本文選題：內(nèi)源性大麻素 + 大麻素受體　； 參考：《第三軍醫(yī)大學(xué)》2009年碩士論文

【摘要】：目的:內(nèi)源性大麻素通過(guò)激活G蛋白耦連的大麻素受體(CB1和CB2)而發(fā)揮對(duì)神經(jīng)、心血管、免疫等系統(tǒng)的調(diào)節(jié)作用。此外,內(nèi)源性大麻素也具有抗炎、鎮(zhèn)痛等多種醫(yī)療用途,但其生理效應(yīng)尚未完全揭示。熱應(yīng)激反應(yīng)是細(xì)胞最重要的應(yīng)激保護(hù)機(jī)制之一,主要通過(guò)激活熱休克轉(zhuǎn)錄因子1而顯著增強(qiáng)包括分子伴侶機(jī)制在內(nèi)的多種保護(hù)機(jī)制。而內(nèi)源性大麻素對(duì)熱應(yīng)激反應(yīng)有無(wú)調(diào)節(jié)效應(yīng)及其機(jī)制如何,迄今國(guó)內(nèi)外尚無(wú)研究報(bào)道。本研究在文獻(xiàn)調(diào)研的基礎(chǔ)上,初步推測(cè)大麻素可能通過(guò)其受體對(duì)熱應(yīng)激反應(yīng)產(chǎn)生調(diào)節(jié)作用。迄今國(guó)內(nèi)外尚無(wú)研究報(bào)道。本研究在文獻(xiàn)調(diào)研的基礎(chǔ)上,初步推測(cè)大麻素可能通過(guò)其受體對(duì)熱應(yīng)激反應(yīng)產(chǎn)生調(diào)節(jié)作用。為證實(shí)這一假設(shè),本研究擬以大鼠膠質(zhì)瘤細(xì)胞株作為研究對(duì)象,觀察內(nèi)源性大麻素是否對(duì)其熱應(yīng)激反應(yīng)具有調(diào)節(jié)效應(yīng),并探索其相應(yīng)的受體機(jī)制;以期初步闡明內(nèi)源性大麻對(duì)熱應(yīng)激反應(yīng)的調(diào)節(jié)效應(yīng)及機(jī)制,為內(nèi)源性大麻素的效應(yīng)及機(jī)制研究提供新思路和突破點(diǎn)。 方法:選用大鼠膠質(zhì)瘤C6細(xì)胞為實(shí)驗(yàn)?zāi)Ｐ?實(shí)驗(yàn)組共分4類,即熱應(yīng)激組(42℃水浴1h)、熱應(yīng)激+內(nèi)源性大麻素(2-AG)處理組、熱應(yīng)激+2-AG + CB1受體特異性拮抗劑(AM251)或CB2受體特異性拮抗劑(AM630)處理組、以及各自的對(duì)照組;設(shè)3h、6h、12h、24h等4個(gè)時(shí)相點(diǎn)。①采用RT-PCR技術(shù),檢測(cè)熱應(yīng)激、熱應(yīng)激+2-AG、熱應(yīng)激+2-AG+大麻素受體抑制劑處理后HSP70 mRNA表達(dá)水平。②采用Western Blot技術(shù),檢測(cè)熱應(yīng)激、熱應(yīng)激+2-AG、熱應(yīng)激+2-AG+抑制劑處理后HSP70蛋白表達(dá)水平。 結(jié)果:①C6細(xì)胞熱應(yīng)激處理后,分別于3h、6h、12h、24h檢測(cè)HSP70 mRNA及蛋白的變化。結(jié)果顯示,熱刺激后3h、6h、12h、24h,HSP70 mRNA和蛋白表達(dá)水平均較對(duì)照組顯著增高,12h時(shí)增加最為顯著。②選擇熱應(yīng)激后12h為觀察時(shí)相點(diǎn),以10-7mol/L、10-6mol/L、10-5mol/L三種不同濃度的2-AG進(jìn)行處理,結(jié)果發(fā)現(xiàn)10-5mol/L 2-AG對(duì)熱刺激1h后HSP70 mRNA、蛋白表達(dá)具有顯著促進(jìn)作用;而10-7mol/L、10-6mol/L2-AG則無(wú)此促進(jìn)效應(yīng)。③觀察熱刺激及10-5mol/L 2-AG處理后,不同時(shí)相點(diǎn)HSP70 mRNA和蛋白的表達(dá)情況,結(jié)果發(fā)現(xiàn)2-AG處理后HSP70 mRNA和蛋白表達(dá)在6h到達(dá)高峰,無(wú)2-AG處理對(duì)照組HSP70 mRNA和蛋白在12h到達(dá)高峰。④以CB1受體特異性拮抗劑(AM251)和CB2受體特異性拮抗劑(AM630)處理細(xì)胞后,檢測(cè)2-AG對(duì)HSP70 mRNA、蛋白表達(dá)的影響,結(jié)果顯示,AM251阻斷了2-AG對(duì)HSP70 mRNA和蛋白的促進(jìn)效應(yīng),而AM630無(wú)此效應(yīng)。 結(jié)論:一定濃度的內(nèi)源性大麻素2-AG對(duì)熱刺激處理的C6細(xì)胞內(nèi)HSP70基因的表達(dá)有明顯促進(jìn)效應(yīng),表明2-AG可以參與調(diào)控細(xì)胞熱應(yīng)激反應(yīng);此促進(jìn)效應(yīng)可被CB1特異性受體抑制劑AM251所抑制,表明2-AG的抑制效應(yīng)是由CB1,而非CB2,信號(hào)通路所介導(dǎo)。本研究結(jié)果揭示了2-AG可能具備調(diào)控?zé)釕?yīng)激效應(yīng)的能力。
[Abstract]:Aim: endogenous cannabinoids regulate the nervous, cardiovascular and immune systems by activating the G protein-coupled cannabinoid receptors CB1 and CB2. In addition, endogenous cannabinoids also have a variety of anti-inflammatory, analgesic and other medical uses, but its physiological effects have not been fully revealed. Heat stress response is one of the most important mechanisms of cell stress protection, mainly through the activation of heat shock transcription factor 1 to significantly enhance a variety of protective mechanisms, including molecular chaperone mechanism. So far, there are no studies on whether endogenous cannabinoids regulate the thermal stress response and how they are related to them. On the basis of literature review, this study preliminarily speculated that cannabinoid may regulate the thermal stress reaction through its receptor. So far, there is no research report at home and abroad. On the basis of literature review, this study preliminarily speculated that cannabinoid may regulate the thermal stress reaction through its receptor. In order to confirm this hypothesis, this study intends to investigate whether endogenous cannabinoids can regulate the heat stress response of rat glioma cells and explore its receptor mechanism. The aim of this study was to elucidate the regulatory effect and mechanism of endogenous cannabis on heat stress and provide new ideas and breakthrough points for the study of endogenous cannabinoid effect and mechanism. Methods: rat glioma C6 cells were selected as the experimental model, the experimental group was divided into 4 groups: heat stress group (42 鈩，

本文編號(hào)：1937718