CAJ | 학술논문

目的探讨中等剂量X射线对小鼠间充质干细胞的损伤及其机制.方法小鼠间充质干细胞株C3H10T1/2行3.5 Gy X射线照射,于照射后3、6、12、24、48和72 h及照射后1周采用Hoechst33258染色和Annexin V-FITC/P1染色检测细胞凋亡.于照射后24、48和72 h及照射后1周采用SA-β-gal染色检测细胞衰老.收集照射后3、6、12、24、48、72 h及1周的细胞,采用Real-TimePCR技术检测Fas及其配体FasL、p53及其下游组分p21WAF1 mRNA水平的表达.采用免疫荧光法检测照射后6、12和24 h细胞Fas蛋白的表达.结果照射后3h细胞凋亡率开始增加11.72％±1.61％(t=9.01,P＜0.01),至12 h达高峰20.52％±1.96％ (t=16.27,P＜0.01),48 h已基本恢复正常4.93％ ±0.46％(t=2.26,P＞0.05).照射后72 h细胞SA-β-gal阳性率为53.33％±5.62％,较正常组3.24％±0.39％明显升高(t=17.77,P＜0.01),即照射后72 h出现细胞衰老高峰.Fas、FasL mRNA在照射后3h开始增高,12h达到高峰,1周后恢复正常.Fas蛋白在照射后12h达到高峰.Fas、FasL mRNA及Fas蛋白的表达高峰时相与细胞的凋亡高峰时相一致.p53、p21WAF1mRNA在照射后12h短暂升高,72 h出现大幅度升高,p53、p21WAF1 mRNA的双峰分别与细胞的凋亡高峰和衰老高峰相吻合,且两者在细胞衰老时相的表达均明显高于在细胞凋亡时相的表达(t=17.85、13.26,P＜0.01).结论小鼠间充质干细胞C3H10T1/2对中剂量X射线较敏感,照射后出现了早期凋亡晚期衰老的损伤规律,Fas/FasL通路和p53/p21WAF1通路参与了中剂量X射线照射后小鼠间充质干细胞C3H10T1/2的损伤过程.
목적 탐토중등제량X사선대소서간충질간세포적손상급기궤제.방법 소서간충질간세포주C3H10T1/2행3.5 Gy X사선조사,우조사후3、6、12、24、48화72 h급조사후1주채용Hoechst33258염색화Annexin V-FITC/P1염색검측세포조망.우조사후24、48화72 h급조사후1주채용SA-β-gal염색검측세포쇠로.수집조사후3、6、12、24、48、72 h급1주적세포,채용Real-TimePCR기술검측Fas급기배체FasL、p53급기하유조분p21WAF1 mRNA수평적표체.채용면역형광법검측조사후6、12화24 h세포Fas단백적표체.결과 조사후3h세포조망솔개시증가11.72％±1.61％(t=9.01,P＜0.01),지12 h체고봉20.52％±1.96％ (t=16.27,P＜0.01),48 h이기본회복정상4.93％ ±0.46％(t=2.26,P＞0.05).조사후72 h세포SA-β-gal양성솔위53.33％±5.62％,교정상조3.24％±0.39％명현승고(t=17.77,P＜0.01),즉조사후72 h출현세포쇠로고봉.Fas、FasL mRNA재조사후3h개시증고,12h체도고봉,1주후회복정상.Fas단백재조사후12h체도고봉.Fas、FasL mRNA급Fas단백적표체고봉시상여세포적조망고봉시상일치.p53、p21WAF1mRNA재조사후12h단잠승고,72 h출현대폭도승고,p53、p21WAF1 mRNA적쌍봉분별여세포적조망고봉화쇠로고봉상문합,차량자재세포쇠로시상적표체균명현고우재세포조망시상적표체(t=17.85、13.26,P＜0.01).결론 소서간충질간세포C3H10T1/2대중제량X사선교민감,조사후출현료조기조망만기쇠로적손상규률,Fas/FasL통로화p53/p21WAF1통로삼여료중제량X사선조사후소서간충질간세포C3H10T1/2적손상과정.
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.