葛根素对腹膜透析液诱导的体外培养人腹膜间皮细胞损伤的保护作用

【目的】了解葛根素对腹膜透析液干预下的体外培养的人腹膜间皮细胞增殖活性及转化生长因子β1（TGF-β1）分泌的影响。【方法】体外培养的人腹膜间皮细胞,分对照组、PDS组、A组（葛根素终浓度为50μg／mL）、B组（葛根素终浓度为100μg／mL）、C组（葛根素终浓度为200μg／mL）五组观察。检测各组上清液中TGF-β1的含量以及间皮细胞的增殖活性并比较。【结果】腹膜间皮细胞在PDS干预下,TGF-β1分泌显著增加,添加葛根素再干预的A、B、C组TGF-β1分泌与PDS组比较有显著下降。与PDS组比较,A、B、c纽间皮细胞增殖活性显著升高。【结论】葛根素可以抑制腹膜间皮细胞TGF-β1分泌,拮抗腹膜透析液对腹膜间皮细胞增殖活性的抑制作用。     

灯盏花素对腹膜透析液诱导人腹膜间皮细胞转化生长因子β1的影响

背景：从不同层面了解灯盏花素阻止/延缓腹膜功能衰竭的作用及其机制,从而在临床上推广使用灯盏花素来阻止/延缓腹膜功能衰竭从而延长终末期肾脏病患者腹膜透析时间、提高透析质量、减少透析失败率,提高腹膜透析远期疗效具有广泛的应用前景。目的：观察灯盏花素对腹膜透析液诱导的人腹膜间皮细胞转化生长因子β1分泌及其增殖活性的影响。方法：体外培养人腹膜间皮细胞,分为5组：分别为对照组、腹膜透析液组、灯盏花素终浓度为5,10,20μmol/L组。检测各组上清液中转化生长因子β1的水平以及间皮细胞的增殖活性。结果与结论：腹膜间皮细胞在腹膜透析液诱导下,转化生长因子β1分泌显著增加、细胞增殖活性显著降低。灯盏花素5μmol/L组转化生长因子β1分泌低于腹膜透析液组（P〈0.05）,细胞增殖活性高于腹膜透析液组（P〈0.05）;灯盏花素10,20μmol/L组转化生长因子β1分泌显著低于腹膜透析液组（P〈0.01）,细胞增殖活性显著高于腹膜透析液组（P〈0.01）。结果显示灯盏花素可以抑制腹膜间皮细胞转化生长因子β1分泌,拮抗腹膜透析液对腹膜间皮细胞增殖活性的抑制作用。     

胰岛素对人腹膜间皮细胞增殖、损伤及凋亡的影响

目的明确胰岛素是否对人腹膜间皮细胞具有毒性或者保护作用。方法使用人腹膜间皮细胞细胞株，传代培养。用四甲基偶氮唑盐比色法(MTT)测定HPMC的增殖程度。采用全自动生化分析仪测定培养液中乳酸脱氢酶LDH水平示细胞损伤，Hoechst33258染色观察细胞凋亡形态学变化及计算凋亡细胞百分比。分为单纯培养基对照组，4．25％葡萄糖组，不同浓度胰岛素组和不同浓度的胰岛素加4．25％葡萄糖组。结果与对照组相比，4．25％葡萄糖致LDH水平明显增高，并显著降低NIT渗入量，明显增加凋亡细胞百分比；不同浓度的胰岛素对HPMC增殖无影响，不同浓度胰岛素24h后可明显升高LDH水平(P＜0．05)，不同浓度胰岛素作用12h均后导致凋亡细胞百分比显著升高(P＜0．05)；不同浓度葡萄糖胰岛素组12h、24hMMT渗入量较葡萄糖组明显升高(P＜0．05)，组间差异无显著性(P＞0．05)，浓度大于25U／L胰岛素加葡萄糖组作用24h后与葡萄糖组相比显著降低LDH水平(P＜0．05)；不同浓度胰岛素加葡萄糖组作用12h后与葡萄糖组相比凋亡细胞百分比明显下降(P＜0．05)。结论胰岛素能导致HPMC损伤、诱导凋亡，同时在一定范围内可缓解高糖对HPMC的毒性作用。     

