Safaralizadeh Reza, Soheili Zahra-Soheila, Deezagi Abdolkhaleg, Pourpak Zahra, Samiei Shahram, Moin Mostafa
Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Iran J Allergy Asthma Immunol. 2009 Dec;8(4):177-83.
FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast cell-mediated allergic reactions.
FcepsilonRI，即IgE的高亲和力受体，在引发过敏反应中起关键作用。当它被过敏原和IgE复合物聚集时，负责诱导肥大细胞脱颗粒并释放过敏介质。肥大细胞上的FcepsilonRI由三个亚基组成；α链直接结合IgE，β链和γ链二聚体共同介导细胞内信号传导。多价抗原使肥大细胞和嗜碱性粒细胞表面结合IgE的FcepsilonRI交联，从而诱导化学介质的释放。本体外研究旨在探讨合成的FcepsilonRI-α siRNA对抗原诱导的MC/9细胞激活的影响。用FcepsilonRI-α siRNA和阴性对照siRNA转染小鼠肥大细胞MC/9细胞。6小时后，用抗二硝基苯基（DNP）IgE对细胞进行致敏。然后用二硝基苯基-人血清白蛋白（DNP-HSA）刺激细胞以诱导肥大细胞脱颗粒，之后收集上清液。分别通过实时PCR和蛋白质印迹分析测量mRNA和蛋白质表达量。通过流式细胞术测定细胞表面FcepsilonRI-α的表达率。随后使用ELISA和分光光度法测量FcepsilonRI-α siRNA对抗原诱导的组胺和β-己糖胺酶释放的影响。与对照细胞相比，FcepsilonRI-α siRNA处理的细胞中FcepsilonRI-α mRNA和蛋白质表达显著降低。FcepsilonRI介导的肥大细胞β-己糖胺酶和组胺释放也受到抑制。本研究表明，FcepsilonRI-α siRNA可抑制FcepsilonRI-α表达，并抑制抗原刺激的MC/9细胞中的脱颗粒和组胺释放。总之，通过siRNA敲低FcepsilonRI-α可能是抑制肥大细胞介导的过敏反应的一种有前景的方法。
