本文選題：木犀草素　切入點(diǎn)：膠質(zhì)母細(xì)胞瘤　出處：《南京大學(xué)》2016年博士論文

【摘要】：膠質(zhì)母細(xì)胞瘤是顱內(nèi)腫瘤中最常見且惡性程度最高的惡性腫瘤,由于腫瘤高度增殖、侵襲以及對(duì)放化療的耐受性,患者即使接受最積極的治療手段,患者依然要面臨手術(shù)療效差、術(shù)后極易復(fù)發(fā)、生存期短的境地。因此,亟需我們開發(fā)新的治療藥物,尋找有效的治療方法。木犀草素是一種天然黃酮類化合物,有文獻(xiàn)報(bào)道其在體外、體內(nèi)都有明確的抗腫瘤作用。研究表明,肺癌中木犀草素是Nrf2的有效的和選擇性抑制劑,能有效抑制肺癌裸鼠種植瘤的生長(zhǎng)并提高化療藥物的敏感性。胰島素樣生長(zhǎng)因子(IGF-1)在多種惡性腫瘤組織中高表達(dá),其介導(dǎo)的信號(hào)通路參與了腫瘤細(xì)胞凋亡調(diào)控。PI3K/AKT/mTOR信號(hào)通路的過(guò)度活化在腫瘤的發(fā)生發(fā)展過(guò)程中具有十分重要的作用,與腫瘤細(xì)胞的惡性生物學(xué)行為侵襲/遷移、凋亡和耐藥等有著密不可分的關(guān)系。內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)是細(xì)胞應(yīng)對(duì)外界刺激的一種保護(hù)性反應(yīng),但是研究表明長(zhǎng)時(shí)間持續(xù)的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)會(huì)誘導(dǎo)細(xì)胞發(fā)生凋亡。因此,本研究以膠質(zhì)母細(xì)胞瘤細(xì)胞系U251MG和U87MG為體外模型,裸鼠皮下種植瘤為體內(nèi)模型,詳細(xì)研究木犀草素的抗膠質(zhì)母細(xì)胞瘤作用,其對(duì)膠質(zhì)母細(xì)胞瘤侵襲/遷移、凋亡的影響,以及對(duì)膠質(zhì)母細(xì)胞瘤替莫唑胺耐藥細(xì)胞的作用,并研究IGF-1R/PI3K/AKT/mTOR通路以及藥物所引發(fā)的腫瘤細(xì)胞的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)在這些過(guò)程中的作用。本課題共分為以下五個(gè)研究部分:第一部分木犀草素對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞遷移的作用目的:研究木犀草素能否抑制膠質(zhì)母細(xì)胞瘤細(xì)胞的遷移。方法:體外培養(yǎng)膠質(zhì)母細(xì)胞瘤細(xì)胞系U251MG和U87MG,接種于96孔,利用CCK-8進(jìn)行細(xì)胞增殖實(shí)驗(yàn),將不同濃度梯度0,5,10,20,40,80μmol/L的木犀草素處理細(xì)胞24h后用CCK-8檢測(cè)增殖,確定藥物的給藥濃度和時(shí)間(0,5,10,20μmol/L,24h)。之后再檢測(cè)對(duì)U251MG和U87MG遷移的影響;利用western blot檢測(cè)侵襲和遷移相關(guān)蛋白MMP-9,MMP-2,TIMP-1,TIMP-2的變化;利用 westernblot 檢測(cè) EMT 相關(guān)蛋白 Vimentin,β-catenin,N-cadherin 和 E-cadherin水平變化;利用羅丹明標(biāo)記的鬼筆環(huán)肽染色細(xì)胞骨架蛋白,檢測(cè)木犀草素對(duì)細(xì)胞形態(tài)和遷移表型的影響。結(jié)果:細(xì)胞增殖實(shí)驗(yàn)結(jié)果顯示木犀草素濃度小于40μmol/L時(shí)對(duì)腫瘤細(xì)胞增殖沒有明顯影響;細(xì)胞劃痕遷移實(shí)驗(yàn)表明木犀草素能顯著抑制膠質(zhì)母細(xì)胞瘤細(xì)胞的遷移;木犀草素能夠明顯下調(diào)侵襲和遷移相關(guān)蛋白MMP-2,MMP-9的表達(dá)而上調(diào)抑制蛋白TIMP-1,TIMP-2的表達(dá);此外木犀草素能夠阻止膠質(zhì)母細(xì)胞瘤細(xì)胞的EMT過(guò)程。結(jié)論:木犀草素可以抑制膠質(zhì)母細(xì)胞瘤細(xì)胞的遷移。第二部分木犀草素對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡的影響目的:確定木犀草素對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞的凋亡的作用。方法:利用膠質(zhì)母細(xì)胞瘤細(xì)胞系U251MG和U87MG以及小鼠原代血管內(nèi)皮細(xì)胞模型,用不同濃度的木犀草素處理細(xì)胞,用CCK-8檢測(cè)細(xì)胞活力確定其對(duì)腫瘤細(xì)胞的殺傷作用;通過(guò)Annexin V和Propidium iodode(PI)染色利用流式細(xì)胞儀檢測(cè)對(duì)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡的影響。同時(shí)用TUNEL染色、免疫熒光和western blot檢測(cè)cleaved-caspase-3的表達(dá)進(jìn)一步確定其對(duì)腫瘤細(xì)胞凋亡的作用。利用裸鼠皮下種植模型,檢測(cè)木犀草素在體內(nèi)的抗腫瘤作用,并用western blot檢測(cè)凋亡相關(guān)蛋白的表達(dá),利用TUNEL檢測(cè)腫瘤組織中的凋亡情況。結(jié)果:我們發(fā)現(xiàn)木犀草素在40,80μmol/L時(shí)在體外具有明顯促進(jìn)腫瘤細(xì)胞凋亡的作用;木犀草素能明顯提高腫瘤細(xì)胞的TUNEL水平,cleaved-caspase3的蛋白水平明顯升高;木犀草素能通過(guò)促進(jìn)凋亡抑制膠質(zhì)母細(xì)胞瘤裸鼠皮下種植瘤的生長(zhǎng)。結(jié)論:木犀草素能促進(jìn)膠質(zhì)母細(xì)胞瘤細(xì)胞的凋亡并抑制體內(nèi)腫瘤生長(zhǎng)。第三部分內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)在木犀草素引發(fā)的膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡中的作用目的:明確內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)是否參與了木犀草素引起的腫瘤的凋亡。