摘要
目的探讨辛伐他汀对体外培养的人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)在肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)诱导后组织型纤溶酶原激活物(tis-sue-type plasminogen activator,t-PA)和纤溶酶原激活物抑制因子1(plasminogen activator inhibitor-1,PAId)表达的影响。方法应用胰蛋白酶消化法分离HPMCs进行原代培养并传代,将第三代分为5组(每组设3个样本):①正常对照组:将HPMCs置于完全培养液中,置于37℃、5%CO2培养箱培养24h;②单纯TNF-α(1μg/L)诱导组:将HPMCs置于浓度为1μg/L的TNF-α诱导缓冲液中,环境温度及处理时间如前;③TNF-α诱导+辛伐他汀(2.5μmol/L)组:将HPMCs置于含有TNF-α(1μg/L)诱导液+辛伐他汀(2.5μmol/L)缓冲液中,余同前;④TNF-α诱导+辛伐他汀(5.0μmol/L)组:将HPMCs置于含有TNF-α(1μg/L)诱导液+辛伐他汀(5.0μmol/L)缓冲液中,余同前;⑤TND-α诱导+辛伐他汀(10.0μmol/L)组:将HPMCs置于含有TNF-α(1μg/L)诱导液+辛伐他汀(10.0μmol/L)缓冲液中,余同前。采用半定量逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)法检测细胞内t-PA和PAI-1 mRNA表达;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测细胞上清液中t-PA和PAI-1的蛋白质水平,用二喹啉甲酸(bicinchoninic acid,BCA)蛋白检测法测定细胞中蛋白质含量,用以校正ELISA结果。结果与正常对照组比较,TNF-α诱导能抑制HPMCS中t-PA表达,增加PAI-1的表达(P〈0.01);与单纯TNF-α诱导组比较,辛伐他汀(2.5μmol/L)能显著增加TNF-α诱导HPMCs中t-PA表达,抑制PAI-1的表达(P〈0.05),t-PAmRNA表达水平显著上升和PAI-1mRNA表达水平显著降低(P〈0.01);辛伐他汀(5.0μmol/L及10.0μmol/L)亦能明显增加TNF-α诱导的HPMCs中t-PA表达,抑制PAI-1的表达(P〈0.01),两者在蛋白质和基因水平均呈量效关系。结论辛伐他汀干预后能促进HPMCs在炎症状态下增加t-PA生成并抑制PAI-1的表达,从而为腹膜透析患者腹膜透析相关腹膜炎发生时使用他汀类药物以预防腹膜纤维化提供理论依据。
Objective To investigate the effects of Simvastatin on tissue-type plasminogen acti- vator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) expression in human peritoneal mesotheli- al ceils (HPMCs) cultured under inflammatory conditions. Methods HPMCs were isolated from hu- man omenta by trypsin digestion method and subcultured. Then, the third-generation HPMCs were divided into 5 groups., normal control group (The HPMCs were cultured in the complete medium); tumor necrosis factor-α(TNF-α) group[-The HPMCs were cultured with TNF-α (1μg/L)]; TNF-α plus Simvastatin (2. 5 μmol/L) group[-The HPMCs were cultured with TNF-α (1 μg/L) and Simvas- tatin (2. 5μmol/L)]; TNF-α plus Simvastatin (5.0 μmol/L) groupEThe HPMCs were cultured with TNF-α (1 μg/L) and Simvastatin (5.0 μmol/L)]; TNF-α plus Simvastatin (10.0 μmol/L) group I-The HPMCs were cultured with TNF-α (1 μg/L) and Simvastatin (10. 0 μmol/L)]. Semi-quantita- tive reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of t- PA and PAI-1 mRNA in HPMCs. The expression of t-PA and PAI-1 proteins in the culture superna- rants was detected by enzyme linked immunosorbent assay (ELISA). Cell protein concentration was measured by trace bicinchoninic acid (BCA) method to correct the ELISA results. Results (1) As compared with the normal control group, TNF-α could significantly decrease the expression of t-PA in HPMCs, meanwhile increase the expression of PAI-1 (P〈0.01). (2) As compared with TNF-α group, Simvastatin (2. 5 μmol/L) significantly decreased PAI-1 expression and increased t-PA expres- sion in HPMCs in a dose-dependent manner both in protein and gene levels (P〈0. 05). Simvastatin (5.0 and 10. 0 μmol/L) also significantly decreased PAI-1 expression and increased t-PA expression in HPMCs in a dose-dependent manner both in protein and gene levels (P〈0. 01). Conclusions Simvas- tatin does not only increase the expression of t-PA, but also inhibit the expression of PAFI in HPMCs cultured under inflammatory conditions. Our findings provide explanation for the anti-inflammatory properties of statins in HPMCs and a rationale for use of these drugs to protect peritoneal dialysis pa- tients from peritoneal fibrosis development during bacterial peritonitis.
出处
《临床肾脏病杂志》
2015年第4期239-243,共5页
Journal Of Clinical Nephrology