Study of anti-laminin-1 autoantibodies in endometriosis-associated infertility

1. 子宫内膜异位症不孕症患者的抗层粘连蛋白-1自身抗体识别小鼠层粘连蛋白-1的n-连锁寡糖链。百分比的患者表达一种识别A - gal （Galal- 3Ga1β1-4G1cNAc-R）糖链表位的天然抗体。然而，从小鼠laminin-1中去除末端唾液酸残基导致大多数患者血清中完全失去抗体结合。为了鉴定这些抗体的真正自身抗原，用Western blot分析检测了异位和异位子宫内膜、卵巢囊肿、腹膜液和子宫内膜异位症不孕患者或健康对照者的血清。由于没有检测到自身抗原，因此我们使用HPLC/MS分析了低聚糖链表位的结构。根据确定的表位结构，我们将预测真正的自身抗原。我们研究了最近建立的抗层粘连蛋白-1小鼠单克隆IgM （AK8）、从子宫内膜异位症不孕症患者血清亲和纯化的抗层粘连蛋白-1 IgG和兔多克隆抗层粘连蛋白IgG对小鼠体外胚胎着床的影响。AK8和兔多克隆抗层粘连蛋白IgG对囊胚的孵化没有影响，但对囊胚的粘附和生长有较强的抑制作用。另一方面，针对laminin-1寡糖链的亲和纯化的IgG不影响囊胚的孵化和粘附，但抑制了约30%囊胚的生长面积。部分子宫内膜异位症不孕患者血清与层粘连蛋白γ - l链肽段发生交叉反应。Laminin-1包含约20个血管生成肽序列。其中，γ - 1链的血管生成肽序列（C16肽）与内皮细胞的整合素α5β1和αvβ3结合，促进血管生成的作用最强。血管生成对子宫内膜异位症的发生和发展至关重要。我们用bsa偶联的C16肽免疫小鼠，建立抗C16单克隆IgM （anti-C16 mAb）。抗c16单抗可抑制人脐内皮细胞（HUVECs）体外成管和裸鼠体内血管生成。我们将在体外研究抗c16单抗对人子宫内膜碎片或子宫内膜异位症病变血管生成的抑制作用。少

1. Anti-laminin-1 autoantibodies in infertile patients with endometriosis recognized N-linked oligosaccharide chains of mouse laminin-1. A percentage of patients expressed a natural antibody that recognizes the a-Gal (Galal- 3Ga1β1-4G1cNAc-R) sugar chain epitope. However, the removal of terminal sialic acid residues from mouse laminin-1 resulted in a complete loss of antibody binding by most patients' sera. To identify real autoantigens of these antibodies, lysates from ectopic and eutopic endometrium, and ovarian cyst, peritoneal fluids, and sera from infertile patients with endometriosis or healthy controls were examined by Western blot analysis. Autoantigens were not detected, therefore, we analyze the structure of the oligosaccharide chain epitope using HPLC/MS. Based on the determined epitope structure, we will predict the real autoantigens.2. We studied the effect of a recently established anti-laminin-1 mouse monoclonal IgM (AK8), an affinity-purified anti-laminin-1 IgG from the … More serum of an infertile patient with endometriosis, and a polyclonal anti-laminin rabbit IgG on mouse embryo implantation in vitro. AK8 and the polyclonal anti-laminin rabbit IgG did not have an effect on blastocyst hatching, but strongly inhibited the blastocyst adhesion and outgrowth in about 100 % of the blastocysts. On the other hand, the affinity-purified IgG specific for the oligosaccharide chains of laminin-1 did not affect blastocyst hatching or adhesion, but inhibited the area of outgrowth in about 30 % of the blastocysts.3. Sera from some infertile patients with endometriosis cross-reacted with the peptide moiety of laminin γl chain. Laminin-1 contains about 20 angiogenic peptide sequences. Among of them, angiogenic peptide sequence of γ1 chain (C16 peptide) binds to integrin α5β1 and αvβ3 of endothelial cells and most potently promotes angiogenesis. Angiogenesis is critical for the initiation and progression of endometriosis. We established an anti-C16 peptide monoclonal IgM (anti-C16 mAb) by immunizing mice with BSA-conjugated C16 peptide. Anti-C16 mAb inhibited in vitro tube formation of human umbilical endothelial cells (HUVECs) and in vivo angiogenesis in nude mice. We will investigate the inhibitory effect of anti-C16 mAb on angiogenesis in human endometrial fragments or endometriotic lesions in vitro. Less