方法:利用熒光檢測(cè)腫瘤細(xì)胞在藥物刺激后ROS的水平情況,并利用western blot檢測(cè)木犀草素(0,40,80μM)作用24h后氧化應(yīng)激相關(guān)蛋白Nrf2、NQO-1、HO-1的水平變化:利用western blot檢測(cè)40 μM木犀草素刺激不同時(shí)間(0,1,3,6,12,24h)及不同濃度(0,5,10,20，40,80μM)作用24h后內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)相關(guān)蛋白 p-PERK,p-eIF2α,ATF4,CHOP,Cleaved-caspase-12 的蛋白水平變化情況;利用JC-1染料檢測(cè)40μM木犀草素處理24h后腫瘤細(xì)胞線粒體的膜電位變化情況,及免疫熒光檢測(cè)處理后細(xì)胞色素c的表達(dá)情況,利用western blot檢測(cè)線粒體凋亡相關(guān)蛋白Bcl-2,Bax的表達(dá)情況;利用western blot檢測(cè)裸鼠皮下種植瘤中內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白ATF4,CHOP,Cleaved-caspase-12的表達(dá)情況。結(jié)果:用藥物木犀草素刺激膠質(zhì)母細(xì)胞瘤細(xì)胞后能夠誘導(dǎo)其ROS水平的升高,降低氧化應(yīng)激相關(guān)蛋白Nrf2、NQO-1和HO-1的水平。進(jìn)而激活內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng),p-PERK、p-eIF2α蛋白在40μM木犀草素處理6h時(shí)最高,而CHOP,Cleaved-caspase-12和Cleaved-capase-3的水平在24h最高,而且在體外以劑量依賴形式促進(jìn)促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)相關(guān)蛋白p-PERK、p-eIF2α、CHOP的表,在裸鼠體內(nèi)同樣具有促進(jìn)ATF4、CHOP、Cleaved-caspase-12的水平。木犀草素的處理能導(dǎo)致腫瘤細(xì)胞內(nèi)線粒體膜電位的下降,并抑制Bcl-2的表達(dá)而促進(jìn)促凋亡蛋白Bax的表達(dá)。結(jié)論:木犀草素通過(guò)內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡。第四部分p-IGF-1Rβ/PI3K/AKT/mTOR信號(hào)通路在木犀草素引起的膠質(zhì)母細(xì)胞瘤細(xì)胞生物行為變化中的作用目的:研究木犀草素是否通過(guò)調(diào)控p-IGF-1Rβ/PI3K/AKT/mTOR信號(hào)通路來(lái)發(fā)揮抗膠質(zhì)母細(xì)胞瘤作用。方法:用不同濃度(0,5,10,20,40,80μM)的木犀草素處理膠質(zhì)母細(xì)胞瘤細(xì)胞U251MG 和 U87MG 24h 后,通過(guò) western blot 檢測(cè)細(xì)胞 p-IGF-1Rβ/PI3K/AKT/mTOR通路上各信號(hào)分子蛋白水平的變化;用p-IGF1R特異性激動(dòng)劑IGF-1(100ng/ml)處理后,再次檢測(cè)這些蛋白的變化,并利用western blot檢測(cè)侵襲/遷移相關(guān)蛋白 MMP-2,MMP-9,TIMP-1,TIMP-2,Cleaved-caspase-3 的水平。結(jié)果:在木犀草素起到抑制膠質(zhì)母細(xì)胞瘤細(xì)胞遷移的條件下,其能顯著抑制細(xì)胞內(nèi)IGF-1Rβ,AKT,mTOR的磷酸化水平,而對(duì)其總蛋白水平?jīng)]有明顯影響,說(shuō)明木犀草素能抑制p-IGF-1Rβ/PI3K/AKT/mTOR信號(hào)通路;此外加入IGF-1后,升高的TIMP-1,TIMP-2，E-cadherin的水平出現(xiàn)下降,而被抑制的MMP-2,MMP-9,Vimentin的蛋白水平重新恢復(fù)。結(jié)論:木犀草素通過(guò)抑制p-IGF-1Rβ/PI3K/AKT/mTOR信號(hào)通路達(dá)到抑制膠質(zhì)母細(xì)胞瘤細(xì)胞遷移和EMT過(guò)程的作用。第五部分木犀草素對(duì)膠質(zhì)母細(xì)胞瘤替莫唑胺耐藥細(xì)胞系的作用目的:通過(guò)建立的膠質(zhì)母細(xì)胞瘤U251MG替莫唑胺耐藥細(xì)胞系,驗(yàn)證其耐藥表型,研究木犀草素對(duì)該耐藥細(xì)胞系的促凋亡作用,及是否能逆轉(zhuǎn)其對(duì)替莫唑胺的敏感性。方法:體外培養(yǎng)U251MG和U251MG-TR細(xì)胞,利用倒置顯微鏡觀察期形態(tài)的變化,利用CCK-8檢測(cè)其對(duì)替莫唑胺IC-50的變化,利用western blot和QRT-PCR檢測(cè)多藥耐藥相關(guān)基因MRP-3的蛋白和mRNA表達(dá);利用CCK-8檢測(cè)不同濃度木犀草素(0,5,10,20,40,80μM)對(duì)耐藥細(xì)胞系的殺傷作用,并與U251MG非耐藥細(xì)胞比較,利用AV/PI染色在流式細(xì)胞儀上檢測(cè)其對(duì)耐藥細(xì)胞凋亡的作用,利用western blot檢測(cè)凋亡相關(guān)蛋白Cleaved-caspase-3,Bcl-2,Bax的蛋白水平變化;利用western blot,QRT-PCR和免疫熒光檢測(cè)耐藥細(xì)胞系中Nrf2的水平,利用western blot,QRT-PCR檢測(cè)耐藥細(xì)胞系中NQO-1,HO-1的蛋白和mRNA水平;利用Nrf2慢病毒轉(zhuǎn)染U251MG耐藥細(xì)胞下調(diào)其Nrf2的表達(dá),再利用CCK-8檢測(cè)木犀草素對(duì)其的殺傷作用。將不同濃度的木犀草素和替莫唑胺聯(lián)用,利用CCK-8檢測(cè)其存活率,用AV/PI標(biāo)記用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,利用western blot檢測(cè)相應(yīng)Cleaved-caspase-3,Bax的蛋白水平。結(jié)果:我們實(shí)驗(yàn)室構(gòu)建的U251MG替莫唑胺耐藥細(xì)胞系(U251MG-TR)具有明顯的耐藥細(xì)胞特征,細(xì)胞變大而且成團(tuán)塊生長(zhǎng);其對(duì)替莫唑胺具有明顯的耐藥性,IC-50值上升9倍左右,多藥耐藥相關(guān)基因MRP-3的mRNA和蛋白水平明顯升高;木犀草素能顯著降低U251MG-TR的存活率,并且耐藥細(xì)胞比非耐藥細(xì)胞對(duì)木犀草素更加敏感,藥物刺激后U251MG-TR細(xì)胞凋亡明顯升高,凋亡相關(guān)蛋白Cleaved-caspase-3,Bax的水平明顯升高,而抑制凋亡蛋白Bcl-2水平明顯下降。