川芎嗪及辛伐他汀对高糖状态下人腹膜间皮细胞t-PA和PAI-1分泌与表达的影响

发布时间：2018-05-16 13:10

本文选题：辛伐他汀 + 川芎嗪　；参考：《南京中医药大学》2015年硕士论文

【摘要】：背景腹膜透析(Peritoneal dialysis, PD)为肾脏替代治疗的一种方式,有较好地保护残余肾功能、中分子物质滤过效果好、可在家自行治疗等优点,近年来成为部分终末期肾衰竭患者的首选肾脏替代治疗方式。但随着腹膜透析的持续进行,腹膜纤维化(peritoneal fibrosis, PF)逐渐出现并日渐加重,为腹膜透析常见并发症,其影响代谢废物及水分的超滤,致使肌酐、尿素、胍类等毒素的清除效率下降,患者最终选择退出腹膜透析,改行其他替代治疗。在腹膜纤维化发生时,腹膜中细胞外基质(extracellular matrix, ECM)大量沉积,腹膜血管过度增生,腹膜间皮细胞损伤,纤维化逐渐加重。截止目前的研究已表明,高糖、低PH值、高渗透压的腹膜透析液及其中的葡萄糖降解产物长期作用于腹膜以及反复发作的腹膜透析相关腹膜炎均可诱导致纤维化细胞因子如TGF-β、FGF、VEGF等的分泌,细胞外基质大量沉积,血管增生,腹膜纤维化出现及加重。既往研究表明,组织型纤溶酶原激活物(t-PA)、纤溶酶原激活物抑制因子-1(PAI-1)是人体正常生命活动所必需的细胞因子,调节体内的纤溶过程,同时其分泌及表达在腹膜纤维化过程中也有重要影响,可干预腹膜纤维化的发生、发展。目的该研究通过观察川芎嗪和辛伐他汀对高糖诱导后的人腹膜间皮细胞(human peritoneal mesothelial cells, HPMCs)中t-PA、PAI-1的分泌及表达,进一步探讨上述两种药物对上述指标变化的影响响机制,为腹膜纤维化的预防及治疗提供理论依据。方法所用人体腹膜来目鼓楼医院腹部手术(非尿毒症、腹膜炎)患者,经胰蛋白酶消化、分离后得到腹膜间皮细胞,再进行原代培养、传代培养,第三代细胞用于本实验,各次实验均使用3名患者的腹膜间皮细胞实施3次独立实验。用培养液同步48h后,将HPMCs分为8组(每组设3个样木)观察：空白对照组(N组,完全培养液),高糖诱导组(H组,2.5%葡萄糖),低剂量辛伐他汀组(高糖+2.5μmol/L辛伐他汀)、中剂量辛伐他汀组(S2组,高糖+5.0μmol/L辛伐他汀)、高剂量辛伐他汀组(S3组,高糖+10.0μmol/L辛伐他汀)、低剂量川芎嗪组(T1组,高糖+川芎嗪10mg/L)、中剂量川芎嗪组(T2组,高糖+川芎嗪20mg/L)、高剂量川芎嗪组(T3组,高糖+川芎嗪40mg/L)。MTT法检测各组HPMCs的活性,RT-PCR法检测细胞内t-PA和PAI-1的表达,ELISA法检测细胞上清液中t-PA和PAI-1蛋白质水平,细胞部分用BCA蛋白检测法测定细胞蛋白质表达量,以此校正ELISA结果。结果① 高糖诱导组和空白组比较,高浓度葡萄糖能明显降低腹膜间皮细胞活性(P0.01),同时腹膜间皮细胞中t-PA生成减少和PAI-1的表达增强(P0.01)；②与高糖诱导组比较,辛伐他汀干预组腹膜间皮细胞的活性明显升高(P0.01), t-PA生成增加、PAI-1生成显著减少(P0.01), t-PA mRNA表达水平显著增加、PAI-1 mRNA表达水平显著降低(P0.01),在蛋白及基因水平上,两者均与辛伐他汀存在量效关系;③与高糖诱导组比较,川芎嗪干预组腹膜间皮细胞活性明显升高(P0.01),川芎嗪各干预组(T1、T2、T3组)腹膜间皮细胞活性明显升高(P0.01), T1、T2、T3组中t-PA的表达量增加,PAI-1的表达量均明显降低(P0.01),t-PA mRNA表达均明显上调、PAI-1 mRNA表达明显降低(P0.01),在蛋白和基因水平上,两者也与川芎嗪有量效关系。结论辛伐他汀和川芎嗪能抑制腹膜间皮细胞中PAI-1的表达,促进t-PA的表达,因此可减少细胞外基质的大量积聚,有预防、抑制腹膜纤维化的作用。
[Abstract]:Peritoneal dialysis (PD) is a way of renal replacement therapy. It has the advantages of better protection of residual kidney function, good filtration effect of medium molecular material and self treatment at home. In recent years, it has become the first choice for renal replacement therapy in patients with partial end-stage renal failure. However, peritoneal dialysis continues to carry out peritoneum. Peritoneal fibrosis (PF) is gradually appearing and aggravating gradually. It is a common complication of peritoneal dialysis, which affects the ultrafiltration of metabolic waste and water. The removal efficiency of creatinine, urea, guanidine and other toxins is decreased. The patients choose to withdraw from peritoneal dialysis and change to other alternative treatments. The matrix (extracellular matrix, ECM) deposits, peritoneal hyperproliferation, peritoneal mesothelial cell damage, and fibrosis progressively. The current study has shown that high glucose, low pH, hypertonic Liquor Dialysisintraperitoneus and glucose degradation products are used for peritoneum and recurrent peritoneal dialysis related peritonitis for a long time. The secretion of fibrotic cytokines such as TGF- beta, FGF, VEGF and so on, a large amount of extracellular matrix deposition, vascular proliferation, and peritoneal fibrosis appear and aggravate. Previous studies have shown that tissue plasminogen activator (t-PA) and plasminogen activator inhibitor -1 (PAI-1) are essential cytokines in human normal life activities, regulatory bodies. The internal fibrinolysis process, and its secretion and expression in the process of peritoneal fibrosis also have important effects, can interfere with the occurrence of peritoneal fibrosis, development. Objective the