STAT6和NF-κB信号通路在U266细胞株产生免疫球蛋白E过程中的作用研究

发布时间：2018-02-13 00:25

本文关键词： 免疫球蛋白E(IgE) 白介素4 CD40抗体信号通路　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的:近年来,过敏性疾病的发生率越来越高,引起了西方国家乃至全球的关注,过敏性疾病对人类健康所造成的影响已成为一个全球性普遍关注的问题。现如今我国有1.3亿人正在饱受过敏性疾病的困扰,而全球人群发病率也呈逐年增高趋势,严重地影响了人们的生活质量。而对于此类疾病的治疗,目前只是通过控制患者的一些过敏性症状,而不能彻底治愈。目前我们所使用的抗过敏治疗大都是以降低血清中的游离血清中游离的免疫球蛋白E(Ig E)的含量为主,如奥马珠单抗治疗过敏性疾病,通过与游离Ig E结合而显著降低游离Ig E水平,阻断Ig E与肥大细胞、嗜酸粒细胞结合,从而抑制肥大细胞和嗜酸粒细胞的活化,减少和防止炎症介质的释放。在过敏性疾病中,免疫球蛋白E(immunoglobulin E,Ig E)主要介导了I型过敏反应。我们所使用的治疗并不能从源头降低免疫球蛋白E(Ig E)的产生。因此,了解Ig E产生的信号通路可能能为过敏性疾病的治疗提供新的靶点和思路。过去的研究表明,IL-4和CD40在Ig E的产生过程中必不可少。有研究指出,在过敏性患者的外周血单核细胞中(PBMc)中,Ig E产生的信号调节与NF-κB和STAT6信号通路有关,但是研究B细胞株中Ig E产生的信号通路调节罕见报导。因此,本研究在此基础上,采用两种特异性的信号通路的抑制剂,了解在B细胞株U266细胞株中免疫球蛋白E(Ig E)产生的信号通路调节,为疾病的治疗提供新的思路和方向。方法:采用不同浓度的白介素4(IL-4)或不同浓度的抗CD40抗体(CD40)诱导B细胞株,使用酶联免疫吸附测定法(ELISA)测定免疫球蛋白E(Ig E)的浓度变化,以及采用q-RTPCR的方法检测诱导的Ig E的重链基因(εGLT)的变化,选择最合适的诱导浓度;采用CCK8细胞增殖、毒性检测的实验方法,检测不同浓度的两种抑制剂对细胞活力的影响;选取合适的抑制剂浓度,并使用STAT6特异性抑制剂(A177-1726)和NF-κB特异性的抑制剂(JSH-23)抑制这两条信号通路,检测上清中免疫球蛋白E(Ig E)和Ig E的重链基因(εGLT),以及某些相关蛋白的变化。结果:(1)不同浓度的IL-4或CD40均可以诱导B细胞株产生免疫球蛋白E(Ig E)以及能够观察到Ig E重链基因的表达变化;(2)使用NF-KB特异性抑制剂JSH-23抑制通路,可以明显观察到免疫球蛋白E表达水平的降低,以及Ig E重链基因表达的变化;(3)同样地,使用STAT6特异性抑制剂,免疫球蛋白E的浓度以及重链基因的表达均降低,以及其相关蛋白的表达也明显下降;结论:本实验结果指出,NF-κB和STAT6信号通路在U266细胞株产生免疫球蛋白E(Ig E)的过程中有重要的作用,两者在Ig E的产生过程中起协同作用,了解B细细胞株中Ig E产生过程的调节,可以为临床治疗过敏性疾病奠定理论研究基础,并为寻找治疗靶点提供参考价值。
[Abstract]:Background and objective: in recent years, the incidence of allergic diseases has become increasingly high, which has attracted the attention of western countries and even the whole world. The impact of allergic diseases on human health has become a global concern. Nowadays, 130 million people in China are suffering from allergic diseases, and the incidence of the global population is also increasing year by year. It seriously affects people's quality of life. And the treatment of these diseases is now done only by controlling some of the allergic symptoms of the patients. At present, most of the antiallergic therapy we use is mainly to reduce the content of free immunoglobulin (Ig) in serum, such as omazumab in the treatment of allergic diseases. By binding with free IgE, the level of free IgE was significantly decreased, and the binding of IgE to mast cells and eosinophils was blocked, thus inhibiting the activation of mast cells and eosinophils. To reduce and prevent the release of inflammatory mediators. In allergic diseases, immunoglobulin Eg Eg) mainly mediates type I allergic reactions. The treatment we use does not reduce the production of immunoglobulin Ig E from the source. Understanding the signal pathway produced by IgE may provide new targets and ideas for the treatment of allergic diseases. Previous studies have shown that IL-4 and CD40 are essential in the production of IgE. The signal regulation of Ig E in peripheral blood monocytes of allergic patients is related to NF- 魏 B and STAT6 signaling pathway, but it is rare to study the regulation of Ig E signaling pathway in B cell line. Two specific signal pathway inhibitors were used to understand the regulation of the signal pathway produced in B cell line U266. Methods: B cell lines were induced by different concentrations of IL-4 (IL-4) or anti-CD40 (anti CD40 antibody CD40). The changes of heavy chain genes (蔚 GLTs) of induced IgE were detected by q-RT PCR and the most suitable concentration was selected, and the effects of two inhibitors of different concentrations on cell viability were detected by CCK8 cell proliferation and toxicity test. STAT6 specific inhibitor A177-1726) and NF- 魏 B specific inhibitor JSH-23) were used to inhibit these two signaling pathways. The heavy chain gene (蔚 GLTG) and the changes of some related proteins in the supernatant were detected. The results showed that different concentrations of IL-4 or CD40 could induce the production of immunoglobulin Eg E from B cell lines and the expression of Ig could be observed. The expression of E heavy chain gene was changed by using JSH-23, a specific inhibitor of NF-KB, to inhibit the transduction pathway. The decrease of immunoglobulin E expression and the change of IgE heavy chain gene expression were also observed. Similarly, the concentration of immunoglobulin E and the expression of heavy chain gene were decreased with the use of STAT6 specific inhibitor. Conclusion: NF- 魏 B and STAT6 signaling pathway play an important role in the production of immunoglobulin Ig E in U266 cell line, and they play a synergistic role in the production of IgE. Understanding the regulation of IgE production in B cell lines can lay a theoretical foundation for clinical treatment of allergic diseases and provide a reference value for finding therapeutic targets.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.1

【相似文献】