本文選題：IgE　切入點(diǎn)：肥大細(xì)胞　出處：《第二軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：研究背景及目的 近幾年隨著空氣污染等環(huán)境變化，過敏性疾病的發(fā)病率顯著上升，其中包括Ⅰ型超敏反應(yīng)。Ⅰ型超敏反應(yīng)發(fā)病突然，嚴(yán)重時可能威脅生命。在自然界廣泛存在能夠致敏的抗原性物質(zhì)，它們可以經(jīng)呼吸道消化道或皮膚接觸等途徑進(jìn)入體內(nèi)，刺激免疫系統(tǒng)產(chǎn)生抗原特異性免疫球蛋白E（IgE）。隨后IgE分子可以同肥大細(xì)胞表面受體FcεRI(high-affinity receptor for the Fc region of immunoglobulin E)穩(wěn)定結(jié)合。FcεRI是一條α鏈、一條β鏈和兩條相同的γ鏈組成的四聚體。α鏈?zhǔn)桥潴w結(jié)合鏈，其胞外區(qū)能和IgE的Fc段結(jié)合；β鏈和γ鏈有ITAM區(qū)，發(fā)生磷酸化后介導(dǎo)下游信號傳遞。當(dāng)特異抗原再次進(jìn)入體內(nèi)，可以同IgE分子結(jié)合并且使相鄰的兩個FcεRI交聯(lián)，引起肥大細(xì)胞活化。肥大細(xì)胞活化后通過脫顆粒和分泌炎性因子等發(fā)揮其生物學(xué)作用。 體內(nèi)的免疫反應(yīng)強(qiáng)度和范圍受到多種機(jī)制調(diào)節(jié)，其中包括細(xì)胞表面激活性受體和抑制性受體平衡所起到的調(diào)控作用。信號調(diào)節(jié)蛋白家族（signal regulatory proteinfamily）就是一類細(xì)胞表面的抑制性受體。信號調(diào)節(jié)蛋白α（signal regulatory protein α，SIRPα）是一種廣泛存在的抑制性受體，屬于免疫球蛋白受體超家族。SIRPα主要表達(dá)在神經(jīng)元細(xì)胞及髓系非淋巴細(xì)胞，如肥大細(xì)胞。SIRP胞外區(qū)具有三個IgSF樣結(jié)構(gòu)域和多個糖基化位點(diǎn)，胞內(nèi)區(qū)帶有免疫受體酪氨酸抑制基序（ITIMs），其中含四個酪氨酸殘基，磷酸化后能夠募集含SH2結(jié)構(gòu)域的分子如蛋白磷酸酶SHP-1和SHP-2。 肥大細(xì)胞作為過敏反應(yīng)的效應(yīng)細(xì)胞，抑制性受體SIRPα對其功能的調(diào)節(jié)尚未見系統(tǒng)性報道。我們研究發(fā)現(xiàn)抗原活化肥大細(xì)胞后SIRPα蛋白表達(dá)水平下降，提示SIRPα對肥大細(xì)胞活化可能存在抑制作用。為進(jìn)一步了解SIRPα在肥大細(xì)胞活化過程以及Ⅰ型超敏反應(yīng)中的作用，本課題通過利用差異表達(dá)SIRPα的細(xì)胞進(jìn)行研究，研究了SIRPα在抗原誘導(dǎo)肥大細(xì)胞活化中的作用。重點(diǎn)是SIRPα對肥大細(xì)胞脫顆粒及分泌細(xì)胞因子的影響及相關(guān)信號通路的調(diào)節(jié)。 實(shí)驗(yàn)方法 1．建立穩(wěn)定高表達(dá)Myc-SIRPα和轉(zhuǎn)染空載體對照的肥大細(xì)胞系。分離培養(yǎng)小鼠骨髓來源肥大細(xì)胞(BMMCs)，合成鑒定具有特異干擾效果siRNA，瞬時干擾SIRPα表達(dá)，構(gòu)建BMMCs siSIRPα和BMMCs control細(xì)胞； 2．采用特異性磷酸化抗體檢測SIRPα差異表達(dá)的肥大細(xì)胞在抗原刺激后p38、JNK、ERK、Akt、NFAT等信號分子的磷酸化水平變化，反映SIRPα對信號通路活化的調(diào)節(jié)； 3．運(yùn)用熒光素酶報告基因系統(tǒng)檢測SIRPα對NF-κB，NFAT轉(zhuǎn)錄活性的調(diào)節(jié)； 4．