本文選題：肥大細(xì)胞脫顆粒 + 微電子細(xì)胞芯片技術(shù)�。� 參考：《浙江大學(xué)》2017年碩士論文

【摘要】：過敏反應(yīng),又稱速發(fā)型超敏反應(yīng),含致敏、激發(fā)和效應(yīng)三個(gè)階段,由多種免疫細(xì)胞共同調(diào)控。其中,肥大細(xì)胞在過敏反應(yīng)的激發(fā)階段起"開關(guān)"作用,占有重要地位。病原體和機(jī)體首次接觸時(shí),B細(xì)胞會(huì)分泌大量IgE結(jié)合在肥大細(xì)胞表面。當(dāng)外界相同的抗原再次入侵機(jī)體,抗原與IgE結(jié)合,引起肥大細(xì)胞活化。肥大細(xì)胞活化后會(huì)脫顆粒分泌大量調(diào)節(jié)介質(zhì),從而殺死病原體、增強(qiáng)血管通透性、募集各種免疫細(xì)胞以及引起支氣管的收縮等。因此,在調(diào)控肥大細(xì)胞脫顆粒的信號(hào)網(wǎng)絡(luò)中找尋潛在靶點(diǎn),后續(xù)研發(fā)針對(duì)性藥物受到制藥業(yè)強(qiáng)烈關(guān)注。在本文中,我們建立了一種利用微電子細(xì)胞芯片技術(shù)分析肥大細(xì)胞脫顆粒的新型方法學(xué),該方法具有實(shí)時(shí)監(jiān)控以及通量篩選的優(yōu)勢(shì),后續(xù)可發(fā)展用于研發(fā)針對(duì)肥大細(xì)胞脫顆粒的抗過敏藥物。微電子細(xì)胞芯片檢測(cè)系統(tǒng)主要由96孔電極板、實(shí)時(shí)電極阻值檢測(cè)儀器和顯示器三個(gè)部分組成。肥大細(xì)胞被培養(yǎng)在96孔電極板中,受到刺激會(huì)在短時(shí)間內(nèi)發(fā)生顯著的形態(tài)學(xué)變化并釋放大量相關(guān)介質(zhì)。這些細(xì)胞生物學(xué)狀態(tài)的變化,會(huì)引起細(xì)胞附著的孔底電阻的阻抗急劇升高,被實(shí)時(shí)電極阻值檢測(cè)儀器捕捉并記錄。因此,DNP-BSA刺激肥大細(xì)胞脫顆粒后,可在顯示器上觀察到細(xì)胞指數(shù)(Cell Index,CI,細(xì)胞電極阻抗的衍生參數(shù))形了成信號(hào)峰。通過其它終點(diǎn)分析方法,我們進(jìn)一步確認(rèn)此CI曲線特異性地代表了肥大細(xì)胞脫顆粒的整個(gè)過程,由此建立了肥大細(xì)胞脫顆粒特異性時(shí)間依賴性細(xì)胞響應(yīng)信號(hào)圖譜(Time-dependent Cellular Responding Profile,TCRP)。隨后,我們用靶向磷酸化的小分子抑制劑預(yù)處理肥大細(xì)胞系,監(jiān)測(cè)細(xì)胞產(chǎn)生的TCRPs。結(jié)果顯示,能夠抑制肥大細(xì)胞脫顆粒的小分子抑制劑可導(dǎo)致細(xì)胞活化后產(chǎn)生的CI曲線上升減緩,峰值降低�；谏鲜鰧�(shí)驗(yàn)結(jié)果,我們確認(rèn)用TCRP分析肥大細(xì)胞脫顆粒的方法可以初步判斷小分子抑制劑對(duì)脫顆粒的作用,并可用于后續(xù)篩選對(duì)肥大細(xì)胞活化產(chǎn)生影響的小分子抑制劑。已有文獻(xiàn)報(bào)道,Stat3磷酸化在肥大細(xì)胞脫顆粒中起關(guān)鍵作用,抑制Stat3的磷酸化會(huì)顯著抑制肥大細(xì)胞脫顆粒。在通量篩選小分子抑制劑的過程中,我們發(fā)現(xiàn)JSI124和Stattic均為p-Stat3抑制劑,然而它們產(chǎn)生的TCRP圖譜模式卻具有很大差異。后續(xù)終點(diǎn)測(cè)定方法的結(jié)果進(jìn)一步證明Stattic阻止了脫顆粒過程,與Stattic的TCRP結(jié)果一致。而常規(guī)分析方法的結(jié)果證明JSI124并不符合預(yù)期,雖然抑制了 Stat3的磷酸化水平但并不對(duì)肥大細(xì)胞脫顆粒過程產(chǎn)生抑制作用。進(jìn)一步研究發(fā)現(xiàn),JSI124實(shí)際上誘導(dǎo)了肥大細(xì)胞的凋亡,它的TCRP圖譜是凋亡模式。此外,我們也發(fā)現(xiàn)Jak2/Stat3抑制劑AG490的TCRP證明AG490對(duì)脫顆粒具有顯著抑制效果。然而,AG490預(yù)處理后的肥大細(xì)胞發(fā)生活化,其β-氨基已糖苷酶的釋放量保持不變。之前已有研究證明AG490并非通過影響酶的分泌,而是通過抑制LTC4釋放來阻礙肥大細(xì)胞脫顆粒。因此,我們認(rèn)為肥大細(xì)胞脫顆粒的TCRP模型不僅可用于通量篩選,且相較于傳統(tǒng)的分析方法,對(duì)于判斷化合物對(duì)整個(gè)脫顆粒過程的影響更能提供精準(zhǔn)的信息。在新藥研發(fā)中,藥物先導(dǎo)化合物不但可在通量篩選中被發(fā)現(xiàn),亦可來源于針對(duì)某個(gè)靶標(biāo)設(shè)計(jì)的特異性化合物。然而,即使該化合物對(duì)靶點(diǎn)具有高特異性和強(qiáng)抑制作用,其最終效果也難盡如預(yù)期,可能會(huì)產(chǎn)生其它作用。常規(guī)檢測(cè)肥大細(xì)胞脫顆粒的方法,一般為終點(diǎn)測(cè)定或者是單點(diǎn)分析方法,難以評(píng)估和實(shí)時(shí)監(jiān)測(cè)細(xì)胞應(yīng)對(duì)于抑制劑產(chǎn)生的反應(yīng)。而本文建立的IgE介導(dǎo)的肥大細(xì)胞脫顆粒相關(guān)TCRP是一種可靠且敏感的方法,可被用于針對(duì)于肥大細(xì)胞脫顆粒的抗過敏藥物的研發(fā)。不僅因?yàn)樗軌蛱峁┖侠淼耐亢Y選,實(shí)時(shí)監(jiān)測(cè)細(xì)胞對(duì)化合物的反應(yīng),更可被用于藥物研發(fā)早期階段,具有預(yù)測(cè)化合物的潛在功能。
[Abstract]:Anaphylaxis, also known as the rapid hypersensitivity, with three stages of sensitization, excitation and effect, is regulated by a variety of immune cells. Among them, mast cells play a "switch" role in the stage of anaphylaxis and play an important role. When the pathogen and the body first contact, B cells will secrete a large number of IgE binding on the surface of mast cells. The same antigen invaded the body again, and the antigen combined with IgE to cause the activation of mast cells. After the mast cells were activated, a large number of mediators were removed and secreted, thus killing pathogens, enhancing vascular permeability, raising various immune cells and causing bronchoconstriction. Therefore, the submersible in the signal network regulating mast cell degranulation was found. In this paper, we have established a novel methodology for the analysis of mast cell degranulation using microelectronic cell chip technology. This method has the advantages of real-time monitoring and flux screening, and subsequent development is used to develop antihypersensitivity for mast cell degranulation. The microelectronic cell chip detection system is mainly composed of 96 holes electrode plate, real-time electrode resistance detection instrument and display three parts. The mast cell is cultured in the 96 hole electrode