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Objective:To explore the effects of ultraviolet B (UVB) on the premature senescence of human immortalized keratinocytes HaCaT cells and the possible underlying molecular mechanism.Methods:HaCaT cells were exposed with UVB of different doses (20, 50, 80 and 100 mJ/cm 2). At 72 h after exposure, cellular morphology was observed by Giemsa staining, cell proliferation was detected by clone formation assay, and the proportion of premature senescence cells was detected by β-galactosidase staining. The number change of lysosomes was detected by Lyso-Tracker Red fluorescence probe at 24, 48 and 72 h after exposure. Cell migration was measured by scratch test at 24 h and 48 h after exposure. The protein expressions of p53 and p16 related to premature senescence were detected by Western blot assay at 72 h after exposure. Results:After UVB exposure, HaCaT cells showed a premature senescence phenotype. At 72 h after exposure, the cell volume increased ( F=115.18, P<0.05), the cell proliferation ability decreased ( F=410.32, P<0.05), the activity of β-galactosidase increased ( F=16.31, P<0.05), and the expressions of P53 and P16 increased. In addition, the number of lysosomes increased at 24, 48, and 72 h after exposure ( F=17.65, 38.36, 13.66, P<0.05), and cell migration capacity was inhibited at 24 and 48 h after exposure ( F=8.21, 11.48, P<0.05). Conclusions:UVB exposure can induce premature senescence of HaCaT cells by increasing the expression of p53 and p16 proteins.
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Objective:To investigate the feasibility of dose estimation based on the dose-response curves established by different types of dicentrics (dic).Methods:Human peripheral blood samples were irradiated by 60Co γ-rays at the doses of 0, 0.5, 1, 2, 3, 4, 5 and 6 Gy with the dose rate of 0.27 Gy/min, and chromosome preparation was carried out. The different types of dics were analyzed. The dose-response curves were fitted with software of Chromosomal Aberration Calculation Software (CABAS). Two samples were used to validate the dose-response curves. Results:The aberration rates of different types of dics increased with the exposure doses ( R2=0.886-0.943, P<0.01). The proportion of typical and single-ended dic components accounted for more than 92% of all types of dic, while the proportion of short-range dic and double-ended dic components was less than 4% at each dose point. The dose-response curves based on different types of dics were established ( R2=0.998, P<0.01). There was no significant difference in the doses estimated by four different dose-response curves ( P>0.05). The relative deviations between actual and predict doses were less than 13.08% by using dose-response curve based on typical dics. Conclusions:Dose-response curves based on different types of dics have the feasibility of dose estimation.
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Objective To explore the influences of the final concentration and adding time of Cytochalasin-B (Cyt-B) on radiation-induced nucleoplasmic bridges (NPB) in cytokinesis-block assay.Methods Hunan peripheral blood samples were divided into 5 final concentration groups (group 2,4,6,8,10 μg/ml) according to different final concentrations of Cyt-B.Moreover,blood samples were divided into 4 adding time groups (group 0,28,40,44 h) according to different adding times of Cyt-B.Blood samples were irradiated with 0 (sham irradiation) and 2 Gy 60Co-rays in vitro,at a dose rate of 1 Gy/min.A cytokinesis-block assay was carried out to prepare NPB samples.The percentages of mononucleated,binucleated and multinucleated cells,as well as the frequencies of NPB and micronucleus (MN) in binucleated cells were analyzed using an optical microscope.Results Nuclear division index (NDI) and the percentages of binucleated cells increased with increased concentration of Cyt-B,and decreased with delayed adding time of Cyt-B (except group 0 h) in both final concentration groups and adding time groups.After exposed to 2 Gy,NPB frequencies were no significant difference (except group 0 h).MN frequencies had the trend of decreased with the increased concentration of Cyt-B,but no significant difference with adding time of Cyt-B.Conclusions In cytokinesis-block assay,different final concentration and adding time of Cyt-B may induce to the variation of NPB frequencies,but there was no significant difference.Appropriate increased final concentration or ahead adding time of Cyt-B can increase the percentage of binucleated cells that help to improve the efficiency of analysis.
