression in the cells were detected by using quantitative real-time PCR at 2,4,8,12,24,48 and 72 h after irradiation.Results The Pokemon mRNA expression levels decreased in the early period after irradiation(except 2 and 4 h after irradiation in 2 Gy group)and then increased in the later stage(48 h after irradiation)with significant statistical differences at the most time points in comparison with the control group(t=3.40-154.76,P<0.05).Conclusions Higher doses of X-rays may degrade the expression of Pokemon mRNA in the human A549 cells and induce apoptosis in the early period,hut also may upgrade its expression in the later period, which might be correlated with the cell cycle regulation and DNA damage repair in the A549 cells.     

辐射诱导人外周血淋巴细胞DNA损伤反应相关基因表达变化的实验研究

Objective To detect the expression of DNA damage response genes induced by radiation in human peripheral blood lymphocyte,and to explore the new biomarkers of radiation.Methods The human peripheral blood cells were irradiated to X-rays at different doses of 0,1,2,3,4,and 5 Gy.The quantitative real.time qPCR wag used to detect the expressions of cyclin-dependent kinase inhibitor l a gene(Cdknl a)and growth arrest and DNA damage inducible gene(Gadd45a)in lymphoeytes at 4 and 24 h post-irradiation,respectively.The method of CB mieronucleus was used to determine the change of micronucleus ratio.Results The expression of Cdknl a in peripheral blood lymphocytes wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy.reached the peak at 4 Gy and began to decrease at 5 Gy,which showed a dose-dependent manner(r=0.946,0.975,P<0.05).Similarly,the expression of Gadd45α in human peripheral blood lymphocytes was also increased significantly at 4 and 24 h post-irradiation to 0-5 Gy in a dose-dependent manner,while the expression of Gadd45a at 4 h wag higher than that at 24 h(r=0.936,0.797,P<0.05).The ratio of micronuclei wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy(r=0.990,0.984,P<0.05).Conciusions Cdknl a and Gadd45α expression could be increaged significandy at 4 and 24 h post-irradiation to 0-5 Gy,showing a good linear relationship.which might be candidate for radiation biological dosimeter.     

Human lymphocyte damage and phosphorylation of H2AX and ATM induced by γ-rays

Objective To investigate 60Co γ-ray induced damage in lymphocytes and the relationship between doses of 60Co γ-ray irradiation and the levels of phosphorylated H2AX and ATM.Methods Cells were irradiated with 60Co γ-rays in the range of 0-8 Gy.The levels of phosphorylated H2AX and ATM were detected by Western blot and FACScan,respectively.The micronucleus(MN)was analyzed by CB method to evaluate DNA damage.Results FACScan results showed the dose-effect relationship of γ-H2AX expression were linear.square at 0.5 h post-irradiation to different doses,and the fitting curve was shown as Y=3.96+11.29D-0.45D2.The level of phosphorylated ATM(p-ATM)was not changed significantly by using the same method.Western blot showed that p-ATM protein expression was significandy increased after irradiation compared with sham.irradiated group.The MN assay which represented DNA damage was sensitive to different doses.Conclusions γ-ray irradiation could induce the phosphorylation of H2AX and ATM,which may play an important role in indicating DNA damage.Both of H2AX and ATM have the potential as sensitive biomarker and biodosimeter for radiation damage.     

低剂量辐射诱导的旁效应及其机制的初步研究

Objective To investigate whether the supernatant (the conditioned fluid) of myeloid cells suspension after low dose radiation (6 cGy) in vitro could resuh in bormesis on the normal or radiation damage cells and its mechanism.Methods Mice myeloid cell suspension was irradiated by 0,2 and 5 Gy,respectively,and cultured in vitro.MTT method was used to measure the reproductive activity of cells.Cytochrome C reduction method was used to determine the concentration of O2-,the immunohistochemical method to test the protein expression of c-fos.Results Co-cultured with the conditioned fluid,the reproductive activity of the myeloid cells after high dose irradiation( P＜0.01 ),while both the concentration of O2- and the protein expression of c-fos were enhanced (P＜0.05 ).Conclusions The conditioned fluid could enhance the proliferation of the myeloid cells after radiation injury.The mechanism of the low dose radiation-induced bystander effect might be correlated with the increase of both the concentration of O2- and the protein expression of c-fos.     

