【作者】 侯涛；

【导师】 胡春宏；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 研究背景：非小细胞肺癌(non-small cell lung cancer, NSCLC)是我国高发肿瘤,其发病率位居男性肿瘤患者的第一位、女性肿瘤患者的第二位。手术、放疗、化疗是NSCLC的传统治疗手段。近年来,随着对NSCLC发病机制研究的深入,表皮生长因子(Epidermal Growth Factor Receptor, EGFR)信号通路在NSCLC的发生发展过程中的重要作用不断被认识。基于此发展起来的靶向EGFR信号通路的靶向治疗已应用于NSCLC的临床治疗。靶向治疗因其治疗效果好,副作用相对较小,已成为NSCLC的一种重要治疗手段。然而,患者接受一定时间的治疗后,几乎不可避免地出现耐药,而使治疗失效,病情进展。因此,寻找延缓、逆转靶向治疗耐药的方法是当前靶向治疗领域的一个重要问题。二甲双胍是目前广泛使用于II型糖尿病治疗的一种胰岛素增敏剂。近年来的研究发现,二甲双胍除对II型糖尿病的治疗作用外,在心血管疾病、多囊卵巢综合症及肿瘤的预防和治疗中可能发挥重要作用。二甲双胍与肿瘤的关系尤其受到重视。越来越多的证据表明,二甲双胍可能具有抑制肿瘤增殖,改善肿瘤对化疗药物敏感性的作用。本实验用二甲双胍作用于人肺腺癌PC9、PC9/GR细胞,观察其对不同EGFR突变状态的肺腺癌细胞增殖抑制作用,及其对PC9/GR细胞吉非替尼敏感性的影响及其可能机制,为进一步研究提供基础。第一章二甲双胍对肺腺癌PC9、PC9/GR细胞的增殖抑制、凋亡诱导作用研究目的：研究二甲双胍对人肺腺癌PC、PC9/GR细胞抑制增殖及诱导凋亡的效应研究方法：以不同浓度二甲双胍干预PC9、PC9/GR细胞,四甲基偶氮唑蓝(MTT)法检测细胞增殖抑制情况,AnnexinV-FITC/PI双染流式细胞术检测细胞凋亡率。结果：1.MTT比色法检测不同浓度二甲双胍作用于PC9,PC9/GR细胞,发现随着药物浓度的提高、作用时间的延长,药物对PC9、PC9/GR细胞的增殖抑制作用逐渐增强。且二甲双胍对PC9的增殖抑制作用与对PC9/GR的增殖抑制作用无明显差异。2.不同浓度二甲双胍(1mmol/L、2mmol/L、4mmol/L、8mmol/L、10mmol/L)干预PC9/PC9/GR细胞48h后,PC9细胞凋亡率由9.59%升高至25.94%,PC9/GR细胞凋亡率由7.28%上升至23.93%。各浓度二甲双胍干预组与对照组比较,差异具有统计学意义(P<0.05)。结论：1、体外实验证明,在一定浓度范围内,二甲双胍对人肺腺癌PC9、 PC9/GR细胞株具有抑制增殖作用,其作用具有浓度和时间依赖性。2、二甲双胍在体外可诱导PC9、PC9/GR发生凋亡,其作用具有浓度依赖性。第二章二甲双胍对肺腺癌PC9/GR细胞株吉非替尼敏感性的影响及其机制研究目的：研究二甲双胍对PC9/GR细胞吉非替尼敏感性的影响及其可能机制。研究方法：以5μmmol/L吉非替尼联合不同浓度二甲双胍作用于PC9/GR细胞48小时,以MTT法检测细胞增殖抑制率,判断不同浓度二甲双胍对PC9/GR细胞吉非替尼敏感性的影响。流式细胞术分析二甲双胍、吉非替尼、二甲双胍+吉非替尼干预PC9/GR细胞后,细胞凋亡率的变化。Western Blot法检测AMPK、p-AMPK、m-TOR、p-mTOR等有关蛋白的表达水平。结果：1.MTT法检测发现,不同浓度二甲双胍与吉非替尼联用后,均能较单药吉非替尼更强地抑制细胞增殖。两药表现为协同作用,且较低剂量的二甲双胍的协同效应较强。2.流式细胞术检测发现,2mmol/L二甲双胍与5μmol/L吉非替尼联用,干预PC9/GR细胞48h后,可较单药吉非替尼导致更高比例的细胞凋亡,细胞凋亡率从22.29%上升至35.98%,差异具有统计学意义(P<0.05)。两药的协同系数Q=1.094,表明两药在诱导PC9/GR细胞凋亡上具有相加作用。3.Western Blot法检测示2mmol/L二甲双胍干预PC9/GR细胞后,引起AMPK、p-AMPK、mTOR表达的上调,p-mTOR表达的下调。5μmol/L吉非替尼干预后,引起AMPK、p-AMPK、mTOR的下调,p-mTOR的上调。而两药联合作用干预PC9/GR细胞后,引起AMPK、 p-AMPK、mTOR表达上调,p-mTOR下调,其表达水平较单独用药组有显著差异(P<0.05)。结论：一定浓度范围内,二甲双胍具有增强PC9/GR细胞对吉非替尼敏感性的作用,其机制可能与二甲双胍影响AMPK/mTOR通路的活性,进而诱导细胞凋亡增加有关。更多还原

【Abstract】 Background:Lung Cancer is the leading cause of death in China, whose incidence ranks No.1in male cancer patients and No.2in female cancer patients. Non-Small Cell Lung Cancer (NSCLC) is the major pathological subtype of lung cancer. Most of the patients are diagnosed at a comparatively late stage and lose the chance to take an operation. Thus, chemotherapy and radiotherapy