purpose of this study through the observation of Tetramethylpyrazine and simvastatin on high glucose induced human peritoneal mesothelial cells (human peritoneal mesothelial cells, HPMCs), t-PA, PAI-1 secretion and expression, Further explore the effect of the above two drugs on the changes of the above indexes, and provide a theoretical basis for the prevention and treatment of peritoneal fibrosis. Methods the human peritoneum in the abdomen of the Drum Tower Hospital (non uremia, peritonitis), the peritoneal mesothelial cells were obtained after trypsin digestion, then the primary culture was carried out and the passage was passed. Culture, third generation of cells used in this experiment, each experiment used 3 patients with peritoneal mesothelial cells to carry out 3 independent experiments. After using the culture fluid to synchronize 48h, HPMCs was divided into 8 groups (each set of 3 samples): blank control group (group N, complete culture fluid), high glucose induction group (H group, 2.5% glucose), low dose simvastatin group (high glucose +2.5 Mu Mo). L/L simvastatin), medium dose simvastatin group (group S2, high glucose +5.0 mol/L simvastatin), high dose simvastatin group (S3 group, high glucose +10.0 mol/L simvastatin), low dose Ligustrazine group (T1 group, high sugar + Ligustrazine 10mg/L), middle dose Ligustrazine group (T2 group, high sugar + Ligustrazine 20mg/L), high dose Ligustrazine group (T3 group, high sugar + Ligustrazine) The activity of HPMCs in each group was detected by TT. The expression of t-PA and PAI-1 in cell was detected by RT-PCR method. The protein level of t-PA and PAI-1 in the cell supernatant was detected by ELISA method. The cell protein expression was measured by the BCA protein detection method. The results were corrected for the ELISA results. The activity of peritoneal mesothelial cells (P0.01) decreased and the expression of t-PA in peritoneal mesothelial cells decreased and the expression of PAI-1 increased (P0.01). Compared with the high glucose induction group, the activity of peritoneal mesothelial cells in the simvastatin group increased significantly (P0.01), the formation of t-PA was increased, the formation of PAI-1 was significantly reduced (P0.01), and the expression level of t-PA mRNA increased significantly, PAI-1. PAI-1 The expression level of mRNA was significantly decreased (P0.01), both in protein and gene level, both of which were related with simvastatin. Compared with the high glucose induction group, the activity of peritoneal mesothelial cells in the tetramethylpyrazine intervention group was significantly increased (P0.01). The activity of peritoneal mesothelial cells in the tetramethylpyrazine intervention group (T1, T2, T3 group) was significantly increased (P0.01), T1, T2, T3 group t-PA. The expression of PAI-1 decreased significantly (P0.01), the expression of t-PA mRNA was obviously up-regulated, and the expression of PAI-1 mRNA decreased significantly (P0.01). In protein and gene levels, both of them also had a quantitative relationship with Ligustrazine. Conclusion simvastatin and ligustrazine could inhibit the expression of PAI-1 in the peritoneal mesothelial cells and promote the expression of t-PA, thus reducing the expression of t-PA. The accumulation of extracellular matrix can prevent and inhibit peritoneal fibrosis.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R692.5

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