Initial research of screening for the differentially expressed proteins in beagles irradiated with 4.5 Gy60 CO γ-rays by two-dimensional gel electrophoresis and mass spectrometry

Objective To explore the mechanisms of cytokines on acute radiation disease in irradiated beagles.Methods The sera of beagles irradiated with 4.5 Gy γ-rays with cytokines treatment was collected at different time points post irradiation.The two-dimensional gel electrophoresis(2-DE)was used to isolate and compare the differentially expressed proteins in sera.HD-MS was used to analyze the differentially expressed proteins with significance,and the amino acid sequences should be determined. Results High resolution 2-DE gel map was obtained.There were six differentially expressed proteins in sera of irradiated beagles at different time points.Four protein spots were successfully identified by MS.A significant spot was identified as serum amyloid A(SAA)by HD-MS,with relative molecular mass of 13 077 and isoelectfie point of 6.26.Expression of SAA was not found 1 d pre-irradiation and 36 d postirradiation,but increased slightly 1 d(0.2166)and significantly 14 d post-irradiation(0.4577). Conclusions The expression of serum amyloid A was consistent with the process of acute radiation injury,which might indicate the turnover of the disease.     

Initial research of screening for the differentially expressed proteins in beagles irradiated with 4.5 Gy60 CO γ-rays by two-dimensional gel electrophoresis and mass spectrometry

Objective To explore the mechanisms of cytokines on acute radiation disease in irradiated beagles.Methods The sera of beagles irradiated with 4.5 Gy γ-rays with cytokines treatment was collected at different time points post irradiation.The two-dimensional gel electrophoresis(2-DE)was used to isolate and compare the differentially expressed proteins in sera.HD-MS was used to analyze the differentially expressed proteins with significance,and the amino acid sequences should be determined. Results High resolution 2-DE gel map was obtained.There were six differentially expressed proteins in sera of irradiated beagles at different time points.Four protein spots were successfully identified by MS.A significant spot was identified as serum amyloid A(SAA)by HD-MS,with relative molecular mass of 13 077 and isoelectfie point of 6.26.Expression of SAA was not found 1 d pre-irradiation and 36 d postirradiation,but increased slightly 1 d(0.2166)and significantly 14 d post-irradiation(0.4577). Conclusions The expression of serum amyloid A was consistent with the process of acute radiation injury,which might indicate the turnover of the disease.     

不同剂量60Co γ 射线部分照射人离体血对染色体畸变形成的影响

目的 分析不同剂量60Co γ射线部分照射人离体血对淋巴细胞染色体畸变形成的影响.方法 用0～8 Gy(剂量率为0.35 Gy/min)60Coγ射线在37 ℃条件下照射3份离体健康人外周血标本,以0.5∶1的比例与同一供血者的未受照血混合,120 min后进行培养、制片,显微镜下分析染色体畸变(双着丝粒+着丝粒环)的变化,借此进行剂量估算.结果 各组的双着丝粒体+着丝粒环和总畸变,以及断片和单体断裂均随着剂量的增加而增加.用双着丝粒+着丝粒环进行的剂量估算,0.5～2 Gy组大于照射剂量的1/3,4～8 Gy组均接近照射剂量的1/3.结论 染色体畸变可以作为估算非均匀照射的生物学指标之一.

