【摘要】：肥大細胞是一類由骨髓來源的造血干細胞分化而來的免疫細胞,主要分布在與外界環(huán)境接觸緊密的皮膚、氣道和腸道等組織的黏膜,是引發(fā)機體I型超敏反應和過敏性反應的極為關鍵的效應細胞。就目前研究認為,肥大細胞是過敏反應的初級效應細胞,在過敏反應中居于十分關鍵的位置[1,2]。活化的肥大細胞通過釋放顆粒中預先合成的化學介質(zhì),并產(chǎn)生多種細胞因子和趨化因子,以及分泌新合成的花生四烯酸和各種蛋白質(zhì)從而導致信號級聯(lián)放大,促發(fā)過敏反應[3]Raf激酶抑制蛋白RKIP是磷脂酰乙醇胺結(jié)合蛋白家族成員之一,能夠抑制MEK的磷酸化而影響下游信號通路的活化,參與調(diào)控腫瘤細胞的轉(zhuǎn)移及心肌細胞的功能。也有研究表明RKIP還可以通過影響NF-κB的活化而在天然免疫反應中發(fā)揮一定的作用。我們最近研究發(fā)現(xiàn)RKIP正向調(diào)控抗病毒免疫反應和促進炎癥性腸炎的發(fā)病。RKIP廣泛表達于各類哺乳動物的腦、肝臟、胃、脾臟及肌肉等組織中,在小鼠肥大細胞中也有較高表達。目前,尚未報道RKIP在肥大細胞及過敏性疾病中作用,我們從數(shù)據(jù)庫中檢索發(fā)現(xiàn)RKIP的表達量在肥大細胞被動致敏和受到抗原刺激后發(fā)生顯著性變化,同時檢測哮喘患者外周血中RKIP基因水平的表達變化,我們發(fā)現(xiàn)RKIP的mRNA表達明顯低于正常人的外周血中RKIP的表達水平。同時,我們在體外培養(yǎng)的小鼠原代肥大細胞中發(fā)現(xiàn),IgE/BSA致敏并刺激小鼠原代肥大細胞后,RKIP表達下降。分離培養(yǎng)野生型WT和RKIP敲除KO原代肥大細胞,進行體外研究,結(jié)果顯示,RKIP敲除后,顯著增加DNP-IgE/DNP-BSA(FcεRI活化)誘導的炎癥性細胞因子和趨化因子產(chǎn)生,同時增強肥大細胞脫顆粒過程。進一步研究發(fā)現(xiàn),而RKIP在小鼠中敲除后,能明顯地促進FCεRI活化的肥大細胞介導的全身性以及局部過敏反應。信號通路實驗發(fā)現(xiàn)RKIP靶向PI3K調(diào)節(jié)亞基p85cα從而負向調(diào)控FcεRI介導的肥大細胞的PI3K/AKT/NF-κB信號通路。進一步機制研究發(fā)現(xiàn),RKIP是通過與Gab2競爭性結(jié)合PI3K調(diào)節(jié)亞基p85a而抑制PI3K活化。綜上所述,本研究結(jié)果揭示了RKIP負向調(diào)控肥大細胞的介導的過敏反應重要作用機制,豐富了RKIP的生理功能,并為機體免疫防御及過敏性疫病疾病的治療提供新的思路。
[Abstract]:Mast cells are a class of immune cells derived from hematopoietic stem cells derived from bone marrow, mainly distributed in the mucous membranes of skin, airways and intestines that are in close contact with the outside environment. It is a key effector cell for type I hypersensitivity and hypersensitivity. It is believed that mast cells are primary effector cells of allergic reaction and play a key role in anaphylaxis [1]. Activated mast cells produce a variety of cytokines and chemokines, as well as newly synthesized arachidonic acid and proteins, resulting in signal cascade amplification by releasing pre-synthesized chemical mediators in the particles. RKIP is a member of phosphatidylethanolamine binding protein family, which can inhibit the phosphorylation of MEK and affect the activation of downstream signal pathway. RKIP is involved in regulating the metastasis of tumor cells and the function of cardiomyocytes. Some studies have also shown that RKIP can also play a role in innate immune response by affecting the activation of NF- 魏 B. We recently found that RKIP positively regulates the antiviral immune response and promotes the pathogenesis of inflammatory enteritis. RKIP is widely expressed in the brain, liver, stomach, spleen and muscle of various mammals, as well as in mouse mast cells. At present, we have not reported the role of RKIP in mast cells and allergic diseases. We found that the expression of RKIP changed significantly after the mast cells were sensitized passively and stimulated by antigen. At the same time, we detected the expression of RKIP gene in the peripheral blood of asthmatic patients. We found that the mRNA expression of RKIP was significantly lower than that of the normal controls. At the same time, we found that IgE / BSA sensitized primary mast cells in vitro and stimulated the expression of RKIP in primary mast cells of mice. Wild type WT and RKIP knockout KO primary mast cells were isolated and cultured in vitro. The results showed that RKIP knockout significantly increased the production of inflammatory cytokines and chemokines induced by DNP-IgE / DNP-BSA (FC 蔚 RI activation) and enhanced the degranulation process of mast cells. Further studies showed that RKIP could significantly promote FC 蔚 RI activated mast cell mediated systemic and local hypersensitivity after knockout in mice. RKIP targeted PI3K regulatory subunit p85c 偽 and negatively regulated the PI3K / AKT / NF- 魏 B signaling pathway of mast cells mediated by FC 蔚 RI. Further studies revealed that RKIP inhibited the activation of PI3K by competing with Gab2 PI3K regulatory subunit p85a. In conclusion, this study revealed the important mechanism of RKIP negative regulation of mast cell mediated allergic reaction, enriched the physiological function of RKIP, and provided a new idea for immune defense and the treatment of allergic blight disease.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392
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本文編號：2126218