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Objective To investigate the dose response of S100A4 gene expression in the irradiated lymphoblastoid cells AHH-1 at different time points post irradiation.Methods AHH-1 cells was exposed to different doses(0,1,3,5,8,10,15 and 18 Gy)of 60Co γ-rays,and its mRNA levels of S100A4 was detected by reverse transcription PCR and real-time PCR at 4,8,12,24,48 and 72 h after irradiation.Results Within the range of applied doses,the level of S100A4 gene expression was upregulated with a good dose-response (R2 =0.79-0.93,P ＜ 0.05) and had obvious difference at different time points (F =8.91,P ＜ 0.01).Conclusion S100A4 gene expression at transcriptional level could be detected easily and had optimum dose-responses at certain time points after irradiation,and hence is applicable as a dosimeter.
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OBJECTIVE:To observe the clinical efficacy and ADR of ultrasound debridement combined with Huangma tinc-ture in the treatment of diabetic foot ulcer. METHODS:90 diabetic foot ulcer patients were randomly divided into combination treatment group(ultrasound debridement combined with Huangma tincture wet compress),Huangma tincture group(Huangma tinc-ture wet compress) and control group (vaseline) with 30 cases in each group. 3 months after treatment,Ulcer healing rate,cure time,effective time,recovery time,cure rate and ADR were observed in each group. RESULTS:The ulcer healing rate of combi-nation treatment group was significantly higher than that of Huangma tincture group and control group,with statistical significance (P<0.05);there was statistical significance between Huangma tincture group and control group(P<0.05). Mean cure time,effec-tive time and recovery time of combination treatment group were all significantly lower than Huangma tincture group and control group,with statistical significance(P<0.05);there was statistical significance between Huangma tincture group and control group (P<0.05). Cure rate of combination treatment group was higher than that of Huangma tincture group and control group,with statis-tical significance (P<0.05);there was statistical significance between Huangma tincture group and control group (P<0.05). No ADR was found in combination treatment group and Huangma tincture group. CONCLUSIONS:The ultrasound debridement com-bined with Huangma tincture in the treatment of diabetic foot ulcer improve healing rate and shorten healing time significantly with-out obvious ADR. It has good clinical efficacy.
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Objective To establish the analysis criteria of nucleoplasmic bridges (NPB) and a dose-response curve of NPB frequencies in human peripheral blood lymphocytes irradiated by 60Co γ-rays.Methods Human peripheral blood samples were collected from three healthy males,and were irradiated with 0,1,2,3,4,5 and 6 Gy of 60Co γ-rays.A cytokinesis-block micronucleus assay was carried out to analyze NPBs and micronuclei (MN) in binucleated cells.Results NPBs in binucleated cells at each dose level of γ-ray was conformed to the Poisson distribution.The dose-response curve of the γ-ray-induced NPB frequencies in human peripheral lymphocytes followed the linear-quadratic model y =(1.39 × 10-3)x2 + (4.94 × 10-3)x (R2 =0.981,P ＜ 0.05).Conclusions The dose-response curve of NPB frequencies in human peripheral blood lymphocytes induced by 0-6 Gy 60Co γ-rays was established.
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Objective For developing safe and effective anti-radiation new drugs,the effects of different lipoic acid amino acid salts on radiosensitivity were investigated.Methods The free radical scavenging ability of the above salts was evaluated in Fenton system.CCK-8 assay and flow cytometry assay were performed to evaluate the survival rate of L02 and apoptosis of AHH-1 after γ-ray irradiation,respectively.Results The lipoic acid arginine salt had the best ability of scavenging free radicals in Fenton system with an IC50 of 8.40 μ moL/ml.The survival assay showed that lipoic acid amino acid salts had better stability and equal ability in radioprotection (P ＞ 0.05) compared with lipoic acid.The apoptosis assay indicated that all lipoic acid amino acid salts could inhibit radiation-induced apoptosis,where lipoic acid arginine salt was more effective (t =-6.67,P ＜ 0.01).Conclusions Lipoic acid arginine salt has good radioprotection effect on L02 and AHH-1 cells by scavenging free radicals.
