系统性红斑狼疮患者外周血B细胞脂筏及细胞骨架蛋白的表达初探

Study on the expression of lipid rafts and F-actin in peripheral blood B lymphocytes from patients with systemic lupus erythematosus

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。

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 Fieoll-Hypaque. B lymphoeytes were isolated by positive selection from PBMCs. Membrane staining for LRs was achieved with FITC-eonjugated cholera toxin B （CTB）. The level and distribution of LRs were studied by flow eytometry and confoeal 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=0.0011. Confoeal 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 levets. 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, the in 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.