肿瘤坏死因子α对人腹膜间皮细胞基质金属蛋白酶及其组织抑制物表达的影响

目的 观察肿瘤坏死因子α（TNF-α）对人腹膜间皮细胞(HPMC) 基质金属蛋白酶（MMP）2、MMP-9及其组织抑制物（TIMP）2、TIMP-1 mRNA和Ⅰ型胶原表达的影响，同时观察TNF-α、TGF-β、IL-1单独或协同作用对HPMC的MMP-9活性的影响。 方法 采用半定量RT-PCR法测定细胞MMP-2、MMP-9、TIMP-2及TIMP-1 mRNA 的表达。采用Biotrak MMP-9 活性检测系统来精确定量测定MMP-9活性及MMP-9原的含量。ELISA法检测Ⅰ型胶原蛋白的表达。 结果 TNF-α(1~10 μg/L)分别刺激HPMC 4、16、24及48 h后， HPMC的MMP-9 mRNA表达显著上调，为基础的2.3～4.9倍（P ＜ 0.05），呈时间依赖性。TNF-α（1 μg/L）作用48 h后显著下调TIMP-1、TIMP-2 mRNA 表达，为基础的77.2％、61.3％（P ＜ 0.05），而MMP-2 mRNA表达没有显著变化。TNF-α+TGF-β１ （1~10 μg/L）、 TNF-α+TGF-β1+IL-1(1~10 μg/L)和TNF-α(5~10 μg/L)刺激HPMC 24 h后，促其分泌MMP-9 的作用最为明显。同时，TNF-α明显上调Ⅰ型胶原蛋白表达（P ＜ 0.05）。 结论 TNF-α 明显上调HPMC的MMP-9 mRNA的表达和MMP-9 的活性；联合其他细胞因子后促MMP-9分泌的作用更明显。TNF-α单独或协同其他因子在腹膜纤维化的过程中可能发挥了重要作用。

Effects of TNF-α on the expression of matrix metalloproteinases and their inhibitors in human peritoneal mesothelial cells

Objective To investigate the effects of TNF-α on the expression of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in human peritoneal mesothelial cells,meanwhile, to investgate the effects of treatment of TNF-α+TGF-β1, TNF-α+TGF-β1+IL-1 or TNF-α alone on the MMP-9 activity of the HPMC. Methods After 24-hour stimulation by different concerntrations of cytokines（TNF-α，TGF-β， IL-1）, the serum-free conditioned HPMC culture supernatants and the HPMC were collected. The mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-1 was detected by semi-quantitative RT-PCR. The activity of active and pro MMP-9 in the conditioned media was quantitatively measured by Biotrak MMP-9 activity assay system. Collagen Ⅰ protein level was examined by ELISA. Results In vitro by the treatment of TNF-α, the expression of MMP-9 mRNA of HPMC was significantly up-regulated in a dose- and time-dependent manner（2.3~4.9 folds of base value, P＜0.05）; the expression of TIMP-1 mRNA and TIMP-2 mRNA was slightly down-regulated（77.2%, 61.3% of base value, P＜0.05）, however the expression of MMP-2 mRNA did not change obviously. Activity and secretion of MMP-9 in the conditioned cell supernatant increased significantly by the treatment of TNF-α+TGF-β１, TNF-α+TGF-β１+IL-1 or TNF-α alone. Collagen Ⅰ protein expression of HPMC increased significantly as well by 24-hour stimulation of TNF-α. Conclusion The increase of MMP-9 activity and expression induced by TNF-α alone and TNF-α with other cytokines may play a role in the peritoneal fibrogenesis process.