本文選題：非小細(xì)胞肺癌　切入點(diǎn)：EGFR-TKI耐藥　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的： 通過沉默或激活PC-9細(xì)胞(攜帶EGFR基因19外顯子突變的NSCLC細(xì)胞株)中的內(nèi)源性FGF2表達(dá),同時(shí)添加吉非替尼與外源性FGF2,誘導(dǎo)PC-9細(xì)胞產(chǎn)生針對(duì)EGFR-TKIs快速獲得性耐藥的狀態(tài),篩選出FGF2介導(dǎo)的EGFR-TKIs快速獲得性耐藥的細(xì)胞模型,以進(jìn)一步明確FGF-2信號(hào)通路介導(dǎo)EGFR-TKIs快速獲得性耐藥這一全新的耐藥模式中所涉及的分子機(jī)制。 研究方法： 1.構(gòu)建編碼靶向FGF2基因shRNA及基于PAS (PCR-based Accurate Synthesis)的慢病毒載體,包裝慢病毒,感染人肺癌細(xì)胞PC-9,以熒光定量PCR檢測(cè)FGF2基因在mRNA水平的變化,以western blot檢測(cè)FGF2蛋白表達(dá)水平的變化,篩選出FGF2沉默表達(dá)/高表達(dá)的細(xì)胞株； 2.PC-9細(xì)胞針對(duì)EGFR-TKIs快速獲得性耐藥狀態(tài)的誘導(dǎo)及鑒定：對(duì)FGF2沉默表達(dá)(PC-9-FGF2-KD)或過表達(dá)細(xì)胞株(PC-9-FGF2-OE)單用吉非替尼或聯(lián)用外源性FGF2,篩選出耐藥細(xì)胞株。進(jìn)一步用相關(guān)的生物學(xué)指標(biāo)驗(yàn)證其耐藥屬性,生物學(xué)指標(biāo)檢測(cè)包括：(1)以MTS法檢測(cè)細(xì)胞增殖情況；(2)以AnnexinV/PI凋亡檢測(cè)試劑盒檢測(cè)時(shí)的細(xì)胞凋亡情況,以PI單染法檢測(cè)細(xì)胞周期分布情況；(3)軟瓊脂實(shí)驗(yàn)測(cè)定細(xì)胞錨定非依賴生長(zhǎng)的影響；(4)Transwell法測(cè)定細(xì)胞遷移和侵襲能力；(5)實(shí)時(shí)熒光定量PCR法檢測(cè)獲得性耐藥相關(guān)突變T790M突變和cMET擴(kuò)增拷貝數(shù)。 3.以Affymetrix人全基因組3'IVT芯片檢測(cè)PC-9-NC+FGF2、PC-9-FGF2-KD+FGF2、PC-9-FGF2-OE+FGF2,以及空白對(duì)照的PC-9細(xì)胞的差異基因表達(dá)改變。 結(jié)果： 1.采用慢病毒載體介導(dǎo)的轉(zhuǎn)基因方法構(gòu)建了FGF2基因沉默和FGF2基因高表達(dá)的PC-9細(xì)胞,Western Blot檢測(cè)進(jìn)一步在蛋白水平對(duì)細(xì)胞內(nèi)FGF2的表達(dá)改變進(jìn)行了驗(yàn)證,成功篩選出穩(wěn)定的FGF2基因沉默(PC-9-FGF2-KD)和FGF2基因高表達(dá)(PC-9-FGF2-OE)的PC-9細(xì)胞株； 2.在FGF2基因過表達(dá)的PC-9細(xì)胞株(PC-9-FGF2-OE)及給予外源性FGF2的PC-9細(xì)胞株中均觀察到細(xì)胞活力的增加,提示內(nèi)外源性的FGF2刺激均有可能誘導(dǎo)出PC-9細(xì)胞針對(duì)EGFR-TKIs的快速獲得性耐藥；細(xì)胞生物學(xué)檢測(cè)進(jìn)一步表明PC-9-FGF2-OE+FGF2細(xì)胞株中出現(xiàn)顯著的細(xì)胞凋亡率下降、細(xì)胞增殖分裂加快、細(xì)胞遷移和侵襲能力增強(qiáng)等耐藥生物學(xué)行為,證實(shí)該細(xì)胞模型為FGF2介導(dǎo)的EGFR-TKIs快速獲得性耐藥的穩(wěn)定的NSCLC細(xì)胞模型； 3.芯片檢測(cè)發(fā)現(xiàn)PC-9-FGF2-OE+FGF2組細(xì)胞與PC-9-NC+FGF2組相比較而言,主要產(chǎn)生了PI3K-AKT通路、MAPK通路、ErbB通路和VEGF通路的上調(diào),其中PI3K-AKT信號(hào)通路對(duì)FGF2介導(dǎo)的EGFR-TKIs快速獲得性耐藥的發(fā)生機(jī)制可能具有重要意義。 結(jié)論： 1.成功構(gòu)建出FGF2介導(dǎo)的EGFR-TKIs快速獲得性耐藥的穩(wěn)定的NSCLC細(xì)胞模型,為進(jìn)一步揭示FGF-2信號(hào)通路介導(dǎo)EGFR-TKIs快速獲得性耐藥的分子機(jī)制奠定了研究基礎(chǔ)； 2. PI3K-AKT信號(hào)通路可能在FGF2介導(dǎo)的EGFR-TKIs快速獲得性耐藥的發(fā)生機(jī)制中發(fā)揮重要作用。
[Abstract]:Objective:. By silencing or activating the expression of endogenous FGF2 in PC-9 cells (NSCLC cell line with exon 19 mutation of EGFR gene) and adding gefitinib and exogenous FGF2, PC-9 cells were induced to develop rapidly acquired drug resistance to EGFR-TKIs. The cell model of EGFR-TKIs rapid acquired resistance mediated by FGF2 was screened to further clarify the molecular mechanism involved in the novel drug resistance model of EGFR-TKIs mediated by FGF-2 signaling pathway. Research methods:. 