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Objective To investigate the dose-effect relationship of mRNA level of sensitive mitochondrial genes in human lymphoblastoid cells induced by ionizing radiation.Methods Seven human sensitive genes,including ND3,Cyt b,COX Ⅰ,COX Ⅱ,COX Ⅲ,ATPase6 and ATPase8 were chosen.Changes of mRNA level of these genes were detected by RT-PCR and Real-Time PCR at 24 h after irradiation in human lymphoblastoid cells,which were exposed to 0 - 15 Gy of 60 Co γ-rays.Results The expression of these 7 genes at mRNA level was up-regulated 24 h after irradiation in human lymphoblastoid cells.The level of gene expression of COX Ⅰ,which belongs to complex Ⅳ of mitochondrial respiratory chain,was most obvious,and the peak occurred after irradiation of 8 Gy,which was 13 times of the control group.A good dose-effect relationship was showed for COX Ⅲ gene expression at dose range of 3 -10 Gy as well as 3 - 15 Gy for other 3 genes including ND3,ATPase6 and ATPase8.Conclusions Gene expression levels of COX Ⅲ,ND3,ATPase6 and ATPase8 24h post-irradiation at certain irradiation dose range could be used for radiation damage biomarkers.
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Objective To reconstruct the absorbed dose for the individual who was accidentally exposed in uterus 16 years ago.Methods Peripheral blood samples were taken from the victim and her mother.The dicentric and centric ring chromosome aberrations were analyzed with conventional method,the micronucleus was observed with cytokinesis-block micronucleus method,and fluorescence in situ hybridization (FISH) with chromosomes 1,2 and 4 painting probes were used for translocation detection.Dose was estimated according to the standard dose-response curves previously established.Results No unstable chromosome aberrations and normal micronucleus frequencies were observed in two persons 16years after the accident.Against the established dose-response curves with FISH,the doses to the mother and her daughter were 0.76 Gy (95% GI 0.41-1.00 Gy) and 0.61 Gy (95% CI 0.44-0.86 Gy),respectively.Because the biological dose estimated for the mother 1 month after the accident was 2.30 Gy (95% CI 2.07-2.50 Gy),the dose correction factor was 3.03 for dose estimation 16 years later.The estimated dose in uterus to the victim was 1.85 Gy (95% CI 1.33-2.61 Gy).Conclusions The estimated dose to the individual accidentally exposed in uterus 16 years ago can be obtained according to the dose correction factor of the mother with FISH method.
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Objective To explore the changes of human mitochondrial COX Ⅰ , COX Ⅱ and COX Ⅲ genes expression induced by ionizing irradiation. Methods Changes of human COX genes expression were detected by RT-PCR and Real-time PCR 8 h after the irradiation in human lymphoblastoid cell lines,which were exposed to 1-10 Gy60Co γ-rays. The protein levels were detected by flow cytometry and the COX activity was measured by colorimetry. The dose-effect relationships between the expression changes of the genes and the doses were established. The changes of these genes expression were also analyzed at different post-radiation time-points between 0. 5 h and 72 h after irradiation of 5 Gy in order to explore the time-effect. Results The expression of 3 genes at mRNA level was up-regulated. A good dose-effect relationship was showed for COXⅠ and COX Ⅲ at dose range of 0-3 Gy and 0-8 Gy for COX Ⅱ ( F COXⅠ=116. 62, FCOXⅡ = 17. 89, FCOXⅢ = 8.20, P ＜ 0. 05). For the time-effect after irradiation, the gene expression levels of COX Ⅱ and COX Ⅲ genes were up-regulated and the peak change occurred at 4 h after irradiation. For COX Ⅰ gene, the mRNA expression levels were down-regulated during 0.5-72 h( FCOXⅠ =31.99, FCOXⅡ = 19.47, FCOXⅢ = 20. 64, P ＜0. 05 ). At the protein level, the levels of COX Ⅰ and COX Ⅱ were lowered in lower doses and enhanced in higher doses, and the levels of COX Ⅲ were decreased at all dose levels (FCOX Ⅰ = 16.96, FCOXⅡ = 32.5, FCOXⅢ = 6. 51, P ＜ 0. 05 ). The protein levels of COX Ⅰ and COX Ⅱ were enhanced during 4-72 h and 8-72 h respectively after 5 Gy irradiation ( FCOX Ⅰ = 14.68,FCOXⅡ = 17. 18, FCOXⅢ =2. 52, P ＜0. 05). The activities of COX were lowered at different dose levels and different time-points. Conclusions Ionizing radiation might induce the changes in mitochondrial COX Ⅰ,COX Ⅱ and COX Ⅲ gene expression, and lead to the reduction of the COX activities.
