本文選題：非小細胞肺癌　切入點：EGFR-TKI耐藥　出處：《浙江大學》2015年博士論文　論文類型：學位論文

【摘要】：目的： 通過沉默或激活PC-9細胞(攜帶EGFR基因19外顯子突變的NSCLC細胞株)中的內源性FGF2表達,同時添加吉非替尼與外源性FGF2,誘導PC-9細胞產生針對EGFR-TKIs快速獲得性耐藥的狀態(tài),篩選出FGF2介導的EGFR-TKIs快速獲得性耐藥的細胞模型,以進一步明確FGF-2信號通路介導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制。 研究方法： 1.構建編碼靶向FGF2基因shRNA及基于PAS (PCR-based Accurate Synthesis)的慢病毒載體,包裝慢病毒,感染人肺癌細胞PC-9,以熒光定量PCR檢測FGF2基因在mRNA水平的變化,以western blot檢測FGF2蛋白表達水平的變化,篩選出FGF2沉默表達/高表達的細胞株； 2.PC-9細胞針對EGFR-TKIs快速獲得性耐藥狀態(tài)的誘導及鑒定：對FGF2沉默表達(PC-9-FGF2-KD)或過表達細胞株(PC-9-FGF2-OE)單用吉非替尼或聯(lián)用外源性FGF2,篩選出耐藥細胞株。進一步用相關的生物學指標驗證其耐藥屬性,生物學指標檢測包括：(1)以MTS法檢測細胞增殖情況；(2)以AnnexinV/PI凋亡檢測試劑盒檢測時的細胞凋亡情況,以PI單染法檢測細胞周期分布情況；(3)軟瓊脂實驗測定細胞錨定非依賴生長的影響；(4)Transwell法測定細胞遷移和侵襲能力；(5)實時熒光定量PCR法檢測獲得性耐藥相關突變T790M突變和cMET擴增拷貝數(shù)。 3.以Affymetrix人全基因組3'IVT芯片檢測PC-9-NC+FGF2、PC-9-FGF2-KD+FGF2、PC-9-FGF2-OE+FGF2,以及空白對照的PC-9細胞的差異基因表達改變。 結果： 1.采用慢病毒載體介導的轉基因方法構建了FGF2基因沉默和FGF2基因高表達的PC-9細胞,Western Blot檢測進一步在蛋白水平對細胞內FGF2的表達改變進行了驗證,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(PC-9-FGF2-OE)的PC-9細胞株； 2.在FGF2基因過表達的PC-9細胞株(PC-9-FGF2-OE)及給予外源性FGF2的PC-9細胞株中均觀察到細胞活力的增加,提示內外源性的FGF2刺激均有可能誘導出PC-9細胞針對EGFR-TKIs的快速獲得性耐藥；細胞生物學檢測進一步表明PC-9-FGF2-OE+FGF2細胞株中出現(xiàn)顯著的細胞凋亡率下降、細胞增殖分裂加快、細胞遷移和侵襲能力增強等耐藥生物學行為,證實該細胞模型為FGF2介導的EGFR-TKIs快速獲得性耐藥的穩(wěn)定的NSCLC細胞模型； 3.芯片檢測發(fā)現(xiàn)PC-9-FGF2-OE+FGF2組細胞與PC-9-NC+FGF2組相比較而言,主要產生了PI3K-AKT通路、MAPK通路、ErbB通路和VEGF通路的上調,其中PI3K-AKT信號通路對FGF2介導的EGFR-TKIs快速獲得性耐藥的發(fā)生機制可能具有重要意義。 結論： 1.成功構建出FGF2介導的EGFR-TKIs快速獲得性耐藥的穩(wěn)定的NSCLC細胞模型,為進一步揭示FGF-2信號通路介導EGFR-TKIs快速獲得性耐藥的分子機制奠定了研究基礎； 2. PI3K-AKT信號通路可能在FGF2介導的EGFR-TKIs快速獲得性耐藥的發(fā)生機制中發(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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R734.2

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