灯盏花素对腹膜透析液诱导人腹膜间皮细胞转化生长因子β1的影响

背景:从不同层面了解灯盏花素阻止/延缓腹膜功能衰竭的作用及其机制,从而在临床上推广使用灯盏花素来阻止/延缓腹膜功能衰竭从而延长终末期肾脏病患者腹膜透析时间、提高透析质量、减少透析失败率,提高腹膜透析远期疗效具有广泛的应用前景。目的:观察灯盏花素对腹膜透析液诱导的人腹膜间皮细胞转化生长因子β1分泌及其增殖活性的影响。方法:体外培养人腹膜间皮细胞,分为5组:分别为对照组、腹膜透析液组、灯盏花素终浓度为5,10,20μmol/L组。检测各组上清液中转化生长因子β1的水平以及间皮细胞的增殖活性。结果与结论:腹膜间皮细胞在腹膜透析液诱导下,转化生长因子β1分泌显著增加、细胞增殖活性显著降低。灯盏花素5μmol/L组转化生长因子β1分泌低于腹膜透析液组(P<0.05),细胞增殖活性高于腹膜透析液组(P<0.05);灯盏花素10,20μmol/L组转化生长因子β1分泌显著低于腹膜透析液组(P<0.01),细胞增殖活性显著高于腹膜透析液组(P<0.01)。结果显示灯盏花素可以抑制腹膜间皮细胞转化生长因子β1分泌,拮抗腹膜透析液对腹膜间皮细胞增殖活性的抑制作用。     

短发夹结构RNA对人腹膜间皮细胞转化生长因子-β1表达和超微结构的影响

目的 研究靶向转化生长因子-β1（TGF-β1）的短发夹RNA（shRNA）表达载体对腹膜透析液诱导的人腹膜间皮细胞（HPMC）表达TGF-β1的影响,并观察其对HPMC超微结构的影响. 方法 利用pcDU6质粒构建两个靶向TGF-β1的shRNA真核表达载体pcDU6-A1-A2和pcDU6-B1-B2,使用脂质体介导转染腹膜透析液刺激下的HPMC,采用半定量逆转录多聚酶链式反应（RT-PCR）和酶联免疫吸附（ELISA）检测shRNA表达载体对于TGF-β1基因和蛋白质表达水平的抑制效果,使用透射电镜观察HPMC超微结构的改变. 结果 HPMC暴露于4.25%腹膜透析液中48小时后,其上清中TGF-β1的浓度与对照组相比显著增高分别为（39.71±6.60）pg/10^5 cells和（16.32±2.71）pg/10^5 cells,P＜0.01）,1.5%腹膜透析液对于TGF-β1蛋白合成无明显影响;而预先转染了shRNA载体的两组与转染pcDU6载体（空载体）的组相比,其由4.25%腹膜透析液诱导的TGF-β1蛋白增高幅度明显减少,分别下降了39.2%和47.2%（P＜0.01）;两组转染不同的shRNA载体的HPMC之间相比TGF-β1浓度无显著差异,pcDU6载体（空载体）转染组与4.25%腹膜透析液刺激组TGF-β1浓度亦无显著差异.透射电镜显示HPMC暴露于4.25%腹膜透析液可导致细胞扁平、微绒毛减少和线粒体水肿等,而转染shRNA载体组细胞的改变相对较轻. 结论 pcDU6质粒载体介导的短发夹结构RNA（shRNA）能够明显抑制腹膜透析液刺激下的人腹膜间皮细胞TGF-β1的高表达,并减轻其超微结构的异常,提示shRNA表达载体可能有益于腹膜纤维化的防治.     

