【摘要】 观察旋毛虫重组热休克蛋白HSP70(rTs-HSP70)对体外培养小鼠巨噬细胞Toll样受体TLR2/4表达的影响。体外常规培养小鼠巨噬细胞系RAW264.7,分别加入不同浓度rTs-HSP70刺激培养24 h,半定量PCR方法检测培养细胞TLR2/4 mRNA表达,流式细胞仪检测表面标志分子CD80和CD86表达。平行设立不加刺激物阴性对照组,细菌脂多糖LPS阳性对照组;同时试验比较5μg·mL-1浓度的HSP70组和HSP70+LPS组(HSP70预刺激后再加入LPS刺激培养12 h)RAW264.7细胞TLR2/4 mRNA表达差异。结果表明,低浓度rTs-HSP70与巨噬细胞活化呈正相关,浓度为5μg·mL-1时,TLR2/4表达水平达到峰值(P<0.05),并可刺激巨噬细胞共刺激分子CD80和CD86高表达,随着HSP70浓度升高(>5μg·mL-1)则对巨噬细胞RAW264.7表现为抑制作用;当对RAW264.7进行旋毛虫HSP70预刺激,可引起免疫抑制效应,抑制LPS对巨噬细胞TLR2/4的活化反应。试验可为旋毛虫相关分子免疫调节机制研究及旋毛虫病防治提供参考。更多还原

【Abstract】 To observe the effect of 70 kDa recombinant heat shock protein from Trichinella spiralis(rTs- HSP70) on expression of TLR2/4(toll- like receptors) in murine macrophage in vitro. Murine macrophage RAW264.7 cell lines were incubated in twelve-well plates and different concentrations of rTs-HSP70 were added, co-culturing for 24 hours. Then the cells’ TLR2/4 expression were tested by semi-quantitative PCR, those of surface markers CD80 and CD86 by flow cytometry. Cells receiving no stimulation were taken as negative control, bacterial lipopolysaccharide(LPS) as positive control. At the same time, comparative studies on incubation with rTs-HSP70 alone, rTs-HSP70 in combination with LPS, were conducted at concentration 5 μg· mL-1of HSP70. The results indicated that lower concentrations of rTs-HSP70 had a positive correlation with macrophages activation, TLR2/4 expression peaked at concentration of 5 μg· mL-1(P<0.05), and up-regulation of co-stimulatory molecules. when rTs-HSP70concentration rose above a certain threshold(>5 μg· mL-1), a inhibitory effect on macrophage could be observed. Pre- stimulation of the macrophage with rTs- HSP70 could result in immunosuppression,generating a suppresive effect on LPS in TLR2/4 activation. The study lay the foundation for the mechanisms of helminth molecules immunomodulation and the therapeutic treatment of different trichinellosis.更多还原