Cocchiara R, Lampiasi N, Albeggiani G, Azzolina A, Bongiovanni A, Gianaroli L, Di Blasi F, Geraci D
Istituto di Biologia dello Sviluppo, CNR, Palermo, Italia.
Mol Hum Reprod. 1996 Oct;2(10):781-91. doi: 10.1093/molehr/2.10.781.
The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.
胚胎 - 母体交流的生化网络的存在意味着各种分泌分子构成了一种信号 - 反应机制，这对哺乳动物的胚胎着床过程很重要。在此，我们报告了一种表观分子量为136 kDa的蛋白质的纯化，该蛋白质可使胚胎源性组胺释放因子（EHRF）活性增加2000倍。通过亲和色谱法从2 - 8细胞期人类胚胎的体外培养液中纯化得到的这种蛋白质，经免疫印迹和酶联免疫吸附测定证明能够结合免疫球蛋白（Ig）E。我们发现EHRF能够在体外诱导妊娠第4天（胚胎发育的着床前期）收集的大鼠子宫组织释放组胺和细胞因子。当使用EHRF作为促分泌剂时，通过酶联免疫吸附测定检测到粒细胞巨噬细胞集落刺激因子（GM - CSF）释放量从3 pg/g增加到55 pg/g（P < 0.01），肿瘤坏死因子 - α（TNF - α）释放量从0增加到2.1 ng/g（P < 0.01）。使用IgE - 琼脂糖亲和色谱柱采用一种简单方法纯化子宫肥大细胞，并用与特异性大鼠IgE抗体偶联的磁珠检查其纯度（90%）。当用EHRF以与子宫外植体相同的方式刺激纯化的肥大细胞时，获得了类似的GM - CSF和TNF - α释放模式。我们还描述了从纯化的子宫肥大细胞中进行GM - CSF和TNF - α mRNA的逆转录 - 聚合酶链反应（RT - PCR）。在妊娠第4天仅发现TNF - α的mRNA，在用EHRF刺激后其增加。总之，所呈现的数据表明，在着床前期分离的子宫肥大细胞在与胚胎因子相互作用后在体外释放细胞因子。
