信号调节蛋白α负性调节IgE诱导的肥大细胞脱颗粒及细胞因子合成

发布时间：2018-04-08 08:35

本文选题：IgE　切入点：肥大细胞　出处：《第二军医大学》2012年硕士论文

【摘要】：研究背景及目的近几年随着空气污染等环境变化，过敏性疾病的发病率显著上升，其中包括Ⅰ型超敏反应。Ⅰ型超敏反应发病突然，严重时可能威胁生命。在自然界广泛存在能够致敏的抗原性物质，它们可以经呼吸道消化道或皮肤接触等途径进入体内，刺激免疫系统产生抗原特异性免疫球蛋白E（IgE）。随后IgE分子可以同肥大细胞表面受体FcεRI(high-affinity receptor for the Fc region of immunoglobulin E)稳定结合。FcεRI是一条α链、一条β链和两条相同的γ链组成的四聚体。α链是配体结合链，其胞外区能和IgE的Fc段结合；β链和γ链有ITAM区，发生磷酸化后介导下游信号传递。当特异抗原再次进入体内，可以同IgE分子结合并且使相邻的两个FcεRI交联，引起肥大细胞活化。肥大细胞活化后通过脱颗粒和分泌炎性因子等发挥其生物学作用。体内的免疫反应强度和范围受到多种机制调节，其中包括细胞表面激活性受体和抑制性受体平衡所起到的调控作用。信号调节蛋白家族（signal regulatory proteinfamily）就是一类细胞表面的抑制性受体。信号调节蛋白α（signal regulatory protein α，SIRPα）是一种广泛存在的抑制性受体，属于免疫球蛋白受体超家族。SIRPα主要表达在神经元细胞及髓系非淋巴细胞，如肥大细胞。SIRP胞外区具有三个IgSF样结构域和多个糖基化位点，胞内区带有免疫受体酪氨酸抑制基序（ITIMs），其中含四个酪氨酸残基，磷酸化后能够募集含SH2结构域的分子如蛋白磷酸酶SHP-1和SHP-2。肥大细胞作为过敏反应的效应细胞，抑制性受体SIRPα对其功能的调节尚未见系统性报道。我们研究发现抗原活化肥大细胞后SIRPα蛋白表达水平下降，提示SIRPα对肥大细胞活化可能存在抑制作用。为进一步了解SIRPα在肥大细胞活化过程以及Ⅰ型超敏反应中的作用，本课题通过利用差异表达SIRPα的细胞进行研究，研究了SIRPα在抗原诱导肥大细胞活化中的作用。重点是SIRPα对肥大细胞脱颗粒及分泌细胞因子的影响及相关信号通路的调节。实验方法 1．建立稳定高表达Myc-SIRPα和转染空载体对照的肥大细胞系。分离培养小鼠骨髓来源肥大细胞(BMMCs)，合成鉴定具有特异干扰效果siRNA，瞬时干扰SIRPα表达，构建BMMCs siSIRPα和BMMCs control细胞； 2．采用特异性磷酸化抗体检测SIRPα差异表达的肥大细胞在抗原刺激后p38、JNK、ERK、Akt、NFAT等信号分子的磷酸化水平变化，反映SIRPα对信号通路活化的调节； 3．运用荧光素酶报告基因系统检测SIRPα对NF-κB，NFAT转录活性的调节； 4．通过检测肥大细胞活化后β-氨基己糖胺酶的释放，，研究SIRPα对肥大细胞脱颗粒的调节； 5．采用Real-time PCR方法检测肥大细胞炎性因子的mRNA水平； 6．采用荧光探针检测SIRPα对细胞骨架F-actin聚合的调节； 7．使用Ca2+指示剂检测SIRPα对抗原引起肥大活化过程中Ca2+流动的调节； 8．用Western blot检测SIRPα对细胞骨架微管形成的影响； 10.将BMMCs siSIRPα及阴性对照细胞输入肥大细胞缺失小鼠（c-kitwsh/wsh）体内，重构肥大细胞系统。进行被动皮肤过敏反应，体内观察SIRPα对肥大细胞功能的调节。 11.采用免疫组织沉淀的方法检测SIRPα与SHP-2的结合，SHP-2与PI3Kp85和IKKβ的相互作用。结果 1. SIRPα参与抗原诱导的肥大细胞活化过程：在肥大细胞活化过程中，SIRPα蛋白表达降低,并呈现抗原剂量与时间依赖性。同时，SIRPα发生磷酸化并募集SHP-1和SHP-2。 2. SIRPα调节肥大细胞活化后分泌功能：SIRPα显著抑制肥大细胞脱颗粒及细胞因子合成。 3. SIRPα抑制肥大细胞活化过程中的细胞骨架改变：SIRPα抑制肥大细胞活化过程中α-tubulin聚合及F-actin解聚。 4. SIRPα抑制肥大细胞活化过程中的Ca2+释放，提示SIRPα通过调节Ca2+释放影响肥大细胞骨架改变及脱颗粒过程。 5. SIRPα对Ⅰ型超敏反应过程中血管渗出的影响：与对照小鼠相比，输入低表达SIRPα肥大细胞的小鼠皮下渗出更加明显。这进一步说明SIRPα抑制肥大细胞分泌功能。 6. SIRPα抑制细胞骨架重构的机制：抗原引起肥大细胞活化过程中，SIRPα明显抑制PLCγ1磷酸化，从而抑制Ca2+释放。此外SIRPα明显抑制Rac-1GTPase及PAK1的活性，进而抑制了微管的形成。 7. SIRPα影响肥大细胞细胞因子合成分泌的机制：抗原诱导的肥大细胞活化过程中，SIRPα通过抑制MAPK信号通路磷酸化以及转录因子NFκB和NFAT活性，抑制肥大细胞由抗原诱导的炎性因子合成与分泌。 8. SIRPα在抗原诱导的肥大细胞活化过程中发挥负性调节作用的机制：肥大细胞活化后，SIRPα通过Src家族激酶发生磷酸化。随后SIRPα通过竞争结合SHP-2，影响SHP-2与PI3Kp85及IKKβ的结合，抑制Akt与NF-κB信号通路。结论本研究从分子－细胞－动物水平较深入研究了信号调节蛋白SIRPα对肥大细胞功能的调节作用，发现SIRPα负性调节肥大细胞活化，包括抑制肥大细胞脱颗粒及炎性因子合成并对体内的过敏反应能力具有负向调控能力。同时本研究进一步探讨了SIRPα调节相关信号转导通路的作用机制，证明SIRPα可能通过抑制SHP-2与IKKβ及PI3Kp85的结合发挥负向调控作用。
[Abstract]:Background and purpose of research