腹膜透析液对腹膜间皮细胞早衰的影响及间充质干细胞干预的研究

目的探讨腹膜透析液对腹膜间皮细胞早衰的影响及骨髓间充质干细胞的干预作用。方法首先从择期行腹膜透析置管手术者大网膜分离培养出腹膜间皮细胞。将腹膜透析液加入常规培养的腹膜间皮细胞,通过腹膜间皮细胞传代数、细胞增殖、细胞周期、端粒的检测以观察腹膜透析液对腹膜间皮细胞衰老的影响及可能机制;通过在培养液中加入骨髓间充质干细胞,观察骨髓间充质干细胞对腹膜间皮细胞早衰的干预作用。结果与仅用RPMI1640培养基培养的腹膜间皮细胞相比,腹膜透析液致腹膜间皮细胞传代数减少4~5代,生长速率降低42%,细胞周期阻滞于G1期[(69.1±0.3)%],端粒长度缩短(4.35±0.12)kb比(5.45±0.10)kb,t=2.228,P=0.000]。腹膜透析患者透出液中分离培养出腹膜的间皮细胞接近衰老细胞,加入骨髓间充质干细胞培养的间皮细胞衰老进程延缓。结论骨髓间充质干细胞可延缓腹膜透析液所诱导的腹膜间皮细胞的衰老进程。为临床上延缓腹膜纤维化,改善腹膜透析的远期疗效提供一种新的理论依据和治疗手段。     

丹参酮ⅡA对腹膜透析液诱导人腹膜间皮细胞氧化应激及其损伤的影响

【目的】研究丹参酮ⅡA对腹膜透析液（PDF）诱导人腹膜间皮细胞（HPMCs）氧化应激及其损伤的影响。【方法】体外培养的HPMCs同步后分为五组：A组即对照组（DMEM培养基＋完全培养基）;B组即腹膜透析液组（含4．25％PDF的完全培养基）;C组即丹参酮组（含100μmmol／L丹参酮的完全培养基）;C1组即丹参酮1组（含50μmol／L丹参酮ⅡA的PDF）、C2组即丹参酮2组（含100μmol／I。丹参酮ⅡA的PDF）,干预72h后（其中B组、C1、C2组为加热的PDF）,流式细胞仪及荧光显微镜检测线粒体膜电位的变化;荧光显微镜检测胞内钙离子浓度的变化。采用活性氧捕获剂二氯二氢荧光素-乙酰乙酸酯（DCFH—DA）孵育细胞,通过流式细胞仪检测细胞内的荧光强度而测得细胞内活性氧水平。并检测上清液中超氧化物歧化酶、谷胱甘肽过氧化物酶、和丙二醛含量;不同浓度丹参酮ⅡA干预48h后,MTT比色法检测不同浓度丹参酮ⅡA干预下细胞增殖活力。【结果】B组上清液丙二醛含量、胞内钙离子、细胞内活性氧水平较对照组及C1、C2组显著增加（P〈0．01）,超氧化物歧化酶、谷胱甘肽过氧化物酶及线粒体膜电位水平显著降低（P〈0．01）。不同浓度丹参酮ⅡA组HPMCs的增殖活性显著高于B组（P〈0．01）,丹参酮ⅡA不同浓度间无显著差异（P〉0．05）。【结论】丹参酮ⅡA可以通过降低PDF干预下丙二醛含量、减轻钙离子负荷、维持线粒体膜电位的水平、保护抗氧化还原酶的活性来拮抗商业性PDF对HPMCs的氧化应激及其损伤。     

葡萄糖腹膜透析液诱导间皮细胞凋亡的体外研究

[目的]了解葡萄糖腹膜透析液(PDS)对体外培养人腹膜间皮细胞凋亡的影响.[方法]通过向培养液中添加1.5%葡萄糖腹膜透析液,观察细胞caspase-3活性变化,应用Annexin V/PI双染色流式细胞仪方法以及TUNEL方法观察腹膜间皮细胞凋亡的情况.[结果]PDS组细胞caspase-3活性较对照组明显上升Annexin V-FITC/PI双标记结果显示PDS组细胞凋亡率为(46.8士14.2)%,显著高于对照组的(9.04士4.86)%(P＜0.05).TUNEL结果显示PDS组细胞凋亡明显高于对照组.[结论]葡萄糖腹膜透析液可以明显诱导腹膜间皮细胞细胞凋亡的发生.     

