Giudice L C, Dsupin B A, Gargosky S E, Rosenfeld R G, Irwin J C
Department of Gynecology and Obstetrics, Stanford University Medical Center, California 94305.
J Clin Endocrinol Metab. 1994 Nov;79(5):1284-93. doi: 10.1210/jcem.79.5.7525631.
Peritoneal fluid (PF) lines the abdomen and pelvis and is believed to contain growth factors that stimulate endometriosis, a benign gynecological condition associated with pelvic pain and infertility, in which endometrial cells proliferate and differentiate on the pelvic peritoneum, outside of their normal location within the uterus. In this study, we examined the insulin-like growth factor (IGF) system in seven paired samples of PF and serum from normally cycling women and examined the mitogenic potential of this fluid on cultured endometrial stromal cells. IGF-I, IGF-II, and IGF-binding protein-1 (IGFBP-1), -2, -3, and -4 were identified in PF by immunoassays. PF IGF levels, determined by RIA, were approximately 60% of paired serum levels, and PF levels of IGFBP-2 and IGFBP-3, determined by Western ligand blotting and RIA, respectively, were approximately half of their serum concentrations. IGFBP-4 was barely detectable by Western ligand blotting in PF, and levels of IGFBP-1, determined by immunoassay, were not appreciably different in PF and serum. Incubation of [125I]IGF-II with serum and PF and subsequent size-exclusion chromatography at neutral pH revealed approximately equal incorporation of radiolabel in the IGFBP regions of 150 and 44 kilodaltons (kDa) in serum and primarily in the 44-kDa region in PF. RIA of IGFBP-3 in the IGFBP regions of column effluent revealed that the majority of IGFBP-3 was in the 150-kDa region in both serum and PF, suggesting the presence of the ternary complex in PF. Western ligand blotting of column effluent samples revealed 37-/43-kDa IGFBP-3 primarily in the 150-kDa complex in serum and a marked reduction in the amount of the 37-/43-kDa IGFBP in PF. Western immunoblotting of column effluent with IGFBP-3 antiserum revealed immunoreactive IGFBP-3 forms of 37-43 kDa (major) and 28 kDa (minor) in serum and almost exclusively the 28-kDa band in PF, suggesting that IGFBP-3 in PF may be proteolytically processed. The presence of an IGFBP-3 protease was confirmed using [125I]IGFBP-3 as substrate and was not appreciably present in paired serum samples. Inhibitor profiles demonstrated that this protease is a metal-independent serine protease, and its approximate relative molecular mass was estimated to be 69 kDa, determined by size-exclusion chromatography. The mitogenic potential of IGF peptides and PF was assessed on cultured endometrial stromal cells to test the hypothesis that IGFs in PF may stimulate the growth of endometrium in the pelvic cavity, for example in the disorder of endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS)
腹膜液（PF）衬于腹部和盆腔，据信其中含有刺激子宫内膜异位症的生长因子。子宫内膜异位症是一种与盆腔疼痛和不孕相关的良性妇科疾病，在此疾病中，子宫内膜细胞在子宫外的盆腔腹膜上增殖和分化。在本研究中，我们检测了7对来自正常月经周期女性的PF和血清样本中的胰岛素样生长因子（IGF）系统，并检测了该液体对培养的子宫内膜基质细胞的促有丝分裂潜力。通过免疫测定在PF中鉴定出IGF-I、IGF-II以及IGF结合蛋白-1（IGFBP-1）、-2、-3和-4。通过放射免疫分析（RIA）测定的PF中IGF水平约为配对血清水平的60%，通过Western配体印迹法和RIA分别测定的PF中IGFBP-2和IGFBP-3水平约为其血清浓度的一半。通过Western配体印迹法在PF中几乎检测不到IGFBP-4，通过免疫测定测定的PF和血清中IGFBP-1水平无明显差异。将[125I]IGF-II与血清和PF一起温育，随后在中性pH下进行尺寸排阻色谱分析，结果显示血清中150和44千道尔顿（kDa）的IGFBP区域中放射性标记的掺入量大致相等，而在PF中主要在44-kDa区域。对柱流出物的IGFBP区域进行IGFBP-3的RIA分析表明，血清和PF中大多数IGFBP-3都在150-kDa区域，这表明PF中存在三元复合物。对柱流出物样品进行Western配体印迹分析显示，血清中150-kDa复合物中主要是37/43-kDa的IGFBP-3，而PF中37/43-kDa的IGFBP量明显减少。用IGFBP-3抗血清对柱流出物进行Western免疫印迹分析显示，血清中有免疫反应性的37 - 43 kDa（主要）和28 kDa（次要）的IGFBP-3形式，而PF中几乎只有28-kDa条带，这表明PF中的IGFBP-3可能经过蛋白水解处理。以[125I]IGFBP-3为底物证实了PF中存在IGFBP-3蛋白酶，而配对血清样品中未明显检测到该蛋白酶。抑制剂分析表明，该蛋白酶是一种不依赖金属的丝氨酸蛋白酶，通过尺寸排阻色谱法估计其近似相对分子质量为69 kDa。在培养的子宫内膜基质细胞上评估了IGF肽和PF的促有丝分裂潜力，以检验PF中的IGF可能刺激盆腔内子宫内膜生长（例如在子宫内膜异位症中）这一假说。（摘要截断于400字）
