Study of the effect of environmental chemicals on the high-affinity IgG receptor-mediated signal transudation of mest cells.

1.犬皮肤肥大细胞瘤(CM-MC)细胞可通过免疫球蛋白和免疫球蛋白E两种途径被激活。Ig G介导的蛋白酪氨酸磷酸化和Ca~(2+)&Gt~(2+)内流与IgE介导的相似。蛋白激酶抑制剂星形孢子素以剂量依赖方式抑制免疫球蛋白G介导的[Ca~(2+)]i升高和组胺释放。2.免疫沉淀法和逆转录聚合酶链式反应检测CM-MC细胞高亲和力免疫球蛋白受体(Fc-γRI)蛋白和基因的表达。然后对FcγRIα亚基的基因序列进行了鉴定。犬FcγRIα全长1119bp(GenBank登录号，AB 101519)。犬FcγRIA基因与人和小鼠的同源性分别为84%和78%。3.将FcγRIα基因导入COS7细胞，检测单体免疫球蛋白与细胞的结合。采用RTPCR法和5‘/3’-RACE方法，从犬CM-MC细胞总RNA中克隆了犬FcγRIα基因。将该基因连接到哺乳动物表达载体上，并导入COS-7细胞。4.P物质(10-30μM)和C5a(0.5-1μM)呈剂量依赖性地激活CM-MC。胰岛激活蛋白(IAP)可抑制这些物质引起的[Ca^^lt；2+]i升高和脱颗粒。因此，这些物质的激活似乎依赖于G蛋白偶联机制。结论：CM-MC细胞可用于研究免疫球蛋白依赖引起的变态反应性炎症机制。影响P物质和C5a产生的环境化学物质可能影响由免疫球蛋白依赖性肥大细胞激活引起的变态反应性炎症。

1.Canine cutaneous mastocytoma-derived (CM-MC) cells were activated both IgG- and IgE-mediated mechanisms. IgG-mediated protein tyrosine phosphorylation and Ca^<2+> influx were similar to those mediated by IgE. Protein kinase inhibitor staurosporine inhibited IgG-mediated [Ca^<2+>]i elevation and histamine release in a dose-dependent manner.2.The presence of high affinity IgG receptors (FcγRI) protein and mRNA in CM-MC cells was determined by immunoprecipitation and RT-PCR methods. Then, the cDNA sequence of FcγRIα subunit was identified. The entire canine FcγRIα cDNA was 1119bp long (GenBank accession number, AB 101519). The overall identity of the canine FcγRIa cDNA to its human and mouse counterparts was 84% and 78%, respectively.3.We transfected cDNA of FcγRIα to COS-7 cells to examine the binding of monomeric IgG to the cells. The cDNA of canine FcγRIα was cloned by RT-PCR and 5'/3'-RACE from total RNA of CM-MC cells. The cDNA was ligated to mammalian expression vector and transfected into COS -7 cells. The binding of IgG onto the COS -7 cells was detected by flow-cytometry.4.CM-MC was activated by Substance P (10-30 μM) and C5a (0.5-1μM) dose-dependently. [Ca^<2+>]i elevation and degranulation by these substances was inhibited by islet- activated protein (IAP). Therefore, the activation of these substances seemed to be dependent on G-protein-coupled mechanism5.In conclusion, CM-MC cells were useful for the study of allergic inflammation caused by IgG-dependent mechanisms. The environmental chemicals that affect Substance P and C5a production might influence allergic inflammation caused by IgG-dependent mast cell activation.