Abstract：

Objective To investigate the effects of 60Co γ-ray partial radiation on chromosome aberration in human peripheral blood in vitro.Methods The samples of heparinized peripheral whole blood from 3 healthy persons were exposed to 60Co γ-rays at the doses between 0 and 8 Gy with the dose rate of 0.35 Gy/min at the temperature of 37 ℃ ,and then mixed with the unirradiated blood samples of the Microscopy was used to observe the chromosome aberration double ( centromere + centromere) and the biological dose was estimated thereby.ResultsThe amounts of double centromere + centromere were increased along with the dose of irradiation in all groups.The estimated biological dose was higher than the 1/3 of the irradiation dose when the dose was between 0.5 to 2 Gy,and was close to the 1/3 of the irradiation dose when the dose was between 4 to 8 Gy.Conclusion Chromosome aberration can be used as a biomarker in estimation of uneven irradiation.     

辐射对体外培养的小鼠成骨细胞核因子κB 受体活化因子配体/骨保护素mRNA表达的影响

目的 通过对成骨细胞的RANKL和OPG的基因表达分析揭示辐射对成骨细胞功能的影响.方法 在体外诱导骨髓基质细胞生成成骨细胞,碱性磷酸酶(ALP)染色对其特性进行确定,用RT-PCR方法分析了0～4 Gy照射的早期成骨细胞和成熟成骨细胞的RANKL和OPG的表达.结果 骨髓基质细胞在体外被诱导成的成骨细胞,在0～4 Gy剂量照射下,早期成骨细胞中RANKL的mRNA表达在1 Gy照射时,与0 Gy时相比表达最高,达到2.83倍,但各剂量组明显高于成熟成骨细胞的表达(t=8.34～103.57,P＜0.05).早期成骨细胞各剂量组的RANKL/OPG比值,在1 Gy照射最高达0.225±0.018,但明显高于晚期成骨细胞(t=2.84～20.99,P＜0.05).结论 辐射能够增强早期成骨细胞对破骨细胞功能的调节作用,加重骨组织的损伤.

Abstract：

Objective To study the influence of irradiation on the osteoblast function by the gene expression changes of RANKL and OPG.Methods Bone marrow stromal cells were induced to develop into early and mature osteoblasts in vitro.The characterization of osteoblasts was indentified by ALP staining.The RANKL and OPG mRNA levels in early and mature osteoblasts, which exposed to 0 -4 Gy radiation were determined by RT-PCR.Results Bone marrow stromal cells had been induced to early and mature osteoblasts by osteoblast differentiation medium in vitro.In early stage of osteoblast, RANKL mRNA expression levels treated with 1Gy irradiation was 2.83-fold higher than those other irradiation dosage groups.The RANKL mRNA expression levels of each group in early stage of osteoblasts were significantly higher than those in the mature counterpart ( t = 8.34 - 103.57, P ＜ 0.05 ).The ratio of RANKL/OPG mRNA was obviously greater in early osteoblast compared with the mature cells ( t = 2.84 - 20.99, P ＜0.05 ), and it was the highest in 1Gy irradiation treated early osteoblast.Conclusions Radiation exposure of the early osteoblasts promotes osteoclasts function and results in the bone loss.     

基因芯片筛选榄香烯乳增强肺腺癌A549 细胞放射敏感性相关基因

目的 利用基因芯片筛选榄香烯乳增加肺腺癌A549细胞放射敏感性的相关基因.方法 MTT法检测榄香烯乳对A549细胞的生长抑制效应,求得,IC50值;克隆形成实验检测榄香烯乳对肺腺癌A549细胞放射增敏作用;实验分为照射组及榄香烯乳联合照射组,寡核苷酸基因芯片筛选两组细胞的差异表达基因;RT-PCR法对差异表达基因进行验证.结果 榄香烯乳对A549细胞24 h的IC50值为120 mg/L;10 mg/L榄香烯乳对A549细胞有放射增敏作用,放射增敏比SERDDO、SERDq为1.54±0.20和1.43±0.15.基因芯片共检测出差异表达基因122个,上调基因89个,基因33个,其功能参与维持细胞结构、细胞代谢、增殖分化、信号传导、物质转运、细胞凋亡、DNA修复和免疫应答等.RT-PCR结果:上调基因Egr-1及下调基因CyclinDl表达与基因芯片结果一致.结论 榄香烯乳对肺腺癌A549细胞的放射增敏作用机制是多基因参与、协同作用的结果,对于新发现的差异基因的进一步研究,将有助于发现榄香烯乳对肺癌A549细胞放射增敏的新靶点.

