Response of mesenchymal stem cells in mice to 3.5 Gy X-ray irradiation

Objective To investigate the response of mesenchymal stem cells in mice to mediumdose X-ray irradiation in vitro.Methods The mouse mesenchymal stem cell line C3H10T1/2 was submitted to 3.5 Gy X-ray irradiation.Hoechst33258 staining of adherent cells and Annexin V-FITC staining and flow cytometry analysis of suspension cells were performed respectively to assess cellular apoptosis at 3,6,12,24,48,72 h and 1 week after irradiation.SA-β-gal staining was performed to analyze the cellular senescence at 24,48,72 h and 1 week after irradiation.The mRNA level of both Fas with its ligand FasL and p53 with its downstream target p21 WAF1 were measured by Real-Time PCR analysis.The expression of Fas protein was determined by immunofluorescence staining.Results An increased apoptosis was observed at 3 h after irradiation with apoptosis rate 11.72％ ± 1.61％ ( t =9.01,P ＜0.01 ),the apoptosis rate reached the peak level at 12 h 20.52％ ± 1.96％ (t =16.27,P ＜ 0.01 ),and then declined progressively to normal level at 48 h 4.93％ ±0.46％ (t =2.26,P ＞0.05).The SA-β-gal positive rate of post-radiation cells at 72 h was 53.33％ ± 5.62％,significantly higher than that of normal control 3.24％ ± 0.39％ (t =17.77,P ＜ 0.01 ).The level of Fas,FasL mRNA was found to be elevated 3 h after irradiation with a peak at 12 h,and no differences were found l week later.The level of Fas protein was observed to reach the peak at 12 h after irradiation.The occurrence of peak level of Fas/FasL mRNA and protein was consistent with that of apoptosis of C3H10T1/2 cell.A transient up-regulation of p53,p21 WAF1 mRNA expression was found at 12 h after irradiation followed by a significant increase later at 72 h after irradiation.The occurrence of the two peaks of p53,p21WAF1 mRNA expression were coincident with that of cellular apoptosis and senescence,respectively.The levels of p53,p21WAF1 mRNA in senescence group were significantly higher than those of apoptosis group ( t =17.85,13.36,P ＜ 0.01 ).Conclusions The MSC cell line C3H10T1/2 was sensitive to medium-dose X-ray irradiation.Cell apoptosis occurred immediately after irradiation and cellular senescence happened at advanced stage.Both Fas/FasL and p53/p21 WAF1 signal pathway mediate the injury of C3H10T1/2 cell to medium-dose X-ray irradiation exposure. Key words: Irradiation ;  Mesenchymal stem cell;  Apoptosis ;  Senescence