钙库调控钙离子通道特异分子的筛选及其相关特性的研究

发布时间：2018-04-11 13:32

  本文选题：SOC通道 + S2细胞　； 参考：《华东师范大学》2010年硕士论文

【摘要】： 钙库调控钙离子通道(store-operated calcium channel, SOC)是一种在多种细胞中存在的钙离子通道。其基本的门控机制是当胞内钙库逐渐排空时,位于钙库膜上的信号分子STIM1(stromal interaction moleculel)将会聚集并与细胞膜上的SOC通道蛋白Orai1相互结合,改变其构象并打开通道。SOC通道可以调控许多生理功能,比如可以调控一些基因的转录、胞内某些酶的活性以及细胞的胞吐作用等等。同时,SOC通道功能的异常也与一些疾病相关,比如重症联合免疫缺陷综合症等。而目前由于已知的SOC通道的拮抗剂和激动剂数量有限,且基本上都没有特异性,使得人们对SOC通道的深入了解受到了阻碍。 本论文针对这一点利用果蝇S2细胞,应用胞内荧光强度检测的实验方法构建了SOC通道特异分子的筛选模型,并利用该模型对部分从中草药中提取的小分子化合物进行筛选。经反复验证,最终得到了一个有拮抗效果的小分子科罗索酸,并初步研究了科罗索酸对SOC的拮抗机制。 1.SOC通道特异分子筛选模型的构建 选择果蝇S2细胞作为SOC通道特异分子筛选的细胞模型。经摸索,发现25℃、无CO2的培养条件是较适宜S2细胞生长的条件。在该培养条件下,应用SOC通道激动剂TG、胞内钙探针Fluo-4AM以及FlexStation细胞内钙检测的技术,构建了SOC通道特异分子筛选的基本细胞模型,并利用该模型对三种已知的钙通道拮抗剂Gd3+、SKF-96365和2APB进行测试。结果显示,基本模型能够很好地检测出这三种拮抗剂对SOC通道的作用特性及效果,表明基本细胞模型是可靠、稳定的；为后续的药物筛选实验打下了基础。 2.天然提取化合物的筛选及活性化合物的作用机理初探 应用所构建的SOC通道特异分子的筛选模型,对本所从中草药中所分离出的小分子化1选。经验证,最终得到了一个对SOC通道有拮抗效果的小分子化合物科罗索酸。之后,我们对科罗索酸的拮抗机理进行了探索,发现科罗索酸能够拮抗内质网释放钙离子,推测科罗索酸可能通过阻碍内质网的排空进而拮抗SOC通道。
[Abstract]:Calcium storage regulated calcium channel store-operated calcium channel (SOCc) is a kind of calcium channel that exists in many kinds of cells.The basic gating mechanism is that when the intracellular calcium reservoir is gradually emptied, the signal molecule STIM1(stromal interaction molecules located on the calcium storage membrane will aggregate and bind to the SOC channel protein Orai1 on the cell membrane.Changing its conformation and opening the channel. SOC channel can regulate many physiological functions, such as the transcription of some genes, the activity of some enzymes and the exocytosis of cells, and so on.At the same time, the abnormal function of SOC channel is also associated with some diseases, such as severe Joint Immunodeficiency Syndrome.However, because of the limited number of known antagonists and agonists of SOC channels and the lack of specificity, the further understanding of SOC channels has been hindered.In order to solve this problem, a screening model of SOC channel specific molecules was constructed by using intracellular fluorescence intensity detection in fruit fly S2 cells, and some small molecular compounds extracted from Chinese herbal medicine were screened by this model.After repeated verification, a small molecule Corozo acid with antagonistic effect was obtained, and the antagonistic mechanism of Corozo acid against SOC was preliminarily studied.Construction of 1.SOC Channel specific Molecular screening ModelDrosophila S2 cells were selected as cell models for SOC channel specific molecular screening.It was found that 25 鈩，

本文编号：1736212