腹膜透析引流液离心法和大网膜消化法原代培养人腹膜间皮细胞

[目的]建立和改善两种人腹膜间皮原代细胞培养的方法.[方法]分别采用离心法从腹膜透析（PD）引流液中及胰蛋白酶-EDTA消化法从人的腹膜组织中分离培养人腹膜间皮细胞（HPMC）,并分别对两种原代培养的HPMC采用相差倒置显微镜和免疫组化染色对培养的细胞进行鉴定.[结果]①两种方法均在体外原代培养成活,在相差倒置显微镜下观察细胞均呈典型的铺路石样外观,免疫组化显示两种方法培养的HPMC胞浆角蛋白、波形蛋白表达阳性,提示为腹膜间皮细胞.②经大网膜消化法获得的HPMC可传至第四代,保持原有铺路石样外观,PD引流液离心法获得的HPMC传代后生长缓慢,长期接受PD后HPMC可呈现成纤维细胞样改变.[结论]成功应用离心法和消化法进行HPMC原代培养,有助于PD研究体外实验的开展.     

肝细胞生长因子对腹膜透析腹膜纤维化防治作用的研究

目的探讨肝细胞生长因子对腹膜透析腹膜纤维化的防治作用及其机制。方法①MTT法测定肝细胞生长因子对腹膜间皮细胞增殖的影响；②酶联免疫（ELISA）法检测细胞培养液中纤连蛋白（FN）、转化生长因子-β1（TGF-β1）及纤溶酶原激活物抑制物（PAI-1）水平；⑧逆转录-聚合酶链反应（RTPCR）法检测FN、PAI-1及TGF-β1 mRNA的表达。结果肝细胞生长因子浓度〉30ng／ml时，可以改善高糖对腹膜间皮细胞增殖的抑制作用。腹膜透析液组FN、PAI-1、TGF-β1蛋白水平显著升高，肝细胞生长因子组FN、PAI-1、TGF-β1蛋门水平均显著减少。腹膜透析液组高糖上调腹膜间皮细胞FN、PAI-1、TGF-β1 mRNA的表达，肝细胞生长因子组使FN、PAI-1、TGF-β1 mRNA的表达水平下调。结论肝细胞生长因子能改善高糖对腹膜间皮细胞增殖的抑制作用，同时可以使腹膜间皮细胞FN、PAI-1、TGF-β1的表达水平下调。     

Improved in vitro biocompatibility of bicarbonate-buffered peritoneal dialysis fluid.

