本文選題：SOC通道 + S2細胞　； 參考：《華東師范大學(xué)》2010年碩士論文

【摘要】： 鈣庫調(diào)控鈣離子通道(store-operated calcium channel, SOC)是一種在多種細胞中存在的鈣離子通道。其基本的門控機制是當(dāng)胞內(nèi)鈣庫逐漸排空時,位于鈣庫膜上的信號分子STIM1(stromal interaction moleculel)將會聚集并與細胞膜上的SOC通道蛋白Orai1相互結(jié)合,改變其構(gòu)象并打開通道。SOC通道可以調(diào)控許多生理功能,比如可以調(diào)控一些基因的轉(zhuǎn)錄、胞內(nèi)某些酶的活性以及細胞的胞吐作用等等。同時,SOC通道功能的異常也與一些疾病相關(guān),比如重癥聯(lián)合免疫缺陷綜合癥等。而目前由于已知的SOC通道的拮抗劑和激動劑數(shù)量有限,且基本上都沒有特異性,使得人們對SOC通道的深入了解受到了阻礙。 本論文針對這一點利用果蠅S2細胞,應(yīng)用胞內(nèi)熒光強度檢測的實驗方法構(gòu)建了SOC通道特異分子的篩選模型,并利用該模型對部分從中草藥中提取的小分子化合物進行篩選。經(jīng)反復(fù)驗證,最終得到了一個有拮抗效果的小分子科羅索酸,并初步研究了科羅索酸對SOC的拮抗機制。 1.SOC通道特異分子篩選模型的構(gòu)建 選擇果蠅S2細胞作為SOC通道特異分子篩選的細胞模型。經(jīng)摸索,發(fā)現(xiàn)25℃、無CO2的培養(yǎng)條件是較適宜S2細胞生長的條件。在該培養(yǎng)條件下,應(yīng)用SOC通道激動劑TG、胞內(nèi)鈣探針Fluo-4AM以及FlexStation細胞內(nèi)鈣檢測的技術(shù),構(gòu)建了SOC通道特異分子篩選的基本細胞模型,并利用該模型對三種已知的鈣通道拮抗劑Gd3+、SKF-96365和2APB進行測試。結(jié)果顯示,基本模型能夠很好地檢測出這三種拮抗劑對SOC通道的作用特性及效果,表明基本細胞模型是可靠、穩(wěn)定的；為后續(xù)的藥物篩選實驗打下了基礎(chǔ)。 2.天然提取化合物的篩選及活性化合物的作用機理初探 應(yīng)用所構(gòu)建的SOC通道特異分子的篩選模型,對本所從中草藥中所分離出的小分子化1選。經(jīng)驗證,最終得到了一個對SOC通道有拮抗效果的小分子化合物科羅索酸。之后,我們對科羅索酸的拮抗機理進行了探索,發(fā)現(xiàn)科羅索酸能夠拮抗內(nèi)質(zhì)網(wǎng)釋放鈣離子,推測科羅索酸可能通過阻礙內(nèi)質(zhì)網(wǎng)的排空進而拮抗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，

本文編號：1736212