电离辐射诱导神经干细胞衰老的模型研究

Establishment of ionizing radiation-induced neural stem cell aging model

目的研究神经干细胞在不同强度电离辐射(ionizing radiation,IR)处理下的衰老表型变化,建立电离辐射诱导神经干细胞衰老的方法。方法对成年鼠来源的神经干细胞进行不同剂量的连续多次电离辐射,细胞分为照射组(0 Gy组),低剂量连续辐照组(0.5 Gy组),中等剂量的隔代辐照组(2 Gy组),高剂量的每隔3代辐照组(8 Gy组),分别在短期处理和长期处理后,收集细胞样本,提取RNA,检测神经干细胞衰老相关基因(p16、Bmi1)的表达,并用Ki67免疫荧光染色检测细胞的增殖,用SA-β-Gal检测细胞的衰老状态。结果在长期处理后,p16在0.5 Gy、8 Gy组细胞中有20倍的表达升高,在2 Gy组细胞中有2倍的表达升高;Bmi1在3组细胞中表达都降低,并随着剂量增加降低幅度增加;Ki67免疫荧光染色结果显示3个处理组的细胞增殖速率都降低,但在长期处理后2 Gy组细胞增殖加快;SA-β-Gal检测结果显示,0.5 Gy组细胞衰老程度不变,8 Gy组衰老加重,2 Gy组衰老程度降低。结论对神经干细胞长期高剂量的每隔3代辐照处理可以诱导神经干细胞衰老,作为研究干细胞衰老的模型。

Objective To study the aging phenotype of neural stem cells under different dosage of ionizing radiation （IR）and to establish a cellular aging model. Methods The neural stem cells derived from adult mouse were divided into four groups： control （0 group, untreated）, low dosage （0.5 Gy group, IR = 0.5 Gy at every passage）, medium dosage （ 2 Gy group, IR = 2 Gy at every other passage） and high dosage（8 Gy group, IR = 8 Gy at every fourth passage）. After short and long term passage, the cell samples were collected. The expression of aging related genes was detected with qRT-PCR, cell proliferation was determined with Ki67 immunostaining and cell senescense was examined with SA-β-Gal staining. Results After long term treatment, compared with 0 group the expression level of p16 mRNA was 20-folder increased in 0. 5 Gy and 8 Gy group while only 2-folder rose in 2 Gy group. Bmil was down regulated in all 3 groups and the extent of decrease was positively related to increased dosage. The Ki67 immunostaining results showed decreased cell proliferation rate in all 3 groups but more dividing cells in 2 Gy group. SA-[^-Gal staining reveled more severe senescense state in 8 Gy group and unchanged state in 0. 5 Gy group while slightly improved condition of 2 Gy group compared to 0 group. Conclusion Ionizing radiation at dosage of 8 Gy at every fourth passage can induce cell aging of neural stem cells from young adult mouse, which may be used as a cellular aging model for further research.