U251MG-TR細(xì)胞中氧化應(yīng)激相關(guān)蛋白Nrf2,NQO-1,HO-1的mRNA和蛋白水平都明顯升高,用慢病毒將U251MG-TR細(xì)胞中Nrf2干擾后能降低耐藥細(xì)胞對(duì)木犀草素的敏感性;木犀草素能夠部分逆轉(zhuǎn)U251MG-TR對(duì)替莫唑胺的耐藥性。結(jié)論:木犀草素能誘導(dǎo)膠質(zhì)母細(xì)胞瘤替莫唑胺耐藥細(xì)胞的凋亡�？偨Y(jié)本課題的研究結(jié)果從體外和體內(nèi)兩個(gè)方面均證實(shí)木犀草素可以通過(guò)p-IGF1R-β/PI3K/AKT/mTOR信號(hào)通路抑制膠質(zhì)母細(xì)胞瘤細(xì)胞的遷移;并能通過(guò)激活持續(xù)的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)而促進(jìn)膠質(zhì)母細(xì)胞瘤細(xì)胞發(fā)生凋亡:此外U251MG替莫唑胺耐藥細(xì)胞對(duì)木犀草素十分敏感,這可能和耐藥細(xì)胞高表達(dá)的Nrf2有關(guān);而且,低劑量木犀草素就能提高耐藥細(xì)胞對(duì)替莫唑胺的敏感性。因而,我們認(rèn)為木犀草素具有多重抗膠質(zhì)母細(xì)胞瘤作用,內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)誘導(dǎo)的凋亡在抗腫瘤過(guò)程中具有重要作用,可以為膠質(zhì)母細(xì)胞瘤的治療提供新的思路。
[Abstract]:Glioblastoma is the most malignant tumor in the most common intracranial tumors and malignant degree of tumor, because of high proliferation, invasion and tolerance to chemotherapy, even the patients accept the most active treatment, patients are still faced with poor operative effect, postoperative recurrence, short survival situation. Therefore, urgent we developed a new drug, to find effective treatment. Luteolin is a natural flavonoid, has been reported to the in vitro, in vivo have clear anti-tumor effect. The results show that lung cancer luteolin is potent and selective Nrf2 inhibitor, can effectively inhibit the lung cancer xenograft tumor growth and improve the sensitivity of chemotherapy drugs. Insulin like growth factor (IGF-1) is highly expressed in a variety of malignant tumors, the signal pathway mediated apoptosis of tumor cells is involved in the regulation of.PI3K/AKT/mTOR. The excessive activation of signaling pathway plays an important role in the process of tumor progression, invasion / migration and the biological behavior of malignant tumor cells, apoptosis and drug resistance are closely related. Endoplasmic reticulum stress is a protective response of cells respond to external stimuli, but studies have shown that endoplasmic reticulum stress in long time persistent will induce apoptosis. Therefore, the study on glioblastoma cell lines U251MG and U87MG as an in vitro model, nude mice subcutaneous tumor model in vivo, a detailed study of luteolin anti glioblastoma cell tumor and its invasion of glioblastoma cell migration, apoptosis, and the effect on glioma neuroblastoma temozolomide resistant cells, endoplasmic reticulum stress and study of IGF-1R/PI3K/AKT/mTOR induced drug pathway and tumor cells in these processes are used. This paper is divided into the following five parts: the first part of luteolin on migration of glioblastoma cells. Objective: To study whether luteolin inhibited the migration of glioblastoma cells. Methods: human glioblastoma cell lines U251MG and U87MG cultured in vitro were inoculated in 96 holes, cell proliferation experiments using CCK-8, will different 0,5,10,20,40,80 concentration gradient mol/L luteolin treated cells 24h after CCK-8 was used to detect the proliferation, determination of drug concentration and time (0,5,10,20 mol/L, 24h). After testing the effects on U251MG and U87MG