FGF-2信号通路介导EGFR-TKIs快速获得性耐药的分子机制研究

发布时间：2018-03-14 02:40

本文选题：非小细胞肺癌　切入点：EGFR-TKI耐药　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：通过沉默或激活PC-9细胞(携带EGFR基因19外显子突变的NSCLC细胞株)中的内源性FGF2表达,同时添加吉非替尼与外源性FGF2,诱导PC-9细胞产生针对EGFR-TKIs快速获得性耐药的状态,筛选出FGF2介导的EGFR-TKIs快速获得性耐药的细胞模型,以进一步明确FGF-2信号通路介导EGFR-TKIs快速获得性耐药这一全新的耐药模式中所涉及的分子机制。研究方法： 1.构建编码靶向FGF2基因shRNA及基于PAS (PCR-based Accurate Synthesis)的慢病毒载体,包装慢病毒,感染人肺癌细胞PC-9,以荧光定量PCR检测FGF2基因在mRNA水平的变化,以western blot检测FGF2蛋白表达水平的变化,筛选出FGF2沉默表达/高表达的细胞株； 2.PC-9细胞针对EGFR-TKIs快速获得性耐药状态的诱导及鉴定：对FGF2沉默表达(PC-9-FGF2-KD)或过表达细胞株(PC-9-FGF2-OE)单用吉非替尼或联用外源性FGF2,筛选出耐药细胞株。进一步用相关的生物学指标验证其耐药属性,生物学指标检测包括：(1)以MTS法检测细胞增殖情况；(2)以AnnexinV/PI凋亡检测试剂盒检测时的细胞凋亡情况,以PI单染法检测细胞周期分布情况；(3)软琼脂实验测定细胞锚定非依赖生长的影响；(4)Transwell法测定细胞迁移和侵袭能力；(5)实时荧光定量PCR法检测获得性耐药相关突变T790M突变和cMET扩增拷贝数。 3.以Affymetrix人全基因组3'IVT芯片检测PC-9-NC+FGF2、PC-9-FGF2-KD+FGF2、PC-9-FGF2-OE+FGF2,以及空白对照的PC-9细胞的差异基因表达改变。结果： 1.采用慢病毒载体介导的转基因方法构建了FGF2基因沉默和FGF2基因高表达的PC-9细胞,Western Blot检测进一步在蛋白水平对细胞内FGF2的表达改变进行了验证,成功筛选出稳定的FGF2基因沉默(PC-9-FGF2-KD)和FGF2基因高表达(PC-9-FGF2-OE)的PC-9细胞株； 2.在FGF2基因过表达的PC-9细胞株(PC-9-FGF2-OE)及给予外源性FGF2的PC-9细胞株中均观察到细胞活力的增加,提示内外源性的FGF2刺激均有可能诱导出PC-9细胞针对EGFR-TKIs的快速获得性耐药；细胞生物学检测进一步表明PC-9-FGF2-OE+FGF2细胞株中出现显著的细胞凋亡率下降、细胞增殖分裂加快、细胞迁移和侵袭能力增强等耐药生物学行为,证实该细胞模型为FGF2介导的EGFR-TKIs快速获得性耐药的稳定的NSCLC细胞模型； 3.芯片检测发现PC-9-FGF2-OE+FGF2组细胞与PC-9-NC+FGF2组相比较而言,主要产生了PI3K-AKT通路、MAPK通路、ErbB通路和VEGF通路的上调,其中PI3K-AKT信号通路对FGF2介导的EGFR-TKIs快速获得性耐药的发生机制可能具有重要意义。结论： 1.成功构建出FGF2介导的EGFR-TKIs快速获得性耐药的稳定的NSCLC细胞模型,为进一步揭示FGF-2信号通路介导EGFR-TKIs快速获得性耐药的分子机制奠定了研究基础； 2. PI3K-AKT信号通路可能在FGF2介导的EGFR-TKIs快速获得性耐药的发生机制中发挥重要作用。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

【共引文献】