中华风湿病学杂志
中華風濕病學雜誌
중화풍습병학잡지
CHINESE JOURNAL OF RHEUMATOLOGY
2011年
4期
229-233
,共5页
孟德芳%刘蕾%汤郁%施冬艳%张继云%王丹丹%孙凌云
孟德芳%劉蕾%湯鬱%施鼕豔%張繼雲%王丹丹%孫凌雲
맹덕방%류뢰%탕욱%시동염%장계운%왕단단%손릉운
红斑狼疮,系统性%衰老%间质干细胞%活性氧
紅斑狼瘡,繫統性%衰老%間質榦細胞%活性氧
홍반랑창,계통성%쇠로%간질간세포%활성양
Lupus erythematosus,systemic%Aging%Mesenchymal stem cells%Reactive oxygen species
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β-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PI3K、NRas、KRas和FoxO3的表达水平;Western blot法检测FoxO3、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BMSCs SA β-gal活性显著高于健康人[(31.8±9.0)%与(12.4±0.7)%,P＜0.05];③SLE患者BMSCs细胞内ROS水平高于健康人(34 600±9600与17 958±5400,P＜0.05);④SLE患者BMSCs基因PI3K、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PI3K/AKT信号通路活化,进而促进FoxO3磷酸化使其活性降低有关.
目的 探討繫統性紅斑狼瘡(SLE)患者骨髓間充質榦細胞(BMSCs)的衰老與細胞內活性氧(ROS)水平的相關性.方法 採用密度梯度離心和貼壁分離法分離培養8例SLE患者及8名健康者BMSCs;免疫熒光法檢測細胞覈大小;通過檢測衰老相關β-半乳糖苷酶(SA β-gal)活性觀察BMSCs體外培養過程中的衰老現象;流式細胞術檢測BMSCs細胞內活性氧ROS錶達水平;實時熒光定量聚閤酶鏈反應(PCR)技術檢測基因PI3K、NRas、KRas和FoxO3的錶達水平;Western blot法檢測FoxO3、燐痠化-FoxO3、AKT和燐痠化-AKT的錶達水平;採用t檢驗或Mann-Whitney秩和檢驗.結果 ①在體外培養過程中,SLE患者和健康人BMSCs的形態和細胞覈大小差異無統計學意義[(31±4)與(28±5)μm,P＝0.628];②SLE患者BMSCs SA β-gal活性顯著高于健康人[(31.8±9.0)%與(12.4±0.7)%,P＜0.05];③SLE患者BMSCs細胞內ROS水平高于健康人(34 600±9600與17 958±5400,P＜0.05);④SLE患者BMSCs基因PI3K、NRas、KRas和FoxO3在mRNA錶達水平與健康人比較差異無統計學意義(P均＞0.05);⑤SLE患者BMSCs燐痠化-FoxO3以及燐痠化-AKT錶達水平較健康人有所升高.結論 SLE患者BMSCs在體外培養過程中,較健康人容易衰老,細胞內ROS錶達水平升高,其機製可能與PI3K/AKT信號通路活化,進而促進FoxO3燐痠化使其活性降低有關.
목적 탐토계통성홍반랑창(SLE)환자골수간충질간세포(BMSCs)적쇠로여세포내활성양(ROS)수평적상관성.방법 채용밀도제도리심화첩벽분리법분리배양8례SLE환자급8명건강자BMSCs;면역형광법검측세포핵대소;통과검측쇠로상관β-반유당감매(SA β-gal)활성관찰BMSCs체외배양과정중적쇠로현상;류식세포술검측BMSCs세포내활성양ROS표체수평;실시형광정량취합매련반응(PCR)기술검측기인PI3K、NRas、KRas화FoxO3적표체수평;Western blot법검측FoxO3、린산화-FoxO3、AKT화린산화-AKT적표체수평;채용t검험혹Mann-Whitney질화검험.결과 ①재체외배양과정중,SLE환자화건강인BMSCs적형태화세포핵대소차이무통계학의의[(31±4)여(28±5)μm,P＝0.628];②SLE환자BMSCs SA β-gal활성현저고우건강인[(31.8±9.0)%여(12.4±0.7)%,P＜0.05];③SLE환자BMSCs세포내ROS수평고우건강인(34 600±9600여17 958±5400,P＜0.05);④SLE환자BMSCs기인PI3K、NRas、KRas화FoxO3재mRNA표체수평여건강인비교차이무통계학의의(P균＞0.05);⑤SLE환자BMSCs린산화-FoxO3이급린산화-AKT표체수평교건강인유소승고.결론 SLE환자BMSCs재체외배양과정중,교건강인용역쇠로,세포내ROS표체수평승고,기궤제가능여PI3K/AKT신호통로활화,진이촉진FoxO3린산화사기활성강저유관.
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 β-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 (34600±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 pAKT at protein levels. These results indicate that bone marrow mesenchymal cell senescence may be associated with the pathogcnesis of SLE by maintaining the lifespan of BMSCs.