migration; use of migration and invasion related protein MMP-9, Western blot, MMP-2 TIMP-1, TIMP-2 detection, using the change; Westernblot detection of EMT related protein Vimentin, beta -catenin, N-cadherin and E-cadherin level changes; stained cells skeleton protein using Luo Danming phalloidin,. Measuring the effect of luteolin on the morphology and migration of cell phenotype. Results: the cell proliferation assay showed that luteolin concentration lower than 40 mol/L has no obvious effect on tumor cell proliferation; cell scratch migration experiments show that luteolin can significantly inhibit the migration of glioblastoma cells; luteolin can significantly decrease the invasion and migration of MMP-2 protein, the expression of MMP-9 while regulation of suppressor protein TIMP-1, expression of TIMP-2; EMT process in addition luteolin can prevent glioblastoma cells. Conclusion: luteolin can inhibit the migration of glioblastoma cells. The second part: the effect of luteolin on glioblastoma cell apoptosis Objective: to determine luteolin on apoptosis of glioblastoma cells. Methods use: Glioblastoma cell lines U251MG and U87MG and primary murine vascular endothelial cell model with Cells treated with different concentrations of luteolin, determine its cytotoxic effect on tumor cells by CCK-8 detection of cell viability by Annexin V and Propidium iodode; (PI) staining using flow cytometry on glioblastoma cell apoptosis by TUNEL staining. At the same time, the expression of immune fluorescence and Western blot detection of cleaved-caspase-3 further confirmed the apoptosis of tumor cells. The nude mice model, detection of luteolin in vivo antitumor effect and expression of Western blot detection of apoptosis related proteins, apoptosis detected by TUNEL in tumor tissue. Results: we found that luteolin in 40,80 mol/L in vitro could obviously promote the apoptosis of tumor cells effect; luteolin can significantly improve tumor cell TUNEL level and protein level of cleaved-caspase3 increased significantly; can through the To promote the apoptosis of glioblastoma grown in nude mice subcutaneous tumor growth. Conclusion: luteolin can promote apoptosis of glioblastoma cells and inhibit tumor growth in vivo. Endoplasmic reticulum induced apoptosis of luteolin in third glioblastoma cells for apoptosis Objective: clear endoplasmic reticulum stress is involved in luteolin caused by the tumor. Methods: the tumor cells detected by fluorescence in the level of ROS after the stimulation of drugs, and the use of Western blot (0,40,80 M) detection of luteolin oxidative stress related protein Nrf2, 24h NQO-1, HO-1 levels by Western blot detection 40 M (0,1,3,6,12,24h) stimulation of luteolin in different time and different the concentration of (0,5,10,20,40,80 M) related proteins in the endoplasmic reticulum stress reaction after 24h p-PERK, ATF4, CHOP, p-eIF2 alpha, Cleaved-caspase-12 protein in water Flat changes; using membrane potential changes in JC-1 dye detection of tumor cells in mitochondrial M 24h 40 after