發(fā)布時間：2018-04-20 15:48

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

【摘要】：研究目的:近年來的研究表明茯苓酸(PA)有多種藥理活性,特別是在抗腫瘤方面,發(fā)現(xiàn)其對多種癌細胞有明顯的抑制作用。然而,對茯苓酸潛在靶點尤其是其抗腫瘤作用靶點的識別以及靶點的后續(xù)驗證研究仍然十分匱乏。本研究擬通過兩個系統(tǒng)性的藥物靶點預測方法,對茯苓酸進行潛在的靶點預測,再經(jīng)映射到蛋白質(zhì)互作后通過構(gòu)建分子網(wǎng)絡篩選關(guān)鍵靶點,最后借助實驗手段進行實驗驗證及機制分析,為尋找藥物抗癌治療靶點提供新思路,進一步給臨床應用提供一定的實驗依據(jù)。方法:首先,以中藥分子茯苓酸為研究對象,分別用SysDT和WES這兩個藥物-靶點預測的計算機模型,來預測茯苓酸的潛在靶點;然后將得到的潛在靶點與胃癌相關(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BioGRID和STRING中,再經(jīng)Cytoscape軟件構(gòu)建分子網(wǎng)絡從中選取中心基因作為可能的抗胃癌作用靶點;最后利用細胞熱轉(zhuǎn)移(CETSA)和western-blot方法在胃癌細胞上進行關(guān)鍵靶標蛋白的結(jié)合實驗驗證及抗癌作用機制的初步探究。結(jié)果:(1)整合Sys DT和WES模型預測結(jié)果,去重后共得到43個茯苓酸潛在靶點。(2)通過GCgene數(shù)據(jù)庫和胃癌biomarker收集到胃癌相關(guān)基因1763個,進一步整合預測的茯苓酸潛在靶點與胃癌相關(guān)基因獲得15個抗胃癌種子靶點。這些靶點映射蛋白質(zhì)互作PPI網(wǎng)絡和分子作用網(wǎng)絡分析,最后根據(jù)網(wǎng)絡拓撲學參數(shù)進行篩選,得到5個關(guān)鍵節(jié)點/靶點。(3)CETSA靶點結(jié)合實驗結(jié)果表明茯苓酸在胃癌細胞中能結(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蛋白表達上調(diào),且具有濃度依賴性;另外,還發(fā)現(xiàn)PPARγ抑制劑GW9662預處理能影響茯苓酸對Cyclin D1、Bcl-2、Bcl-x L、CDK4、p-ERK1/2、p-p38的表達。結(jié)論:茯苓酸在胃癌細胞中能結(jié)合PPARγ,進一步上調(diào)其蛋白表達。PPARγ作為茯苓酸的抗胃癌作用靶點,其涉及的胃癌治療機制很可能是通過調(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預測提供新的策略,對中藥成分抗癌的機制研究具有重要的指導意義,將促進中藥的現(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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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