系统性红斑狼疮女性患者骨髓间充质干细胞衰老与细胞内活性氧的相关性研究

Association of bone marrow mesenchymal stem cells and intracellular reactive oxygen species levels in patients with systemic lupus erythematosus

目的探讨系统性红斑狼疮（SLE）患者骨髓间充质干细胞（BMSCs）的衰老与细胞内活性氧（ROS）水平的相关性。方法采用密度梯度离心和贴壁分离法分离培养8例SLE患者及8名健康者BMSCs；免疫荧光法检测细胞核大小；通过检测衰老相关B-半乳糖苷酶（SA β-gal）活性观察BMSCs体外培养过程中的衰老现象；流式细胞术检测BMSCs细胞内活性氧ROS表达水平；实时荧光定量聚合酶链反应（PCR）技术检测基因P13K、NRas、KRas和FoxO3的表达水平；Western blot法检测Fox03、磷酸化－FoxO3、AKT和磷酸化－AKT的表达水平；采用t检验或Mann—Whitney秩和检验。结果①在体外培养过程中，SLE患者和健康人BMSCs的形态和细胞核大小差异无统计学意义[（31±4）与（28±5）μm，P=0．628]；②SLE患者BMSCsSAβ-gal活性显著高于健康人[（31．8±9．0）％与（12．4±0．7）％，P〈0．05]；⑧SLE患者BMSCs细胞内ROS水平高于健康人（34600±9600与17958±5400，P〈0．05）；④SLE患者BMSCs基因P13K、NRas、KRas和FoxO3在mRNA表达水平与健康人比较差异无统计学意义（P均〉0．05）；⑤SLE患者BMSCs磷酸化-FoxO3以及磷酸化-AKT表达水平较健康人有所升高。结论SLE患者BMSCs在体外培养过程中，较健康人容易衰老，细胞内ROS表达水平升高，其机制可能与P13K／AKT信号通路活化，进而促进FoxO3磷酸化使其活性降低有关。

Objective To explore the role of intracellular reactive oxygen species （ROS） in the senescence of bone marrow mesenchymal stem cells （BMSCs） in patients with systemic lupus erythematosus （SLE） and the underlying mechanisms that controls the intracellular ROS levels in vitro. Methods Human bone marrow aspirates were collected from iliac of eight donors and eight SLE patients and cultured in vitro. Morphological appearance of BMSCs at different passages was examined by inverted microscope. Nuclear size was measured by fluorescence microscope. BMSCs were monitored using the senescence associated β-galacto- sidase （SAβ-gal） assay to characterize senescence in vitro. The quantification of intracellular ROS production was detected by flow cytometry. Real-time PCR technique was used to determine the gene expressions of PI3K, KRas, NRas and FoxO3 at transcription level. The expression of FoxO3, phospho-FoxO3 （p-FoxO3）, AKT and phospho-AKT （p-AKT） protein were determined by Western blot analysis. Statistical analysis was conducted with t-test and Mann-Whitney rank test. Results There were no differences in morphology and nuclear size [ （31.4 ±4.5 ） vs （28.2±4.8） μm, P=0.628 ] of BMSCs between SLE patients and normal controls. The percentage of SA [3-gal positive BMSCs from SLE patients was higher than that from healthy controls [（31.8±9.0）% vs （12.4±0.7）%, P〈0.05]. Intracellular ROS levels of BMSCs from SLE patients increased more significantly than healthy donors in vitro （34 600±9600 vs 17 958±5400, P〈0.05）. No significant differences in the expression of PI3K, NRas, KRas and FoxO3 from SLE subjects were observed at mRNA levels compared with normal controls, though all showed a similar upward trend. The expression of p-FoxO3 and p-AKT of BMSCs from SLE patients increased significantly compared with healthy controls at protein levels. Conclusion These data suggest that BMSCs from SLE patients aged more quickly, with high SA β-gal activity and up-regulation of intracellular ROS, which is associated with up-regulation of p-FoxO3 and p- AKT at protein levels. These results indicate that bone marrow mesenchymal cell senescence may be associated with the pathogenesis of SLE by maintaining the lifespan of BMSCs.