TLR4在anti-β2GPⅠ/β2GPⅠ复合物诱导THP-1细胞表达TF中的作用探讨

The roles of TLR4 in anti-β2GPⅠ/β2GPⅠ-induced tissue factor expression on THP-1 cells

目的:探讨TLR4及相关信号分子在anti-β2GPⅠ/β2GPⅠ复合物诱导单核细胞株THP-1表达组织因子(TF)中的作用。方法:利用荧光定量PCR(Real-timePCR)检测anti-β2GPⅠ/β2GPⅠ诱导THP-1细胞TFmRNA表达,采用试剂盒检测细胞TF活性;利用自制的β2GPⅠ胶联亲和层析柱(β2GPⅠ-Affi-Gel)分析β2GPⅠ与THP-1细胞表面相应受体结合情况;Real-timePCR及Western蛋白印迹检测anti-β2GPⅠ/β2GPⅠ复合物诱导细胞表达TLR4、MyD88、MD-2情况;观察TLR4途径抑制物——紫杉醇是否干预anti-β2GPⅠ/β2GPⅠ复合物对细胞的作用。结果:Anti-β2GPⅠ/β2GPⅠ复合物(100μg/ml)诱导THP-1细胞TF表达显著增加(P<0.05);THP-1细胞表面的TLR4能够结合于β2GPⅠ-Affi-Gel柱;Anti-β2GPⅠ/β2GPⅠ复合物(100μg/ml)刺激THP-1细胞表达TLR4、MyD88、MD-2显著升高(P<0.05);紫杉醇(1μmol/L)能够抑制anti-β2GPⅠ/β2GPⅠ复合物对细胞的刺激效应。结论:TLR4及相关信号分子在anti-β2GPⅠ/β2GPⅠ复合物诱导THP-1细胞表达TF中具有重要作用。

Objective：To investigate the roles of TLR4 in anti-β2GPⅠ/β2GPⅠ-induced tissue factor expression on THP-1 cells.Methods：TF expression in THP-1 cells was detected by Real-time PCR or TF activity kit after stimulating with anti-β2GPⅠ/β2GPⅠ complex.The binding of TLR4 to β2GPⅠ was analyzed with β2GPⅠ-Affi-Gel columns.The TLR4,MyD88,MD-2 expression induced by anti-β2GPⅠ/β2GPⅠ complex was investigated by Real-time PCR and Western blot.Whether TLR4 pathway inhibitor（paclitaxel） could inhibit the effects of anti-β2GPⅠ/β2GPⅠ on THP-1 cells was also evaluated.Results：TF expression（both mRNA and protein） in THP-1 cells could be significantly increased with treatment of anti-β2GPⅠ/β2GPⅠ complex（100 μg/ml）.The TLR4 on THP-1 cell membrane could bind to β2GPⅠ-Affi-Gel columns.After THP-1 cells was stimulated with anti-β2GPⅠ/β2GPⅠ complex,the TLR4,MD-2 and MyD88 expression（both mRNA and protein） on THP-1 cells was significantly increased by stimulation of anti-β2GPⅠ/β2GPⅠ complex.All the stimulating effects of anti-β2GPⅠ/β2GPⅠ on THP-1 cells could be inhibited by paclitaxel（1 μmol/L）.Conclusion：TLR4 and some molecules of the pathway play an important role in anti-β2GPⅠ/β2GPⅠ-induced TF expression on THP-1 cells.