luteolin treatment, the expression of cytochrome c and immunofluorescence assay after treated by Western blot detection of mitochondrial apoptosis related protein Bcl-2, Bax expression; the endoplasmic reticulum stress Western blot grown in nude mice subcutaneous tumor detection ATF4 protein, CHOP, expression of Cleaved-caspase-12. Results: the drug luteolin stimulation of glioblastoma cells to induced increase of ROS level, reduce the oxidative stress associated protein Nrf2, NQO-1 and HO-1 level. Then activation of endoplasmic reticulum stress, p-PERK, p-eIF2 protein in 40 M 6h treatment of luteolin the highest, while CHOP, Cleaved-caspase-12 and Cleaved-capase-3 levels in the highest 24h, and in vitro in a dose-dependent way to promote endoplasmic reticulum stress P-PERK protein, p-eIF2 alpha, CHOP, CHOP in nude mice with the same Cleaved-caspase-12, promoting ATF4 level. Luteolin treatment can lead to the decline of mitochondrial membrane potential in tumor cells, and inhibit and promote the expression of Pro apoptotic protein Bax Bcl-2 expression. Conclusion: luteolin induced apoptosis of tumor cells through endoplasmic reticulum changes in response to stress. The biological behavior of glioblastoma cells in fourth p-IGF-1R beta /PI3K/AKT/mTOR signaling pathway in the role of luteolin induced Objective: To study whether luteolin regulates p-IGF-1R beta /PI3K/AKT/mTOR signaling pathways play a role of anti glioma cells. Methods: different concentrations (0,5,10,20,40,80 M) of luteolin treatment of glioblastoma cells U251MG and U87MG 24h, by Western blot detection of cell p-IGF-1R beta /PI3K/AKT/mTOR pathway on the signal Molecular changes in protein levels; with p-IGF1R specific agonist IGF-1 (100ng/ml) after the treatment, again change detection of these proteins, and using Western blot to detect invasion / migration related proteins MMP-2, MMP-9, TIMP-1, TIMP-2, Cleaved-caspase-3 level. Results: in the luteolin to inhibit glioblastoma cell migration under the condition that it can significantly inhibit the intracellular IGF-1R beta, AKT, phosphorylation of mTOR, but has no effect on the total protein level, luteolin can inhibit p-IGF-1R beta /PI3K/AKT/mTOR signaling pathway; plus IGF-1, elevated TIMP-1, TIMP-2, E-cadherin levels declined, but was inhibited by MMP-2 MMP-9, the protein level of Vimentin recovered. Conclusion: luteolin can inhibit glioblastoma cell migration and EMT process by inhibiting the p-IGF-1R beta /PI3K/AKT/mTOR signal pathway. Fifth Effect of luteolin on points of glioblastoma temozolomide resistant cell line Objective: through the establishment of U251MG glioma cells of temozolomide resistant cell line, verify the resistance phenotype of luteolin induced apoptosis of the resistant cell line, and whether it can reverse its sensitivity to temozolomide. Methods: U251MG and U251MG-TR cells were cultured in vitro and observe the change of phase morphology by inverted microscope, to detect the changes of temozolomide IC-50, using CCK-8 western and QRT-PCR blot expression detection