本文選題：茯苓酸 + 胃癌�。� 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：研究目的:近年來(lái)的研究表明茯苓酸(PA)有多種藥理活性,特別是在抗腫瘤方面,發(fā)現(xiàn)其對(duì)多種癌細(xì)胞有明顯的抑制作用。然而,對(duì)茯苓酸潛在靶點(diǎn)尤其是其抗腫瘤作用靶點(diǎn)的識(shí)別以及靶點(diǎn)的后續(xù)驗(yàn)證研究仍然十分匱乏。本研究擬通過(guò)兩個(gè)系統(tǒng)性的藥物靶點(diǎn)預(yù)測(cè)方法,對(duì)茯苓酸進(jìn)行潛在的靶點(diǎn)預(yù)測(cè),再經(jīng)映射到蛋白質(zhì)互作后通過(guò)構(gòu)建分子網(wǎng)絡(luò)篩選關(guān)鍵靶點(diǎn),最后借助實(shí)驗(yàn)手段進(jìn)行實(shí)驗(yàn)驗(yàn)證及機(jī)制分析,為尋找藥物抗癌治療靶點(diǎn)提供新思路,進(jìn)一步給臨床應(yīng)用提供一定的實(shí)驗(yàn)依據(jù)。方法:首先,以中藥分子茯苓酸為研究對(duì)象,分別用SysDT和WES這兩個(gè)藥物-靶點(diǎn)預(yù)測(cè)的計(jì)算機(jī)模型,來(lái)預(yù)測(cè)茯苓酸的潛在靶點(diǎn);然后將得到的潛在靶點(diǎn)與胃癌相關(guān)基因的整合結(jié)果映射到蛋白質(zhì)相互作用數(shù)據(jù)庫(kù)BioGRID和STRING中,再經(jīng)Cytoscape軟件構(gòu)建分子網(wǎng)絡(luò)從中選取中心基因作為可能的抗胃癌作用靶點(diǎn);最后利用細(xì)胞熱轉(zhuǎn)移(CETSA)和western-blot方法在胃癌細(xì)胞上進(jìn)行關(guān)鍵靶標(biāo)蛋白的結(jié)合實(shí)驗(yàn)驗(yàn)證及抗癌作用機(jī)制的初步探究。結(jié)果:(1)整合Sys DT和WES模型預(yù)測(cè)結(jié)果,去重后共得到43個(gè)茯苓酸潛在靶點(diǎn)。(2)通過(guò)GCgene數(shù)據(jù)庫(kù)和胃癌biomarker收集到胃癌相關(guān)基因1763個(gè),進(jìn)一步整合預(yù)測(cè)的茯苓酸潛在靶點(diǎn)與胃癌相關(guān)基因獲得15個(gè)抗胃癌種子靶點(diǎn)。這些靶點(diǎn)映射蛋白質(zhì)互作PPI網(wǎng)絡(luò)和分子作用網(wǎng)絡(luò)分析,最后根據(jù)網(wǎng)絡(luò)拓?fù)鋵W(xué)參數(shù)進(jìn)行篩選,得到5個(gè)關(guān)鍵節(jié)點(diǎn)/靶點(diǎn)。(3)CETSA靶點(diǎn)結(jié)合實(shí)驗(yàn)結(jié)果表明茯苓酸在胃癌細(xì)胞中能結(jié)合到PPARγ從而改變其熱穩(wěn)定性;Western-blot分析顯示經(jīng)茯苓酸處理后的PPARγ蛋白表達(dá)上調(diào),且具有濃度依賴性;另外,還發(fā)現(xiàn)PPARγ抑制劑GW9662預(yù)處理能影響茯苓酸對(duì)Cyclin D1、Bcl-2、Bcl-x L、CDK4、p-ERK1/2、p-p38的表達(dá)。結(jié)論:茯苓酸在胃癌細(xì)胞中能結(jié)合PPARγ,進(jìn)一步上調(diào)其蛋白表達(dá)。PPARγ作為茯苓酸的抗胃癌作用靶點(diǎn),其涉及的胃癌治療機(jī)制很可能是通過(guò)調(diào)控一些周期和凋亡相關(guān)因子及絲裂原活化蛋白激酶(MAPK)信號(hào)通路相關(guān)蛋白;而這些因子或蛋白在胃癌的發(fā)生與發(fā)展有著重要的作用。本研究的成功開展將為中藥成分的靶點(diǎn)預(yù)測(cè)提供新的策略,對(duì)中藥成分抗癌的機(jī)制研究具有重要的指導(dǎo)意義,將促進(jìn)中藥的現(xiàn)代化發(fā)展。
[Abstract]:Objective: in recent years, studies have shown that PAA has many pharmacological activities, especially in the aspect of anti-tumor, and it has obvious inhibitory effect on many kinds of cancer cells. However, the identification and subsequent verification of the potential targets of Poria cocos, especially their antitumor effects, are still scarce. In this study, two systematic drug target prediction methods were used to predict the potential targets of Poria cocos, and then to screen the key targets by constructing a molecular network after mapping to protein interaction. Finally, experimental verification and mechanism analysis were carried out by means of experimental means, which provided a new idea for finding the target of anticancer therapy and provided certain experimental basis for clinical application. Methods: firstly, the computer models of SysDT and WES were used to predict the potential targets of Poria cocos. Then the integration results of the potential target and gastric cancer related gene were mapped to the protein interaction database BioGRID and STRING, and then the molecular network was constructed by Cytoscape software to select the center gene as the possible anti-gastric cancer target. Finally, the binding experiments of key target proteins in gastric cancer cells were carried out by cell heat transfer assay (CTSA) and western-blot method, and the mechanism of anticancer action was explored. Results the predicted results of Sys DT and WES model were integrated, and 43 potential targets of Poria were obtained. 1763 genes related to gastric cancer were collected by GCgene database and gastric cancer biomarker. Further integration of the predicted potential target of Poria and gastric cancer related genes to obtain 15 anti-gastric cancer seed targets. These target mapping protein interaction PPI networks and molecular interaction networks were analyzed, and finally selected according to the network topology parameters. The results of five key nodes / target. The results showed that Poria cocos acid could bind to PPAR 緯 in gastric cancer cells and change its thermal stability. Western-blot analysis showed that the expression of PPAR 緯 protein was up-regulated and concentration-dependent after the treatment of Poria cocos acid. In addition, it was found that GW9662 pretreatment with PPAR 緯 inhibitor could affect the expression of p-ERK1 / 2p38 in Cyclin D1Bcl-2Bcl-x CDK4T. Conclusion: Poria cocos acid can bind to PPAR 緯 in gastric cancer cells, and further upregulate its protein expression. The mechanism involved in the treatment of gastric cancer is probably through the regulation of some cycle and apoptosis-related factors and mitogen-activated protein kinase MAPK signaling pathway related proteins which play an important role in the occurrence and development of gastric cancer. The successful development of this study will provide a new strategy for target prediction of Chinese traditional medicine components, and will be of great significance for the study of anticancer mechanism of Chinese traditional medicine ingredients, and will promote the modernization of traditional Chinese medicine.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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