辐射导致小鼠骨髓基质细胞衰老的机制研究

Mechanism of radiation induced premature senescence of bone marrow stromal cells: experiment with murine bone marrow stromal cells

目的探讨辐射导致骨髓基质细胞损伤的机制。方法应用BALB/c小鼠骨髓基质细胞体外放射损伤模型,分别于照射后24h、72h、1周、2周用四甲基偶氮唑盐比色法(MTT法)检测细胞增殖率;用Giemsa染色法观察细胞形态;用流式细胞仪检测细胞周期;用细胞化学法检测衰老相关β半乳糖苷(SA-β-gal)阳性细胞百分率;用实时荧光定量逆转录-聚合酶链反应(RT-PCR)分别检测周期蛋白激酶抑制因子p21Cip1/Waf1和p53的基因表达。结果8Gy60Co-γ射线照射后骨髓基质细胞增殖降低;流式细胞仪结果显示照射后骨髓基质细胞发生G0/G1期阻滞,照射后1周G0/G1期细胞百分率由正常的66·95%±0·36%增加至89·83%±0·05%(P<0·01);细胞体积增大变平;照射后24h、72h、1周、2周SA-β-gal染色阳性细胞百分率照射组(20·33%±0·03%,34·33%±0·03%,86·33%±0·02%,95·67%±0·02%)显著高于正常组(P<0·01);p53、p21Cip1/Waf1基因表达水平分别于照射后24h或72h即见升高,一直持续至照射后两周,与正常对照组相比差异均有统计学意义(P<0·01)。结论γ射线作用于小鼠骨髓基质细胞后,导致小鼠骨髓基质细胞早期衰老,p53-p21Cip1/Waf1通路在其中可能发挥重要的作用。

Objective To investigate the mechanism of radiation induced premature senescence of bone marrow stromal cells induced by γ-radiation. Methods Bone marrow stromal cells were isolated from the bones of mice, cultured, and divided into 2 groups： control group and irradiation group. The cells of the irradiation group were exposed to ^60Coγ-rays of the dose of 8.0 Gy. 24 h, 72 h, 1 week, and 2 weeks after irradiation MTT method was used to test the proliferation of the cells, Giemsa staining was used to observe the morphology, the percentage of cells positive in beta-galactosidase （SA-beta-Gal）, a senescence associated biomarker, was detected by cytochemistry, and RT-PCR was used to detect the mRNA expression of p21^Cipl/Wafl and p53 gene, both associated with senescence. One week after the irradiation flow cytometry was used to analyze the cell cycle distribution. Results Characteristics of mature senescence were seen in the cells of the irradiation group. One week after the irradiation the percentage of G0/G1 of the irradiation group was 89.83% ±0.05%, significantly higher than that of the control group （66. 95% ±0. 36%, P 〈 0. 01 ）. 24 h, 72 h, 1 week, and 2 weeks after irradiation the percentages of SA-beta-gal positive cells in the irradiation group were 20.33% ± 0.03% , 34.33% ± 0.03%, 86.33% ± 0.02% , and 95.67% ± 0.02% respectively, significantly higher than those of the control group （2.33% ±0. 006%, 14.33% ± 0. 02%, 24.67% ± 0.02%, and 45.00% ± 0.02% respectively, all P 〈 0.01 ）. The mRNA expression of p53 and p21^Cipl/Wafl mRNA expressions in the irradiation group increased 24 h after the irradiation and lasted for two weeks. Conclusion γ-radiation induces changes of premature senescence in bone marrow stromal cells in which p53-p21 ^Cipl/Wafl manner pathways may play an important role.