In recent years, with the air pollution and other environmental changes, the incidence of allergic diseases increased significantly, including type I hypersensitivity. Type I hypersensitivity sudden onset, severe life-threatening. Widespread allergens sensitization to in nature, they can breathe the gastrointestinal tract or the skin contact way enter the body, stimulate the immune system to produce antigen specific immunoglobulin E (IgE). Then IgE molecules can with mast cell surface receptor Fc (high-affinity receptor for the e RI Fc region of immunoglobulin E.Fc RI) with stable epsilon is an alpha chain, four dimer a beta chains and two identical the gamma chain composition. Alpha chain is the ligand binding chain, Fc and IgE to the extracellular binding; beta chain and gamma chain ITAM region, occurred after phosphorylation mediated downstream signaling. When specific antigen again into the body, with IgE molecules And the two adjacent Fc epsilon RI were crosslinked and the mast cells were activated. The mast cells were activated by degranulation and secretion of inflammatory factors.
The immune response intensity and range of the body by a variety of mechanisms including regulation, regulation of the activation of cell surface receptors and inhibitory receptors play balance. Signal regulated protein family (signal regulatory proteinfamily) is a kind of cell surface inhibitory receptors. Signal regulatory protein (signal regulatory protein SIRP is alpha, alpha) a widespread inhibitory receptor, belonging to the immunoglobulin superfamily.SIRP alpha receptor expressed mainly in neurons and non myeloid cells, such as mast cells in the extracellular domain of.SIRP with three IgSF like domains and multiple glycosylation sites, the intracellular region with immunoreceptor tyrosine based inhibitory motif (ITIMs), which contains four tyrosine residues, phosphorylation can raise SH2 domain containing protein phosphatase molecules such as SHP-1 and SHP-2.
Mast cells as effector cells in allergic reactions, inhibitory receptor SIRP alpha to regulate its function have not been reported. We found that the SIRP protein expression level decreased antigen activated mast cells, suggesting that SIRP may have inhibitory effect on alpha activation of mast cells. To further understand the SIRP alpha activation and type I hypersensitivity the reaction in the role of mast cells, this study was carried out by using the difference of the expression of SIRP cells on SIRP induced mast cell activation in antigen. Focused on the regulation effect of SIRP on mast cell degranulation and cytokine secretion and related signaling pathways.
