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目的探讨氯化锂(Li Cl)激活Wnt/β-catenin信号通路致小鼠Sca-1+造血干/祖细胞(Sca-1+HSC/HPC)衰老及其机制。方法免疫磁珠法分离纯化小鼠Sca-1+HSC/HPC。纯化后细胞分为:正常对照组,常规培养;Li Cl组,正常对照组基础上,加入Li Cl(终浓度10mmol/L);D-半乳糖致衰组,正常对照组基础上,加入D-半乳糖(终浓度166mmol/L),各组培养48h。造血祖细胞混合集落(CFU-Mix)培养检测Sca-1+HSC/HPC多向分化潜能,细胞计数试剂盒-8(CCK-8)检测Sca-1+HSC/HPC增殖能力,衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老,免疫细胞化学染色和Western blotting检测细胞内β-catenin、糖原合成激酶3β(GSK-3β)、P53、P21蛋白表达,ELISA检测细胞胞质内8羟基脱氧鸟苷(8-OH-d G)含量。结果与正常对照组相比,Li Cl组与D-半乳糖致衰组Sca-1+HSC/HPC增殖能力下降,形成CFU-Mix数量下降,SA-β-Gal染色阳性细胞百分率增加,β-catenin、P53、P21蛋白表达上调,GSK-3β蛋白表达下调,细胞内8-OH-d G水平升高。结论 Li Cl可通过激活Wnt/β-catenin信号通路,使细胞DNA氧化损伤,上调P53/P21途径,这可能是导致小鼠造血干/祖细胞衰老的机制之一。
Objective To investigate the mechanism of Sca-1 + HSC / HPC senescence induced by lithium chloride (LiCl) activation of Wnt / β-catenin signaling pathway. Methods Mouse Sca-1 + HSC / HPC was isolated and purified by immunomagnetic beads method. After purification, the cells were divided into: normal control group, routine culture; LiCl group, normal control group, adding LiCl (final concentration 10mmol / L); D-galactose induced decline group, - galactose (final concentration 166mmol / L), each group cultured for 48h. The multipotential differentiation ability of Sca-1 + HSC / HPC was detected by CFU-Mix. The proliferation of Sca-1 + HSC / HPC, The cell senescence was detected by the method of SA-β-Gal staining. The expression of β-catenin, GSK-3β, P53 and P21 were detected by immunocytochemistry and Western blotting. Cytoplasmic 8-hydroxy-2-deoxyguanosine (8-OH-d G) content. Results Compared with the normal control group, the proliferation of Sca-1 + HSC / HPC in LiCl group and D-galactose group decreased, the number of CFU-Mix formation decreased, the percentage of positive cells in SA- catenin, P53, P21 protein expression, GSK-3β protein expression downregulation of intracellular 8-OH-d G levels. Conclusion Li Cl can up-regulate the P53 / P21 pathway by activating Wnt / β-catenin signaling pathway and oxidative DNA damage, which may be one of the mechanisms that lead to the aging of mouse hematopoietic stem / progenitor cells.