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Objective To explore the changes of human mitochondrial COXI,ND1 and ND6 genes expression induced by ionizing irradiation.Methods Changes of human COXI,ND1 and ND6 gene expression were detected by RT-PCR and Real-time PCR 8 h after the irradiation in human lymphoblastoid cell lines,which were exposed to 1-10 Gy 60Co γ-rays.And the dose-effect relationships between expression changes of the genes and the doses were analyzed.The changes of these three genes expression were also analyzed at different post-radiation time-points between 0.5 h and 72 h after irradiation of 5 Gy in order to explore the time-effect.Results The expression of three genes COXI,ND1 and ND6,showed either the dose-effect or the time-effect after irradiation.The gene expression levels of three genes up-regulated generally and the peak change time-point was 4 h after irradiation.Conclusion Ionizing radiation,msht induce the changes of mitochondrial gene expression,and the gene expression level is up-regulated.
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Objective To establish the immortalized cell lines of peripheral blood lymphocytes for old male residents in high background radiation area(HBRA)in Guangdong,China,in order to preserve the specific genomic resources of residents in HBRA for the further genetic and molecular biological study on HBRA.Methods The peripheral blood samples of 20 old male residents in HBRA were collected after informed consent.The immortalized B lymphoblastoid cell lines,2 for each resident,were established with Epstein-Barr virus.After being frozen and recovered,the cell viability,the contamination of bacterium and mycoplaama were analyzed.The stabilization of cell lines was decided by comparing the karyotypes of the peripheral blood lymphocytes and the cell lines.Results 40 cell lines for 20 residents in HBRA were successfully established.The recovery rate of cell lines after being frozen was 100%.All the cell viablity after recovery was higher than 90%.and no contamination of bacteria and mycoplasma occurred.The karyotypes of the 20th generation cell lines were not change.Conclusion The immortalized cell lines established in this study could provide biological resources for further study on genetics and molecular biology in HBRA.
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Objective To explore if the occupational exposure to low dose thorium could induce adaptive response in peripheral lymphocytes.Methods 40 individuals.who exposed to thorium and rare earth mixed dust(exposure group) or control in Baotou Steel Plant, were selected, and chromosome aberrations were analyzed.Then the peripheral blood samples were irradiated in vitro with 2 Gy60Co γ-rays,and unstable chromosome aberration or DNA stand breakage analysis using single cell gel electrophoresis was performed. Results The dicentrics before 2 Gy exposure in exposure group was higher than that in control(P>0.05). But the dicentries after 2 Gy exposure in exposure group was lower than that in control,but not significantly (P>0.05).The tricentrics in exposure group was significantly lower than that in control(U=3.1622, 0.0ol<P<0.002).The DNA strand breakage in control group was significantly higher than that in exposure group(t=25,P<0.001).Conclusions Occupational exposure to low dose thorium could induce the adaptive response on chromosome aberration and DNA strand breakage in peripheral lymphocytes.