of multidrug resistance related gene MRP-3 and mRNA protein; using CCK-8 to detect different concentrations of luteolin (0,5,10,20,40,80 M) killing effect on drug resistance cell lines, and compared with non U251MG resistant cells, AV/PI staining was used to detect the drug resistance of cell apoptosis by flow cytometer, using Western blot to detect apoptosis related protein White Cleaved-caspase-3, Bcl-2, Bax protein level changes; using Western blot, Nrf2 QRT-PCR and immunofluorescence detection of drug resistance cell line level, using Western blot, NQO-1 QRT-PCR detection of drug-resistant cell lines, HO-1 and mRNA protein level; down regulated the expression of Nrf2 by lentivirus transfected Nrf2 resistant U251MG cells, using CCK-8 the killing effect of luteolin on detection. Different concentrations of luteolin and Temozolomide Combined with the use of CCK-8 to detect the survival rate of apoptosis was assayed by flow cytometry with AV/PI markers detection using western corresponding Cleaved-caspase-3 blot, the protein level of Bax. Results: We constructed U251MG temozolomide resistance cell line (U251MG-TR) has the obvious characteristics of the resistant cells, the cells became larger and mass growth; the temozolomide has obvious resistance, IC-50 value increased 9 times more. Drug resistance related gene MRP-3 and mRNA protein levels were significantly increased; luteolin can significantly reduce the survival rate of U251MG-TR, and resistant cells than non resistant cells more sensitive to luteolin, apoptosis of U251MG-TR cells after stimulation with drugs significantly increased the expression of apoptosis related proteins Cleaved-caspase-3, Bax level increased significantly, and the inhibition of apoptosis protein Bcl-2 decreased oxidative stress.U251MG-TR cell associated protein Nrf2, NQO-1, HO-1, mRNA and protein levels were significantly increased by lentiviral Nrf2 interference in U251MG-TR cells can decrease the sensitivity of resistant cells to luteolin; luteolin can partially reverse the U251MG-TR resistance to temozolomide. Conclusion: luteolin can induce the apoptosis of glioma cells to temozolomide drug resistant cells. Summarize the results of this research from two aspects of in vitro and in vivo showed that luteolin Can inhibit the migration of glioblastoma cells by p-IGF1R- beta /PI3K/AKT/mTOR signaling pathway; and through the activation of endoplasmic reticulum stress sustained and promote glioma cell apoptosis in U251MG cells to temozolomide resistance of luteolin is very sensitive, high expression and this may be related to Nrf2 resistant cells and low dose; luteolin can improve the resistance sensitivity of cells to temozolomide. Therefore, we believe that luteolin has the effect of multiple anti glioblastoma, apoptosis induced by endoplasmic reticulum stress plays an important role in the anti-tumor process, to provide new ideas for the treatment of glioblastoma.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.41

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