1. Construct the lentivirus vector which encodes FGF2 gene shRNA and PAS PCR-based Accurate synthesis, package lentivirus, infect human lung cancer cell PC-9, detect the change of FGF2 gene in mRNA level by fluorescence quantitative PCR, and detect the change of FGF2 protein expression level by western blot. The cell lines with silencing / high expression of FGF2 were screened out. 2.Induction and identification of EGFR-TKIs rapid acquired drug resistance in PC-9 cells: the drug resistant cell lines were screened by silencing the expression of FGF2 or over-expressing cell line pPC-9-FGF2-OE) with either gefitinib or exogenous FGF2. The standard validates its drug resistance, Biological indicators include: 1) MTS assay was used to detect cell proliferation. (2) AnnexinV/PI apoptosis assay kit was used to detect cell apoptosis. Detection of Cell cycle Distribution by Pi Monostaining) effect of Cell Anchorage on Non-dependent growth by soft Agar Assay; determination of Cell Migration and invasion ability by Transwell method; Real-time fluorescence quantitative PCR Assay for Detection of acquired Drug-Related Mutant T790M processes. Variation and cMET amplification of copy number. 3. The differentially expressed genes in PC-9-NC FGF2P PC-9-FGF2-KD PC-9-FGF2-OE FGF2 cells and blank control PC-9 cells were detected by Affymetrix human genome 3IVT microarray. Results:. 1. FGF2 gene silencing and FGF2 gene overexpression in PC-9 cells were constructed by lentivirus vector-mediated transgenic method. Western Blot detection further verified the change of FGF2 expression at protein level. The stable FGF2 gene silencing PC-9 cell lines PC-9-FGF2-KD and FGF2 gene overexpression PC-9-FGF2-OEwere successfully screened. 2. The increase of cell viability was observed in PC-9 cell line (PC-9-FGF2-OE) and PC-9 cell line treated with exogenous FGF2, suggesting that both exogenous and exogenous FGF2 stimuli might induce rapid acquired drug resistance to EGFR-TKIs in PC-9 cells. Cell biology further showed that the cell apoptosis rate decreased significantly, cell proliferation and division accelerated, cell migration and invasion increased in PC-9-FGF2-OE FGF2 cell line. It is confirmed that this cell model is a stable NSCLC cell model of EGFR-TKIs rapid acquired resistance mediated by FGF2. 3.Compared with PC-9-NC FGF2 group, PC-9-FGF2-OE FGF2 cells mainly produced the up-regulation of PI3K-AKT pathway, ErbB pathway and VEGF pathway. PI3K-AKT signaling pathway may play an important role in the pathogenesis of EGFR-TKIs rapid acquired drug resistance mediated by FGF2. Conclusion:. 1. The stable NSCLC cell model of EGFR-TKIs rapid acquired resistance mediated by FGF2 was successfully constructed, which laid a foundation for further study on the molecular mechanism of EGFR-TKIs rapid acquired resistance mediated by FGF-2 signaling pathway. 2. PI3K-AKT signaling pathway may play an important role in the pathogenesis of EGFR-TKIs rapid acquired drug resistance mediated by FGF2.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2

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