肥大细胞脱颗粒的动态表型特征与靶向磷酸化抗炎调控机制研究

发布时间：2018-06-21 05:20

  本文选题：肥大细胞脱颗粒 + 微电子细胞芯片技术　； 参考：《浙江大学》2017年硕士论文

【摘要】：过敏反应,又称速发型超敏反应,含致敏、激发和效应三个阶段,由多种免疫细胞共同调控。其中,肥大细胞在过敏反应的激发阶段起"开关"作用,占有重要地位。病原体和机体首次接触时,B细胞会分泌大量IgE结合在肥大细胞表面。当外界相同的抗原再次入侵机体,抗原与IgE结合,引起肥大细胞活化。肥大细胞活化后会脱颗粒分泌大量调节介质,从而杀死病原体、增强血管通透性、募集各种免疫细胞以及引起支气管的收缩等。因此,在调控肥大细胞脱颗粒的信号网络中找寻潜在靶点,后续研发针对性药物受到制药业强烈关注。在本文中,我们建立了一种利用微电子细胞芯片技术分析肥大细胞脱颗粒的新型方法学,该方法具有实时监控以及通量筛选的优势,后续可发展用于研发针对肥大细胞脱颗粒的抗过敏药物。微电子细胞芯片检测系统主要由96孔电极板、实时电极阻值检测仪器和显示器三个部分组成。肥大细胞被培养在96孔电极板中,受到刺激会在短时间内发生显著的形态学变化并释放大量相关介质。这些细胞生物学状态的变化,会引起细胞附着的孔底电阻的阻抗急剧升高,被实时电极阻值检测仪器捕捉并记录。因此,DNP-BSA刺激肥大细胞脱颗粒后,可在显示器上观察到细胞指数(Cell Index,CI,细胞电极阻抗的衍生参数)形了成信号峰。通过其它终点分析方法,我们进一步确认此CI曲线特异性地代表了肥大细胞脱颗粒的整个过程,由此建立了肥大细胞脱颗粒特异性时间依赖性细胞响应信号图谱(Time-dependent Cellular Responding Profile,TCRP)。随后,我们用靶向磷酸化的小分子抑制剂预处理肥大细胞系,监测细胞产生的TCRPs。结果显示,能够抑制肥大细胞脱颗粒的小分子抑制剂可导致细胞活化后产生的CI曲线上升减缓,峰值降低。基于上述实验结果,我们确认用TCRP分析肥大细胞脱颗粒的方法可以初步判断小分子抑制剂对脱颗粒的作用,并可用于后续筛选对肥大细胞活化产生影响的小分子抑制剂。已有文献报道,Stat3磷酸化在肥大细胞脱颗粒中起关键作用,抑制Stat3的磷酸化会显著抑制肥大细胞脱颗粒。在通量筛选小分子抑制剂的过程中,我们发现JSI124和Stattic均为p-Stat3抑制剂,然而它们产生的TCRP图谱模式却具有很大差异。后续终点测定方法的结果进一步证明Stattic阻止了脱颗粒过程,与Stattic的TCRP结果一致。而常规分析方法的结果证明JSI124并不符合预期,虽然抑制了 Stat3的磷酸化水平但并不对肥大细胞脱颗粒过程产生抑制作用。进一步研究发现,JSI124实际上诱导了肥大细胞的凋亡,它的TCRP图谱是凋亡模式。此外,我们也发现Jak2/Stat3抑制剂AG490的TCRP证明AG490对脱颗粒具有显著抑制效果。然而,AG490预处理后的肥大细胞发生活化,其β-氨基已糖苷酶的释放量保持不变。之前已有研究证明AG490并非通过影响酶的分泌,而是通过抑制LTC4释放来阻碍肥大细胞脱颗粒。因此,我们认为肥大细胞脱颗粒的TCRP模型不仅可用于通量筛选,且相较于传统的分析方法,对于判断化合物对整个脱颗粒过程的影响更能提供精准的信息。在新药研发中,药物先导化合物不但可在通量筛选中被发现,亦可来源于针对某个靶标设计的特异性化合物。然而,即使该化合物对靶点具有高特异性和强抑制作用,其最终效果也难尽如预期,可能会产生其它作用。常规检测肥大细胞脱颗粒的方法,一般为终点测定或者是单点分析方法,难以评估和实时监测细胞应对于抑制剂产生的反应。而本文建立的IgE介导的肥大细胞脱颗粒相关TCRP是一种可靠且敏感的方法,可被用于针对于肥大细胞脱颗粒的抗过敏药物的研发。不仅因为它能够提供合理的通量筛选,实时监测细胞对化合物的反应,更可被用于药物研发早期阶段,具有预测化合物的潜在功能。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.1
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本文编号：2047407