CAJ | 학술논문

目的了解系统性红斑狼疮(SLE)患者外周血B细胞脂筏及细胞骨架蛋白的表达及分布.方法密度梯度离心法分离SLE和健康对照者外周血单个核细胞,磁珠分选纯化B细胞.用异硫氰酸荧光素(FITC)标志的霍乱毒素B亚单位(CTB)对B细胞脂筏染色,流式细胞仪及激光共聚焦显微镜分析；用罗丹明标志的鬼笔环肽对B细胞骨架蛋白染色,激光共聚焦显微镜观察.并设空白对照组、来氟米特组干预培养活动期SLE患者B细胞4h,流式细胞术检测B细胞脂筏的改变.同时记录受试患者的临床资料SLE疾病活动指数(SLEDAI).计量资料进行方差分析或配对t检验,相关性分析采用Pearson法分析.结果 SLE活动组、治疗缓解组CTB-FITC与外周血B细胞脂筏结合率较健康对照组明显增高[(59±4)％,(51±5)％,(33±4)％,F=9.21,P=0.001].共聚焦显微镜观察B细胞脂筏及骨架蛋白的分布及表达时发现,在健康对照组中,脂筏主要均匀分布于B细胞膜中,散在出现小范围聚集；骨架蛋白主要分布于B细胞的外层.而在SLE组中,B细胞脂筏可出现表达和分布的异常,呈现细胞膜荧光染色厚度的增加、聚集范围扩大及荧光强度的增强；同时伴随着B细胞膜骨架蛋白染色荧光强度的减弱.在SLE活动组中,患者外周血B细胞脂筏表达水平与其SLEDAI呈正相关(r=0.632,P=0.028).并且,与空白对照相比,干预培养后来氟米特能降低CTB与活动期SLE患者B细胞脂筏结合率[(48±5)％和(39±5)％,t=2.29,P=0.048)].结论 SLE患者外周血B细胞脂筏和骨架蛋白的表达和分布存在异常,B细胞脂筏和骨架蛋白表达的调节可能在治疗SLE中具有一定的作用.
목적 료해계통성홍반랑창(SLE)환자외주혈B세포지벌급세포골가단백적표체급분포.방법 밀도제도리심법분리SLE화건강대조자외주혈단개핵세포,자주분선순화B세포.용이류청산형광소(FITC)표지적곽란독소B아단위(CTB)대B세포지벌염색,류식세포의급격광공취초현미경분석；용라단명표지적귀필배태대B세포골가단백염색,격광공취초현미경관찰.병설공백대조조、래불미특조간예배양활동기SLE환자B세포4h,류식세포술검측B세포지벌적개변.동시기록수시환자적림상자료SLE질병활동지수(SLEDAI).계량자료진행방차분석혹배대t검험,상관성분석채용Pearson법분석.결과 SLE활동조、치료완해조CTB-FITC여외주혈B세포지벌결합솔교건강대조조명현증고[(59±4)％,(51±5)％,(33±4)％,F=9.21,P=0.001].공취초현미경관찰B세포지벌급골가단백적분포급표체시발현,재건강대조조중,지벌주요균균분포우B세포막중,산재출현소범위취집；골가단백주요분포우B세포적외층.이재SLE조중,B세포지벌가출현표체화분포적이상,정현세포막형광염색후도적증가、취집범위확대급형광강도적증강；동시반수착B세포막골가단백염색형광강도적감약.재SLE활동조중,환자외주혈B세포지벌표체수평여기SLEDAI정정상관(r=0.632,P=0.028).병차,여공백대조상비,간예배양후래불미특능강저CTB여활동기SLE환자B세포지벌결합솔[(48±5)％화(39±5)％,t=2.29,P=0.048)].결론 SLE환자외주혈B세포지벌화골가단백적표체화분포존재이상,B세포지벌화골가단백표체적조절가능재치료SLE중구유일정적작용.
Objective To investigate the expression of lipid rafts (LRs) and actin cytoskeleton (F-actin) in the peripheral blood B lymphocytes of patients with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque.B lymphocytes were isolated by positive selection from PBMCs.Membrane staining for LRs was achieved with FITC-conjugated cholera toxin B (CTB).The level and distribution of LRs were studied by flow cytometry and confocal microscopy.Staining for F-actin was carried out with Rhodamine phalloidin.The expression of F-actin was analyzed by confocal microscopy.In an in vitro examination,the effect of Leflunomide on lipid rafts in B lymphocytes from SLE was analyzed.Disease carried out was measured using the SLE disease activity index (SLEDAI).Analysis of the enumerical data was performed using ANOVA or paired-samples t test.Correlation was examined by Pearson's rank correlation test.Results The number of CTB-binding lipid rafts in B cell from active SLE patients or from SLE patients in disease remission.who were treated with immunosuppressive drugs was higher than B cells from healthy controls [(59+4)％,(51±5)％,(33±4)％,F=9.21,P=0.001].Confocal microscopy revealed that in B cell from healthy controls,lipid raft was found to be small and uniformly distributed on the plasma membrane.F-actin was found mainly in the cortical region of the cells.This pattern was different from the pattern seen in B cells from patients with SLE,which presented with stronger staining and irregular large clustering of LRs,with a decrease in F-actin levels.In addition,the number of CTB-binding LRs in B cells from the active SLE patients was correlated significantly with the SLEDAI score (r=0.632,P=0.028).Furthermore,thein vitro results showed that leflunomide treatment reduced the number of CTB-binding LRs in B cell from SLE patients [(48±5)％ vs (39±5)％,t=2.29,P=0.048].Conclusion The altered expression of Lipid raft and F-actin can been seen in B lymphocytes in SLE,and modulation of LRs and F-actin expression may be a potential approach in the treatment of SLE.