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(duì)人肺癌A549細(xì)胞系蛋白質(zhì)表達(dá)譜的影響,并對(duì)差異表達(dá)的蛋白質(zhì)進(jìn)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(xì)胞A549凋亡的潛在作用靶點(diǎn)和分子機(jī)制。方法1、以肺癌細(xì)胞A549為研究對(duì)象,CCK-8實(shí)驗(yàn)篩選雷公藤甲素(6.25、12.5、25、50、100、200和400 ng/ml)作用于A549細(xì)胞后抑制細(xì)胞增殖的最佳作用濃度和時(shí)間。2、流式細(xì)胞術(shù)檢測(cè)雷公藤甲素(12.5、50、200 ng/mL)對(duì)肺癌A549細(xì)胞周期和細(xì)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(xì)胞A549中的差異表達(dá)蛋白;并對(duì)差異表達(dá)蛋白進(jìn)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(cè)雷公藤甲素抗腫瘤作用的潛在靶點(diǎn)和分子機(jī)制。4、通過(guò)Western blot方法驗(yàn)證部分差異蛋白(PHB、CDH1、AIFM1、MTA2和EIF4A3)在肺癌細(xì)胞A549中的表達(dá)情況。結(jié)果1.雷公藤甲素能夠劑量依賴(lài)性地抑制肺癌A549細(xì)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(lài)性方式阻滯人肺癌細(xì)胞A549細(xì)胞周期于G2/M期(P0.01),并誘導(dǎo)其發(fā)生凋亡(P0.01)。實(shí)驗(yàn)采用200ng/mL的雷公藤甲素和36h作用時(shí)間進(jìn)行后續(xù)的蛋白質(zhì)組學(xué)實(shí)驗(yàn)。2.運(yùn)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(xì)胞A549內(nèi)的差異蛋白結(jié)果顯示:雷公藤甲素組與空白對(duì)照組相比,雷公藤甲素干預(yù)可以引起A549細(xì)胞中的312種蛋白質(zhì)發(fā)生顯著的差異性表達(dá)(P0.05),其中141個(gè)蛋白質(zhì)表達(dá)上調(diào),171個(gè)蛋白質(zhì)表達(dá)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(xì)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(yàn)證實(shí)驗(yàn)結(jié)果顯示:在人肺癌細(xì)胞A549中,雷公藤甲素可以上調(diào)PHB、CDH1和AIFM1蛋白質(zhì)表達(dá)(P0.05),下調(diào)MTA2和EIF4A3蛋白質(zhì)表達(dá)(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(duì)A549細(xì)胞有顯著的細(xì)胞毒性作用,可以抑制細(xì)胞增殖,顯著阻滯細(xì)胞周期于G2/M期并誘導(dǎo)細(xì)胞凋亡(P0.01)。2.雷公藤甲素可以影響肺癌細(xì)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(diǎn)。
[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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳將華;鄭維威;姜旭東;陸曉雅;徐榮臻;;雷公藤甲素通過(guò)抑制逆轉(zhuǎn)錄病毒HERV-K Np9基因轉(zhuǎn)錄誘導(dǎo)人急性T淋巴細(xì)胞白血病Jurkat細(xì)胞凋亡[J];南方醫(yī)科大學(xué)學(xué)報(bào);2015年05期

2 潘霞;王智慧;雷小勇;;microRNAs與ABC轉(zhuǎn)運(yùn)蛋白介導(dǎo)的腫瘤多藥耐藥研究進(jìn)展[J];中華腫瘤防治雜志;2015年08期

3 林高陽(yáng);徐克;;MicroRNA調(diào)控腫瘤耐藥的研究進(jìn)展[J];中國(guó)肺癌雜志;2014年10期

4 何涵;柳約堅(jiān);陳菲莉;查潔;周勇;周淑蕓;徐兵;;低濃度雷公藤甲素增強(qiáng)去甲氧柔紅霉素殺傷急性髓細(xì)胞白血病干細(xì)胞的能力[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2014年18期

5 鄭忠信;柳約堅(jiān);陳菲莉;易正山;董慧娟;周勇;周淑蕓;徐兵;;低濃度雷公藤甲素聯(lián)合伊達(dá)比星誘導(dǎo)急性髓系白血病干細(xì)胞凋亡及其機(jī)制[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2014年05期

6 任麗琴;王成楓;王金海;;雷公藤內(nèi)酯醇聯(lián)合奧沙利鉑抗人結(jié)腸癌細(xì)胞SW480增殖的體外研究[J];海峽藥學(xué);2014年08期

7 卜強(qiáng);曾明輝;蔣華;蔣東方;秦鎖炳;陳巧云;李皓;;吡柔比星聯(lián)合雷公藤甲素對(duì)人膀胱癌T24細(xì)胞生長(zhǎng)的影響[J];腫瘤;2014年06期

8 陳菲莉;柳約堅(jiān);李蓉蔚;王詩(shī)韻;董慧娟;周淑蕓;徐兵;;雷公騰內(nèi)酯與多柔比星誘導(dǎo)耐藥白血病細(xì)胞凋亡協(xié)同作用及其機(jī)制的探討[J];中華腫瘤防治雜志;2014年10期

9 孫運(yùn)良;吳紅玉;金晶;滿(mǎn)曉華;李淑德;;雷公藤內(nèi)酯醇聯(lián)合吉西他濱對(duì)胰腺癌細(xì)胞增殖和凋亡的影響[J];重慶醫(yī)學(xué);2014年05期

10 馬薇;魏柏;熊枝繁;;雷公藤內(nèi)酯醇聯(lián)合熱療對(duì)MKN28細(xì)胞增殖抑制作用的研究[J];現(xiàn)代腫瘤醫(yī)學(xué);2014年01期

，

本文編號(hào)：1811500