通過檢測肥大細(xì)胞活化后β-氨基己糖胺酶的釋放，，研究SIRPα對肥大細(xì)胞脫顆粒的調(diào)節(jié)； 5．采用Real-time PCR方法檢測肥大細(xì)胞炎性因子的mRNA水平； 6．采用熒光探針檢測SIRPα對細(xì)胞骨架F-actin聚合的調(diào)節(jié)； 7．使用Ca2+指示劑檢測SIRPα對抗原引起肥大活化過程中Ca2+流動的調(diào)節(jié)； 8．用Western blot檢測SIRPα對細(xì)胞骨架微管形成的影響； 10.將BMMCs siSIRPα及陰性對照細(xì)胞輸入肥大細(xì)胞缺失小鼠（c-kitwsh/wsh）體內(nèi)，重構(gòu)肥大細(xì)胞系統(tǒng)。進(jìn)行被動皮膚過敏反應(yīng)，體內(nèi)觀察SIRPα對肥大細(xì)胞功能的調(diào)節(jié)。 11.采用免疫組織沉淀的方法檢測SIRPα與SHP-2的結(jié)合，SHP-2與PI3Kp85和IKKβ的相互作用。 結(jié)果 1. SIRPα參與抗原誘導(dǎo)的肥大細(xì)胞活化過程：在肥大細(xì)胞活化過程中，SIRPα蛋白表達(dá)降低,并呈現(xiàn)抗原劑量與時間依賴性。同時，SIRPα發(fā)生磷酸化并募集SHP-1和SHP-2。 2. SIRPα調(diào)節(jié)肥大細(xì)胞活化后分泌功能：SIRPα顯著抑制肥大細(xì)胞脫顆粒及細(xì)胞因子合成。 3. SIRPα抑制肥大細(xì)胞活化過程中的細(xì)胞骨架改變：SIRPα抑制肥大細(xì)胞活化過程中α-tubulin聚合及F-actin解聚。 4. SIRPα抑制肥大細(xì)胞活化過程中的Ca2+釋放，提示SIRPα通過調(diào)節(jié)Ca2+釋放影響肥大細(xì)胞骨架改變及脫顆粒過程。 5. SIRPα對Ⅰ型超敏反應(yīng)過程中血管滲出的影響：與對照小鼠相比，輸入低表達(dá)SIRPα肥大細(xì)胞的小鼠皮下滲出更加明顯。這進(jìn)一步說明SIRPα抑制肥大細(xì)胞分泌功能。 6. SIRPα抑制細(xì)胞骨架重構(gòu)的機(jī)制：抗原引起肥大細(xì)胞活化過程中，SIRPα明顯抑制PLCγ1磷酸化，從而抑制Ca2+釋放。此外SIRPα明顯抑制Rac-1GTPase及PAK1的活性，進(jìn)而抑制了微管的形成。 7. SIRPα影響肥大細(xì)胞細(xì)胞因子合成分泌的機(jī)制：抗原誘導(dǎo)的肥大細(xì)胞活化過程中，SIRPα通過抑制MAPK信號通路磷酸化以及轉(zhuǎn)錄因子NFκB和NFAT活性，抑制肥大細(xì)胞由抗原誘導(dǎo)的炎性因子合成與分泌。 8. SIRPα在抗原誘導(dǎo)的肥大細(xì)胞活化過程中發(fā)揮負(fù)性調(diào)節(jié)作用的機(jī)制：肥大細(xì)胞活化后，SIRPα通過Src家族激酶發(fā)生磷酸化。隨后SIRPα通過競爭結(jié)合SHP-2，影響SHP-2與PI3Kp85及IKKβ的結(jié)合，抑制Akt與NF-κB信號通路。 結(jié)論 本研究從分子－細(xì)胞－動物水平較深入研究了信號調(diào)節(jié)蛋白SIRPα對肥大細(xì)胞功能的調(diào)節(jié)作用，發(fā)現(xiàn)SIRPα負(fù)性調(diào)節(jié)肥大細(xì)胞活化，包括抑制肥大細(xì)胞脫顆粒及炎性因子合成并對體內(nèi)的過敏反應(yīng)能力具有負(fù)向調(diào)控能力。同時本研究進(jìn)一步探討了SIRPα調(diào)節(jié)相關(guān)信號轉(zhuǎn)導(dǎo)通路的作用機(jī)制，證明SIRPα可能通過抑制SHP-2與IKKβ及PI3Kp85的結(jié)合發(fā)揮負(fù)向調(diào)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

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