Experimental method
1., we established a stable mast cell line with high expression of Myc-SIRP alpha and transfected empty vector. A murine bone marrow derived mast cell (BMMCs) was isolated and cultured, and the specific interference effect siRNA was synthesized and identified. The expression of SIRP alpha was transiently interfered, and BMMCs siSIRP alpha and BMMCs control cells were constructed.
2., we used specific phosphorylated antibody to detect the change of phosphorylation level of p38, JNK, ERK, Akt, NFAT and other signal molecules after SIRP stimulation, which reflected the regulation of SIRP alpha on activation of signaling pathway.
3. the regulation of the transcriptional activity of NF- - kappa B and NFAT was detected by the luciferase reporter gene system.
4. the regulation of SIRP - alpha on degranulation of mast cells was studied by detecting the release of beta aminohexaminase after activation of mast cells.
5. the mRNA level of mast cell inflammatory factors was detected by Real-time PCR method.
6. the fluorescence probe was used to detect the regulation of SIRP - alpha on cytoskeleton F-actin polymerization.
7. the Ca2+ indicator was used to detect the regulation of SIRP alpha on the Ca2+ flow during the hypertrophy activation process.
8. Western blot was used to detect the effect of SIRP alpha on the formation of cytoskeleton microtubules.
10., BMMCs siSIRP alpha and negative control cells were injected into mast cell deficient mice (c-kitwsh/wsh) to reconstruct mast cell system. The passive cutaneous anaphylaxis was observed, and the regulation of SIRP alpha on mast cell function was observed in vivo.
11. the binding of SIRP alpha to SHP-2 and the interaction between SHP-2 and PI3Kp85 and IKK beta were detected by immuno tissue precipitation.
Result
1. SIRP alpha participates in the process of antigen induced mast cell activation: in the process of mast cell activation, the expression of SIRP alpha protein decreases, and shows an antigen dose and time dependence. At the same time, SIRP alpha is phosphorylated and SHP-1 and SHP-2. are raised.
2. SIRP alpha regulates the secretory function of mast cell activation: SIRP a significantly inhibits the degranulation of mast cells and the synthesis of cytokines.
3. SIRP alpha inhibits the cytoskeleton changes during the activation of mast cells: SIRP alpha inhibits the aggregation of alpha -tubulin and the depolymerization of F-actin during the activation of mast cells.
4. SIRP alpha inhibits the release of Ca2+ during the activation of mast cells, suggesting that SIRP alpha affects the changes in the cytoskeleton and degranulation of mast cells by regulating the release of Ca2+.
5., the effect of SIRP alpha on vascular exudation in type I hypersensitivity reaction: compared with control mice, the mice with low expression of SIRP alpha mast cells showed a more obvious subcutaneous exudation, which further indicated that SIRP alpha inhibited the secretion function of mast cells.
6., the mechanism of SIRP alpha inhibiting cytoskeletal remodeling is that SIRP alpha significantly inhibits PLC Ca2+ 1 phosphorylation and inhibits Ca2+ release during antigen induced mast cell activation. In addition, SIRP alpha significantly inhibits Rac-1GTPase and PAK1 activity, thereby inhibiting the formation of microtubules.
The mechanism of 7. SIRP alpha on mast cell cytokine secretion: antigen induced activation of mast cells in the process of SIRP alpha through inhibition of the MAPK signaling pathway phosphorylation and transcription factor NF kappa B and NFAT activity, inflammatory cytokines inhibit mast cell induced by antigen synthesis and secretion.
The mechanism of negative regulation function 8. SIRP alpha activation in mast cell antigen induced in mast cells after the activation of SIRP alpha by Src kinase phosphorylation. Then SIRP alpha through competition with SHP-2, SHP-2 and PI3Kp85 binding and IKK beta, Akt and NF- inhibited B signaling pathway.
conclusion
This study from the molecular cellular animal level is in-depth study of signal regulated protein SIRP alpha regulates the function of mast cells, found SIRP a negative regulator of mast cell activation, including inhibition of mast cell degranulation and synthesis of inflammatory factors and allergic reaction ability in vivo has negative regulation ability. At the same time this study further to investigate the mechanism of SIRP regulating alpha related signal transduction pathway, SIRP may prove a negative role played by combined inhibition of SHP-2 and IKK beta and PI3Kp85.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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