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Objective:To analyze the damage in hippocampal tissues of mice after whole-body irradiation with high- or low-dose ionizing radiation and to investigate the roles of microglia/macrophages polarization in the injury.Methods:C57BL/6 mice were randomly divided into three groups: sham irradiation group, low-dose group (0.05 Gy) and high-dose group (7 Gy). Low- and high-dose groups were respectively treated by whole-body irradiation with single dose of 60Co γ-rays. Hippocampal tissues of the mice were collected at 6 h, 1 d, 3 d and 7 d after irradiation. The morphology, structure and apoptosis of neurons were detected by HE staining, Nissl staining and Tunnel staining, respectively. RT-PCR and immunofluorescence assay were performed to detect the expression of M1 and M2 microglial markers at mRNA and protein levels in hippocampus tissues. The cognitive and emotional behaviors of mice were evaluated one month after the irradiation by Morris water maze, open field test, elevated plus maze and tail suspension test. Results:There were morphological and structural changes in the nerve cells in the hippocampus region of mice after irradiation, accompanied by apoptosis. Acute injuries occurred at 6 h after radiation, alleviated at 1 d and 3 d, and persisted at 7 d in a dose-dependent manner. The results of immunofluorescence staining and confocal imaging analysis showed that compared with the sham irradiation group, the high-dose group showed increased number of microglia, down-regulated expression of M1 microglial markers and up-regulated expression of M2 microglial markers in the hippocampus at 6 h and 1 d after radiation, while M2 microglial markers decreased at 3 d and 7 d after irradiation. PCR results showed that the expression of M1 and M2 microglial markers at mRNA level in the irradiation groups increased at 6 h after irradiation, but there was no statistical significance. The expression of related proinflammatory/anti-inflammatory factors was significantly up-regulated. The results of behavioral experiments showed that compared with the sham irradiation group, there was no statistical difference in cognitive or emotional behaviors at one month after irradiation.Conclusions:60Co γ-rays could damage mouse hippocampal tissues and result in the overexpression and different polarization patterns of microglia/macrophages in mice.
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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 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 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 elucidate the function and regulatory mechanism of LyGDI involved delayed cell death in the human K562 cells and HL-60 cells induced by 60Co γ-rays. Methods Erythrosine B cells staining was used to count the apoptosis rate. PI staining and flow cytometry were applied to check the cell cycle. The expression of LYGDI and Rac1 was resolved by Western blot by using monoclonal antibody of LyGDI and Racl. The distribution of Racl protein in cells was observed with immunofluorescence by using the confocal microscope. Results The K562 cells showed G2/M phase arrest and the percent age was 71.3%. The apoptosis rate was very low at early post-irradiation stage in the K562 cells. The apoptosis rate was 14% in the K562 cells at 24 h post-irradiation with 8 Gy of γ-rays, and delayed cell apoptosis was present. LyGDI was cleaved in the K562 cells irradiated by 4 Gy 60 Co γ-rays after 24 hours post-irradiation. The expression of Racl protein was not altered at all, but the distribution was changed in the irradiated cells while the Racl protein moved to cell membrane and a little in cell nucleus. The Racl was activated with the losing the binding affinity with the LyGDI. Conclusion LyGDI could promote the delayed cell apoptosis, which is through the activation of the Rac1.
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Objective To study the bystander effects and associated mechanisms through irradiated conditioned medium(1CM). Methods Natural kilhr(NK) cells were obtained from peripheral blood samples. ICM irradiated with different doses of 60Coγ-rays was used for culturing K562 cell strain. The degree of injury of K562cells by activated NK cells was observed, as well as the apoptosis frequency of K562 cell was investigated. Results Severe injury was induced in K562 cells cultured in ICM than the control (sham-irradiated) as shown by increased sensitivity to NK cells (P < 0.05). The apoptosis frequency of K562 cell was increased significantly compared with the control cells (P < 0.05). Conclusions The bystander effect induced by irradiation is existent. ICM can trigger the bystander effect on K562 cell strains.
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Objective To study the effect and mechanism of polypeptide from Chlamys farreri(PCF) on the thymocytes damaged by 60Co γ-rays. Methods The ceils were randomly divided into six groups: control group, 60Co groups (2 Gy, model),60Co + 0.5% PCF, 60Co+ 0.25% PCF, 60Co + 0.125% PCF, 60Co +0.1% VitC. The concentration of GSH-Px, ROS, A-SAC, T-AOC and the cells' viability were determined. The mitochondria membrane potential were tested. The expressions of P53, Bax and Bel-2 proteins were examined.Results The activities f GSH-Px, A-SAC, T-AOC in cells were enhanced, and the amounts of ROS were decreased by PCF. The expression of Bcl-2 gene was up-regulated, and down-regulated for the expression of P53 and Bax. All observed indexes of the PCF groups were significantly different compared with model group (P <0.05). Conclusions PCF has the protective effects on damages of thymocytes caused by 60Co irradiation. The mechanisms might be related to inhibiting of lymphocyte apoptosis and enhancing the activities of lymphocytes.