BACKGROUND: Conventional peritoneal dialysis fluids (PDFs) have been shown to damage the mesothelial layer and are associated with the development of peritoneal fibrosis and neoangiogenesis. New-generation PDFs have therefore been developed with physiological pH and reduced levels of glucose degradation products (GDPs), precursors of advanced glycation end products (AGEs). In this work, we evaluated and compared the improved biocompatibility of two new-generation PDFs (Balance and bicaVera) using mesothelial cell biology; we also compared them to a standard PDF (stay.safe) (all PDFs by Fresenius Medical Care, Fresnes, France). METHODS: stay.safe, Balance, and bicaVera were tested for their effect on human peritoneal mesothelial cell (HPMC) viability by measuring cell proliferation and apoptosis, and oncosis induction. The formation of AGEs was evaluated by immunoassay. Transforming growth factor beta-1 and vascular endothelial growth factor (VEGF) were immunoassayed in HPMC supernatants exposed to the above PDFs. RESULTS: At 15 g/L glucose concentration, HPMC exposure to bicaVera resulted in higher cell proliferation compared to Balance (p < 0.001) and stay.safe (p < 0.001). Compared to the lactate-buffered PDFs (Balance and stay.safe), oncosis was significantly lower in cells exposed to bicaVera (p < 0.05). bicaVera, containing lower amounts of GDPs, generated less AGE formation (p < 0.05) and VEGF production (p < 0.05) than either Balance or stay.safe. CONCLUSIONS: New-generation PDFs with physiological pH and lower GDP levels, especially if bicarbonate-buffered (bicaVera), have fewer in vitro toxic effects on mesothelial cells and may contribute to peritoneal preservation, thus improving long-term treatment of PD patients.     

Effect of iron sucrose on human peritoneal mesothelial cells

BACKGROUND: Iron supplementation is often required in uraemic patients with anaemia. Peritoneal cavity was proposed as an alternative intravenous route for iron infusion in patients treated with peritoneal dialysis. We studied the effect of iron sucrose (Venofer) on the function of human peritoneal mesothelial cells maintained in in vitro culture. MATERIALS AND METHODS: In in vitro experiments on human peritoneal, the mesothelial effect of elemental iron (in conc. 0.0001-1 mg mL-1) present in Venofer on their viability, growth and synthesis of IL-6 was studied. Additionally we evaluated with a fluorescent probe (2',7'-dichlorodihydro-fluorescein diacatate) generation of reactive oxygen species in cells exposed to iron sucrose. We also measured accumulation of iron in the cytoplasm of mesothelial cells after their in vitro exposure to Venofer. RESULTS: In in vitro conditions iron induces a dose-dependent inhibition of viability of the mesothelial cells as reflected by inhibition of the cells growth by 34% at Fe 0.1 mg mL-1 vs. control (P < 0.05) increased release of lactate dehydrogenase (LDH) from the cytosol: 67.1 +/- 30.3 mU mL-1 at Fe 1 mg mL-1 vs. 7.9 +/- 6.4 in control group (P < 0.001), and reduced synthesis of IL-6: 209 +/- 378 pg mg-1 cell protein at Fe 1 mg mL-1 vs. 38674 +/- 4146 pg mg-1 cell protein in controls (P < 0.001). Cytotoxicity of iron towards mesothelial cells was enhanced in vitro when it was tested in presence of the dialysis fluid. Iron used in vitro at concentration 0.0001 mg mL-1 and greater induces generation of oxygen-derived free radicals in mesothelial cells. Furthermore, iron is taken by these cells and stored in their cytosol, resulting in stimulation of the intracellular generation of free radicals. CONCLUSIONS: We conclude that iron used in the form of iron sucrose is cytotoxic to human peritoneal mesothelial cells. Accumulation of iron sucrose within cytoplasm of these cells may lead to induction of its chronic cytotoxic effect.     

Effects of peritoneal dialysis solutions on the secretion of growth factors and extracellular matrix proteins by human peritoneal mesothelial cells.

