本文選題：雷公藤甲素 + 肺癌��； 參考：《浙江中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的研究雷公藤甲素對人肺癌A549細胞系蛋白質(zhì)表達譜的影響,并對差異表達的蛋白質(zhì)進行GO功能注釋、KEGG通路富集和蛋白質(zhì)相互作用網(wǎng)絡(luò)構(gòu)建(PPI),探討雷公藤甲素誘導(dǎo)肺癌細胞A549凋亡的潛在作用靶點和分子機制。方法1、以肺癌細胞A549為研究對象,CCK-8實驗篩選雷公藤甲素(6.25、12.5、25、50、100、200和400 ng/ml)作用于A549細胞后抑制細胞增殖的最佳作用濃度和時間。2、流式細胞術(shù)檢測雷公藤甲素(12.5、50、200 ng/mL)對肺癌A549細胞周期和細胞凋亡的影響。3、采用體外iTRAQ標(biāo)記結(jié)合納升液相色譜串聯(lián)質(zhì)譜(NanoLC-MS/MS)的定量蛋白質(zhì)組學(xué)技術(shù),并利用PrOteinPi1Ot4.5軟件篩選雷公藤甲素作用前后肺癌細胞A549中的差異表達蛋白;并對差異表達蛋白進行GO功能注釋、KEGG通路富集和蛋白質(zhì)相互作用網(wǎng)絡(luò)(PPI)構(gòu)建,預(yù)測雷公藤甲素抗腫瘤作用的潛在靶點和分子機制。4、通過Western blot方法驗證部分差異蛋白(PHB、CDH1、AIFM1、MTA2和EIF4A3)在肺癌細胞A549中的表達情況。結(jié)果1.雷公藤甲素能夠劑量依賴性地抑制肺癌A549細胞增殖,24h和36h的IC50分別是273ng/mL和210ng/mL。雷公藤甲素(12.5、50、200ng/mL)能以劑量 依賴性方式阻滯人肺癌細胞A549細胞周期于G2/M期(P0.01),并誘導(dǎo)其發(fā)生凋亡(P0.01)。實驗采用200ng/mL的雷公藤甲素和36h作用時間進行后續(xù)的蛋白質(zhì)組學(xué)實驗。2.運用iTRAQ定量蛋白質(zhì)組學(xué)技術(shù)篩選雷公藤甲素處理前后人肺癌細胞A549內(nèi)的差異蛋白結(jié)果顯示:雷公藤甲素組與空白對照組相比,雷公藤甲素干預(yù)可以引起A549細胞中的312種蛋白質(zhì)發(fā)生顯著的差異性表達(P0.05),其中141個蛋白質(zhì)表達上調(diào),171個蛋白質(zhì)表達下調(diào)。生物信息學(xué)分析表明這些蛋白質(zhì)參與多種生物學(xué)途徑,包括真核生物核糖體的生物合成,RNA加工,核糖核蛋白復(fù)合物生物合成,rRNA代謝,rRNA加工,ncRNA加工,細胞組分生物合成等,涉及226種不同的KEGG通路并且彼此相互聯(lián)系構(gòu)成網(wǎng)絡(luò)。5.Western Blot驗證實驗結(jié)果顯示:在人肺癌細胞A549中,雷公藤甲素可以上調(diào)PHB、CDH1和AIFM1蛋白質(zhì)表達(P0.05),下調(diào)MTA2和EIF4A3蛋白質(zhì)表達(P0.05),與蛋白質(zhì)組學(xué)結(jié)果一致。結(jié)論1.雷公藤甲素(200ng/mL,36h)對A549細胞有顯著的細胞毒性作用,可以抑制細胞增殖,顯著阻滯細胞周期于G2/M期并誘導(dǎo)細胞凋亡(P0.01)。2.雷公藤甲素可以影響肺癌細胞A549內(nèi)的多種生物學(xué)途徑,包括真核生物中的核糖體生物合成,剪接體,mRNA監(jiān)控途徑,PARP1/AIF途徑,代謝途徑,上皮間質(zhì)轉(zhuǎn)化(EMT)和其他重要的分子靶標(biāo)等,這些途徑很可能是雷公藤甲素抗腫瘤作用的有效靶點。
[Abstract]:Objective to study the effect of triptolide on protein expression profile of human lung cancer cell line A549. The differentially expressed proteins were enriched in the KEGG pathway and constructed by protein interaction network to explore the potential target and molecular mechanism of triptolide induced apoptosis in lung cancer cell line A549. Methods 1. The best concentration and time of triptolide on A549 cells was selected by CCK-8 assay. Flow cytometry was used to detect the effect of triptolide on A549 cell line. Flow cytometry was used to detect the effect of triptolide (12.5ngmL) on A549 lung cancer cell line (A549 cells) treated with triptolide (6.25ng / ml) and 400ng / ml respectively. Flow cytometry (FCM) was used to detect the effect of triptolide (12.5ngmL) on lung cancer cell line A549. The effects of cell cycle and apoptosis on cell cycle. 3. Quantitative proteomics technique using in vitro iTRAQ labeling combined with NanoLC-MS / MS (NanoLC-MS / MS) was used. PrOteinPi1Ot4.5 software was used to screen differentially expressed protein in lung cancer cell line A549 before and after treatment with triptolide, and to construct the differential expression protein, which was enriched by go functional annotated KEGG pathway and constructed by protein interaction network. The potential target and molecular mechanism of triptolide were predicted, and the expression of partial differential proteins (PHB-CDH1, AIFMTA2 and EIF4A3) in lung cancer cell line A549 was confirmed by Western blot method. Result 1. Triptolide inhibited the proliferation of lung cancer A549 cells in a dose-dependent manner. The IC50 of 24 h and 36 h were 273ng/mL and 210 ng / mL, respectively. Tripterygium wilfordii 12.5ng / mL could block the cell cycle of human lung cancer cell A549 in a dose-dependent manner and induce apoptosis in A549 cells in G _ 2 / M phase. 200ng/mL triptolide and 36 h action time were used to carry out the subsequent proteomics experiment. 2. ITRAQ quantitative proteomics was used to screen the differentially expressed proteins in human lung cancer cell line A549 before and after triptolide treatment. Tripterygium wilfordii could induce 312 proteins in A549 cells to be significantly differentially expressed, of which 141 proteins were up-regulated and 171 proteins were down-regulated. Bioinformatics analysis shows that these proteins are involved in many biological pathways, including the biosynthesis of eukaryotic ribosomes, the biosynthesis of ribosomal protein complexes, the metabolism of rRNA and the processing of ncRNA, and the biosynthesis of cell components. Western Blot verification results show that in human lung cancer cell line A549, there are 226 different KEGG pathways involved and they are connected with each other to form a network. 5. Western Blot verification results show that in human lung cancer cell line A549, Tripterygium wilfordii can up-regulate the expression of AIFM1 and CDH1 protein and down-regulate the expression of MTA2 and EIF4A3 protein, which is consistent with the results of proteomics. Conclusion 1. Tripterygium wilfordii (200ng / mL for 36h) has a significant cytotoxic effect on A549 cells, which can inhibit cell proliferation, significantly block cell cycle at G _ 2 / M phase and induce apoptosis (P _ (0.01) P _ (0.01) 路2). Triptolide can affect many biological pathways in lung cancer cell A549, including ribosomal biosynthesis in eukaryotes, splicing mRNA monitoring pathway, PARP1 / AIF pathway, metabolic pathway, epithelial mesenchymal transformation (EMTT) and other important molecular targets. These pathways may be an effective target for triptolide's anti-tumor effect.
【學(xué)位授予單位】：浙江中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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