Nachtsheim Robert, Dudley Ben, McNeil Paul L, Howdieshell Thomas R
Department of Surgery, University of New Mexico HSC, Albuquerque, New Mexico 87131-0001, USA.
J Surg Res. 2006 Jul;134(1):28-35. doi: 10.1016/j.jss.2006.03.008. Epub 2006 May 2.
This study was designed to analyze porcine plasma and peritoneal fluid for concentration differences of angiogenic molecular mediators and to determine local peritoneal sites of production of these molecules.
The peritoneum is now recognized as a dynamic cellular membrane with important functions, including antigen presentation; transport and movement of fluid, solutes, and particulate matter across serosal cavities; and secretion of glycosaminoglycans, extracellular matrix proteins, proinflammatory cytokines, and growth factors. The mechanisms of the peritoneal response to injury and the factors that determine the outcome of the reactive or reparative processes of the peritoneum remain poorly defined.
Domestic swine (n = 12) underwent percutaneous diagnostic peritoneal lavage to obtain preincision peritoneal fluid for biochemical analysis. Open biopsy samples of parietal peritoneum and omentum were obtained for immunochemical and molecular analysis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels were quantitated by enzyme-linked immunosorbent assay, and nitrite/nitrate (NOx) measured by nonenzymatic assay. Sections of formalin-fixed tissue were stained for immunoreactivity to VEGF, bFGF, and nitric oxide synthase (NOS). Frozen homogenized peritoneum and omentum were prepared for isolation of protein and RNA. An endothelial growth assay was created using human umbilical vein endothelial cells cultured with peritoneal fluid with or without anti-VEGF or anti-bFGF antibodies.
The mean plasma concentrations of VEGF, bFGF, and NOx were 20 +/- 5 pg/mL, 35 +/- 9 pg/mL, and 4.5 +/- 1.3 microm, compared with mean peritoneal fluid concentrations of 395 +/- 75 pg/mL, 486 +/- 72 pg/mL, and 35.0 +/- 8.8 mum respectively (P < 0.05 for each molecule). Immunochemistry demonstrated VEGF, bFGF, and NOS protein in mesothelium, submesothelium, and omentum. The use of Western blotting and reverse transcription polymerase chain reaction confirmed peritoneal and omental presence of VEGF and NOS-2. The use of endothelial bioassay documented peritoneal fluid angiogenic activity, which was inhibited by addition of neutralizing antibody to VEGF or bFGF.
Peritoneal compartmentalization of angiogenic mediators important in wound healing, inflammation, and tumor growth suggests that the plasma concentrations of these mediators do not reflect their tissue concentrations or local biological activity.
本研究旨在分析猪血浆和腹腔液中血管生成分子介质的浓度差异，并确定这些分子在腹腔内的产生部位。
目前认为腹膜是一种具有重要功能的动态细胞膜，包括抗原呈递；液体、溶质和颗粒物在浆膜腔之间的运输和移动；以及糖胺聚糖、细胞外基质蛋白、促炎细胞因子和生长因子的分泌。腹膜对损伤的反应机制以及决定腹膜反应性或修复性过程结果的因素仍不清楚。
对12头家猪进行经皮诊断性腹腔灌洗，以获取切开前的腹腔液进行生化分析。获取壁层腹膜和大网膜的开放活检样本进行免疫化学和分子分析。通过酶联免疫吸附测定法定量血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）水平，通过非酶法测定亚硝酸盐/硝酸盐（NOx）。对福尔马林固定组织切片进行VEGF、bFGF和一氧化氮合酶（NOS）免疫反应性染色。制备冷冻匀浆的腹膜和大网膜用于蛋白质和RNA的分离。使用人脐静脉内皮细胞与添加或不添加抗VEGF或抗bFGF抗体的腹腔液一起培养建立内皮生长测定法。
VEGF、bFGF和NOx的平均血浆浓度分别为20±5 pg/mL、35±9 pg/mL和4.5±1.3 μmol，而腹腔液的平均浓度分别为395±75 pg/mL、486±72 pg/mL和35.0±8.8 μmol（每种分子P<0.05）。免疫化学显示间皮、间皮下和大网膜中有VEGF、bFGF和NOS蛋白。蛋白质印迹法和逆转录聚合酶链反应证实腹膜和大网膜中存在VEGF和NOS-2。内皮生物测定法证明腹腔液具有血管生成活性，添加VEGF或bFGF中和抗体可抑制该活性。
在伤口愈合、炎症和肿瘤生长中起重要作用的血管生成介质在腹腔内存在分隔，这表明这些介质的血浆浓度不能反映其组织浓度或局部生物学活性。