Abstract：

Objective To screen radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip. Methods MTT test was used to calculate the IC50 of elemene. ①The effect of radiosensitivity was detected by colony forming assay. A549 cells were divided into 2 groups: radiation group and radiation + elemene group. Oligonucleotide chip was used to screen the gene expression changes of A549 cells from these 2 groups. The up-regulated gene Egr-1 and the down-regulated gene CyclinDl were selected to undergo RT-PCR so as to confirm the reliability of the result. Results MTT test showed the elemene inhibited the proliferation of the A549 cells dose-dependently. The IC50 value of elemene on the A549 cells was 120 mg/L. ②10 mg/L elemene had radiosensiting effect on A549 cells.The values of SERDO and SERDq obtained from the survival curve were (1.54±0. 20) and (1.43±0. 15 )respectively. Gene chip screened 122 differentially-expressed genes, including 89 up-regulated genes and33 down-regulated genes. ③These altered genes could be related to cell structure, substance metabolism,cell proliferation, cell differentiation, signal transduction, material transport, DNA repair, apoptosis,immune response and so forth. The RT-PCR results of Egr-1 and Cyclin D1 were consistent with the genenchip analysis.Conclusions The mechanism of elemene enhancing the radiosensitivity of lung adenoearcinoma A549 cells is the result of participation and collaboration of multiple genes. Further study of the newly-discovered differentially-expressed gene helps find out new radiosensitizational targets of elemene.     

少突胶质细胞电离辐射后早期基因表达谱的检测

目的 检测电离辐射后早期少突胶质细胞基因表达的变化特征.方法 将离体培养的少突胶质细胞经10 Gy照射后在1和4 h时间点,采用Affymetrix RAT 230 2.0基因表达谱芯片进行检测,并用实时荧光定量RT-PCR验证髓鞘碱性蛋白(MBP)和神经细胞间黏附分子1(NCAM-1)的测量结果.结果 少突胶质细胞经辐照后1和4 h内,其基因表达谱发生了明显的变化,这些基因有1079个已经命名并可以作功能分类.定量PCR表明MBP mRNA表达明显下调,而NCAM-1则上调,照射后4与1 h相比,表达量上调了3倍以上.结论 电离辐射后少突胶质细胞在几小时内就会发生一系列基因表达的变化,其中MBP和NCAM-1的改变可能在放射性脱髓鞘病变的发病机制中起重要作用.

Abstract：

ObjectiveTo characterize the gene expression in acute phase of irradiated oligodendrocytes (OL) in vitro.Methods The total RNA was extracted from irradiated OLs with 10 Gy by 6 MV X-rays at 1 and 4 h.The Affymetrix RAT 230 2.0 microarray were used to evaluate and screen the gene expression profile.The quantitative real-time RT-PCR was performed to validate the microarray results of selected myelin basic protein (MBP) and neural cell adhesion molecule 1 ( NCAM-1 ) genes.Results Compared with un-irradiated OLs,there were 1079 different expressed genes in irradiated cells.Those genes were classified in 79 categories based on the functional classification.Some familiar genes associated with OL cellular physiological process,apoptosis,cell cycle control,metabolism,cell communication and receptor binding were included.Compared with the microarray results,the coincidence rate of real-time RT-PCR was 91.7%.The down-regulation of MBP and up-regulation of NCAM 1 gene expression were confirmed.Conclusions Radiation-induced changes in gene expression in OLs took place in acute phase and influenced by time-course.The changes of MBP and NCAM1 gene expression may play a key role in the pathogenesis of radiation-induced demyelination.     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.