OBJECTIVE: To compare the effects of different peritoneal dialysis solutions (PDS) on secretion of vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGFbeta1), procollagen I C-terminal peptide (PICP), procollagen III N-terminal peptide (PIIINP), and fibronectin by cultured human peritoneal mesothelial cells (HPMC). DESIGN: Using M199 culture medium as control, commercial PDS containing 1.5% or 4.25% glucose and 40 mmol/L lactate [Dianeal 1.5 (D 1.5) and Dianeal 4.25 (D 4.25), respectively; Baxter Healthcare, Deerfield, Illinois, USA]; PDS containing 1.5% or 4.25% glucose with 25 mmol/L bicarbonate and 15 mmol/L lactate [Physioneal 1.5 (P 1.5) and Physioneal 4.25 (P 4.25), respectively; Baxter]; and PDS containing 7.5% icodextrin [Extraneal (E); Baxter] were tested. Growth-arrested and synchronized HPMC were continuously stimulated for 48 hours by test PDS diluted twofold with M199, TGFbeta1 1 ng/mL, or different concentrations of icodextrin. VEGF, TGFbeta1, and fibronectin secreted into the media were analyzed by ELISA, and PICP and PIIINP by radioimmunoassay. RESULTS: Dianeal 1.5, D 4.25, and P 4.25, but not P 1.5 and E, significantly increased VEGF secretion compared with control M199. D 4.25- and P 4.25-induced VEGF secretion was significantly higher than induction by D 1.5 and P 1.5, respectively, suggesting that high glucose may be involved in the induction of VEGF. Physioneal 1.5- and P 4.25-induced VEGF secretion was significantly lower than induction by D 1.5 and D 4.25, respectively, suggesting a role for glucose degradation products (GDP) in VEGF production. TGFbeta1 secretion was significantly increased by D 4.25 and E. Icodextrin increased TGFbeta1 secretion in a dose-dependent manner. All PDS tested significantly increased secretion of PIIINP compared with control. D 1.5- and D 4.25-induced PIIINP secretion was significantly higher than P 1.5, P 4.25, and E. Physioneal 4.25-induced PIIINP secretion was significantly higher than P 1.5, again implicating high glucose and GDP in PIIINP secretion by HPMC. There was no significant increase in PICP or fibronectin secretion using any of the PDS tested. Addition of TGFbeta1 1 ng/mL into M199 control significantly increased VEGF, PICP, PIIINP, and fibronectin secretion by HPMC. CONCLUSIONS: The present study provides direct evidence that HPMC can secrete VEGF, TGFbeta1, and PIIINP in response to PDS, and that HPMC may be actively involved in the development and progression of the peritoneal membrane hyperpermeability and fibrosis observed in long-term PD patients. This study also suggests that both high glucose and GDP in PDS may play important roles in inducing VEGF and PIIINP production/secretion by HPMC.     

Dialysate cancer antigen 125 concentration as marker of peritoneal membrane status in patients treated with chronic peritoneal dialysis.

OBJECTIVE: This study reviews publications on the history of cancer antigen 125 (CA125), the background of its use as a marker of mesothelial cell mass, determination in peritoneal effluent, and its practical use in both the follow-up of peritoneal dialysis (PD) patients and as a marker of in vivo biocompatibility of dialysis solutions. DESIGN: Review article. RESULTS: CA125 is a high molecular weight glycoprotein. Previous studies in ascites suggested its release by mesothelial cells. In vitro studies with cultured mesothelial cells showed constitutive production, the majority of which was dependent on mesothelial cell mass. Serum CA125 is normal in PD patients, but its concentration in peritoneal dialysate suggests local release, probably from mesothelial cells. Effluent CA125 can be considered a marker of mesothelial cell mass in stable PD patients, but large amounts are found during peritonitis, due probably to necrosis of mesothelial cells. The majority of studies found no relationship between dialysate CA125 and peritoneal transport parameters. Some cross-sectional studies reported a relationship with duration of PD, but others were unable to confirm this, due probably to the large interindividual variability. Longitudinal follow-up has shown a decrease in dialysate CA125, indicating loss of mesothelial cell mass. Application of theoretically more-biocompatible PD solutions causes an increase in dialysate CA125. CONCLUSIONS: Dialysate CA125 is a mesothelial cell mass marker. The concentration of CA125 should be determined after a standardized dwell. A single low value is not informative. A decrease with time on PD suggests loss of mesothelial cell mass. Dialysate CA125 is a marker of in vivo biocompatibility of (new) dialysis solutions. More research is necessary on the best methodology for measuring low concentrations and establishing normal values and a significant change.