plate. By stimulation, significant morphological changes will occur in a short time and a large number of related medials are released. The changes in biological state of these cells will lead to the change of the biological state of these cells. The impedance of the cell base resistance increases rapidly and is captured and recorded by a real-time electrode resistance detection instrument. Therefore, after DNP-BSA stimulates the degranulation of mast cells, the cell index (Cell Index, CI, derived parameters of cell electrode impedance) can be observed to form a signal peak. It is confirmed that the CI curve specifically represents the whole process of mast cell degranulation, and thus the specific time dependent cell response signal Atlas of the mast cell degranulation (Time-dependent Cellular Responding Profile, TCRP) is established. Then, we pretreat the mast cell line and monitor the cell line with the targeted phosphorylated small molecular inhibitors. TCRPs. results showed that a small molecular inhibitor that could inhibit the degranulation of mast cells could lead to a slow rise in the CI curve resulting from cell activation and a decrease in its peak value. Based on the above experimental results, we confirm that the analysis of the degranulation of mast cells by TCRP can preliminarily determine the effect of small molecular inhibitors on degranulation and can be used for the analysis of the degranulation of small molecules. Subsequent screening of small molecular inhibitors affecting mast cell activation. It has been reported that Stat3 phosphorylation plays a key role in mast cell degranulation. Inhibition of phosphorylation of Stat3 can significantly inhibit mast cell degranulation. In the process of screening small molecular inhibitors, we found that both JSI124 and Stattic are p-Stat3 inhibitors. However, the pattern of TCRP atlas produced by them was very different. The results of subsequent end-point determination further demonstrated that Stattic prevented the degranulation process from the TCRP results of Stattic. The results of the conventional analysis proved that JSI124 did not conform to the expectation, although the phosphorylation level of Stat3 was inhibited, but the mast cell was not removed. Further studies have found that JSI124 actually induces the apoptosis of mast cells, and its TCRP map is a mode of apoptosis. Furthermore, we also found that TCRP of the Jak2/Stat3 inhibitor AG490 has proved that AG490 has a significant inhibitory effect on degranulation. However, the AG490 pretreated mast cells live, and their beta amino groups have been transformed. The release of glucosidase remains unchanged. Previous studies have shown that AG490 does not impede the degranulation of mast cells by inhibiting LTC4 release. Therefore, we believe that the TCRP model of mast cells degranulation can be used not only for flux screening, but also in judging the whole of the whole compound compared with the analytical method. In the development of new drugs, drug pilot compounds can be found not only in flux screening, but also from specific compounds designed for a target. However, even if the compound has a high specific and strong inhibitory effect on the target, the final effect is difficult as expected. The method of routine detection of mast cell degranulation, usually end-point determination or single point analysis, is difficult to assess and monitor the response to the cells in real time. The IgE mediated mast cell degranulation related TCRP is a reliable and sensitive method, which can be used for targeted treatment. On the degranulation of mast cells, anti allergic drugs. Not only because it can provide reasonable throughput screening, real-time monitoring of cell reaction compounds, can be used in early stage of drug development, the potential function prediction of compounds.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.1
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本文編號(hào)：2047407