| 苏文霞,刘荟敏,陈永红,曾雯,刘文励,孙汉英.3.5 Gy X射线对小鼠间充质干细胞的损伤及其机制的研究[J].中华放射医学与防护杂志,2011,31(6):644-648 |
| 3.5 Gy X射线对小鼠间充质干细胞的损伤及其机制的研究 |
| Response of mesenchymal stem cells in mice to 3.5 Gy X-ray irradiation |
| 投稿时间：2010-12-27 |
| DOI：10.3760/cma.j.issn.0254-5098.2011.06.006 |
| 中文关键词: 照射 间充质干细胞 凋亡 衰老 |
| 英文关键词:Irradiation Mesenchymal stem cell Apoptosis Senescence |
| 基金项目:教育部博士点基金(200804870008);湖北省卫生厅基金(JX4B04) |
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| 中文摘要: |
| 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/PI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-Time PCR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3 h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3 h开始增高,12 h达到高峰,1周后恢复正常。Fas蛋白在照射后12 h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12 h短暂升高,72 h出现大幅度升高,p53、p21WAF1mRNA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Χ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Χ射线照射后小鼠间充质干细胞C3H10T1/2的损伤过程。 |
| 英文摘要: |
| Objective To investigate the response of mesenchymal stem cells in mice to medium-dose Χ-ray irradiation in vitro. Methods The mouse mesenchymal stem cell line C3H10T1/2 was submitted to 3.5 Gy Χ-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 p21WAF1 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 1 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, p21WAF1 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 Χ-ray irradiation. Cell apoptosis occurred immediately after irradiation and cellular senescence happened at advanced stage. Both Fas/FasL and p53/ p21WAF1 signal pathway mediate the injury of C3H10T1/2 cell to medium-dose X-ray irradiation exposure. |
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