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目的探讨辐射损伤导致骨髓造血干/祖细胞(HSC/HPC)衰老的可能机制。方法雄性C57BL/6J小鼠随机分为辐照组和假辐照组,辐照组小鼠经6.5 Gy的X射线全身一次性辐照,假辐照组小鼠处理同辐照组,但不辐照。辐照后24 h免疫磁珠分选法分离并计数两组小鼠的Sca-1+造血干/祖细胞细胞(Sca-1+HSC/HPC),流式细胞术检测细胞周期,β-半乳糖苷酶(SA-β-Gal)染色检测衰老细胞百分率;混合造血祖细胞集落(CFU-Mix)培养观察分选细胞增殖分化能力,单细胞凝胶电泳(SCGE)检测辐照导致细胞的DNA损伤,RT-PCR检测细胞衰老相关基因p16INK4a、p19Arf、p53、p21Cip1/Waf1mRNA的表达;Westernblot法检测p16INK4a、p21Cip1/Waf1蛋白表达。结果免疫磁性分析法分离纯度的Sca-1+HSC/HPC可达94%,辐照后小鼠每支股骨的Sca-1+HSC/HPC数量急剧下降,细胞出现G1期阻滞;形成CFU-Mix集落数量和形成集落的细胞数明显降低;SA-β-Gal染色阳性率显著增高,彗星实验显示细胞拖尾明显延长;p16INK4a、p19Arf、p53、p21Cip1/Waf1mRNA表达明显增强,p16INK4a、p21Cip1/Waf1蛋白的表达水平上调。结论辐射导致HSC/HPC DNA的损伤和衰老相关生物学改变,p16INK4a-Rb和p19Arf-p53-p21Cip1/Waf1信号通路可能起一定作用。
Objective To investigate the possible mechanism of aging of bone marrow hematopoietic stem / progenitor cells (HSC / HPC) induced by radiation. Methods Male C57BL / 6J mice were randomly divided into irradiation group and sham irradiation group. The irradiated mice were irradiated with 6.5 Gy X-ray whole body. The mice in sham irradiation group were treated with irradiation group, but not Irradiation. The Sca-1 + HSC / HPC cells were isolated and counted by immunomagnetic bead sorting 24 h after irradiation. The cell cycle, beta-half The percentage of senescent cells was detected by the method of SA-β-Gal staining. The proliferation and differentiation of the sorted cells were observed by CFU-Mix culture. The single cell gel electrophoresis (SCGE) The expression of p16INK4a, p19Arf, p53 and p21Cip1 / Waf1 mRNA were detected by RT-PCR. The protein expression of p16INK4a and p21Cip1 / Waf1 were detected by Western blot. Results The purity of Sca-1 + HSC / HPC reached 94% after immunomagnetic analysis. The number of Sca-1 + HSC / HPC in each femur decreased sharply after irradiation, The number of colonies and the number of colonies were significantly decreased; the positive rate of SA-β-Gal staining was significantly increased; the expression of p16INK4a, p19Arf, p53, p21Cip1 / Waf1 mRNA was significantly increased in comet assay; Protein expression is upregulated. Conclusions Radiation can cause damage and aging-related biological changes of HSC / HPC DNA, and p16INK4a-Rb and p19Arf-p53-p21Cip1 / Waf1 signaling pathway may play a role.