OX-LDL诱导活化的巨噬细胞对肾小球系膜细胞TGF-β与Fn基因表达的影响及水蛭素的干预作用

OX-LDL-Activated Macrophages Modulate TGF-β and Fibronectin Gene Expression in Glomerular Mesangial Cells and the Inhibition of Hirudin

目的:研究氧化低密度脂蛋白(OX-LDL)诱导活化的巨噬细胞对肾小球系膜细胞转化生长因子(TGF-β)、纤连蛋白(Fn)基因表达的影响以及水蛭素的干预作用,探讨炎症状态下肾硬化发生的病理机制和水蛭素的有效作用。方法:体外培养大鼠肾小球系膜细胞,将OX-LDL诱导活化的大鼠腹腔巨噬细胞转入细胞培养小室,或采集其条件培养基与肾小球系膜细胞共培养;水蛭素干预组在分层共培养系统的培养有系膜细胞的下层培养液中加入水蛭素,浓度分别为2.5U/ml、5.0U/ml、10U/ml。逆转录-聚合酶链扩增反应(RT-PCR)检测肾小球系膜细胞TGF-β、Fn的基因表达。结果:OX-LDL刺激后,大鼠腹腔巨噬细胞上清液中IL-1浓度明显升高;无论是活化的巨噬细胞还是其条件培养基均能上调TGF-β和FnmRNA的表达,但以分层共培养作用最为显著;水蛭素在一定浓度下可显著下调TGF-β和Fn的基因表达。结论:(1)在分层共培养条件下,OX-LDL诱导活化的巨噬细胞与系膜细胞处于一个整体系统中,巨噬细胞不仅可通过分泌细胞因子等病理产物,而且两种细胞还可能通过相互作用增强对系膜细胞增殖和TGF-β和Fn分泌等的促进作用。(2)水蛭素可能通过抑制病理条件下硬化因子和细胞外基质的基因表达起到干预肾硬化的作用。

Objective：To observe the effects of Hirudin on the mRNA expression of transform growth factor -β （TGF-β） and Fibronectin （Fn） in glomerular mesangial cells（MC） stimulated by OX- LDL activated macrophages（Mφ）. Methods：Rat peritoneal macrophages were obtained 24hours after abdominal injection of OX - LDL. The concentration of IL- 1 in Mφ supematant was detected by ELISA to prove the activity of Mφ. MC were co - cultured with OX - LDL activated Mφ or the supernatant. Hirudin was added in the underlayer of culture solution with a concentration of 2.5 U/ml,5.0 U/ml, 10 U/mlrespectively. TGF-β and Fn mRNA were detected by using reverse transcription- polymerase chain reaction analyses （RT- PCR）. Results： OX- LDL activated peritoneal Mφ and enhanced IL- 1 secreting; OX- LDL activated Mφ and its conditional medium could all upregulate TGF-β and Fn mRNA expression in the MC, but the role of OX - LDL activated Mφ is more efficient than its conditional medium. 10 U/ml of Hirudin could reduce the TGF-β mRNA expression,2.5 U/ml and 5 U/ml of Himdin could reduce Fn mRNA expression.Conclusion： Hirudin reduced renal injury by decreasing TGF-β and Fn mRNA expression in mesangial cell.