is the major approaches for treatment. However, the efficacy of these approaches reaches a plateau; even though researchers from all over the world have given great effort to improve it. The5-year survival rate of NSCLC is less than15%. With the deepening understanding of NSCLC tumorgenesis, the importance of Epidermal Growth Factor Receptor (EGFR) signal pathway is unveiled. The target therapy based on EGFR signal pathway (eg. EGFR-TKI) is now widely used in the treatment of EGFR mutant positive NSCLC patients and achieves a great success. Nowadays, target therapy is a new and important treatment approach of NSCLC patients. However, nearly all the patients become resistant to the target therapy, which results in the progression of disease, with a median time-to-progression of about10months. Therefore, it is a challenging and important issue for both physician and researcher to find ways to delay or reverse the resistance.Metformin is a widely used insulin-sensitizer for the treatment of type II diabetes mellitus. However, recent studies showed that, besides its role in diabetes, metformin seems to play a protective role in the cardiovascular disease, poly-cystic ovary syndrome (PCOS) and cancer. The relation between metformin and cancer is emphasized. There have been pre-clinical evidences supporting that metformin can inhibit the proliferation of some kinds of cancer, including breast cancer, ovary cancer, and endometrial cancer, and increase their sensitivity to certain kinds of chemotherapy drugs. However, the exact mechanism of metformin’s effect is not fully understood yet. It is now widely accepted that metformin can influent the intracellular proliferative signal transduction and induce apoptosis via the activation of adenosine monophosphate activated protein kinase (AMPK). The influence of metformin on the sensitivity of lung adenocarcinoma to EGFR-TKI is not reported. In this study, we use human lung adenocarcinoma cell lines PC9and PC9/GR as a model to evaluate the anti-proliferation, apoptosis-inducing, and EGFR-TKI sensitivization effect of metfotmin. Our study may provide a basis for the further study of the exact mechanism of metformin’s effect and the possibility of using metformin as a EGFR-TKI sensitizer for NSCLC patient. Part I Anti-proliferation and apoptosis-inducing effect of metformin on PC9and PC9/GR cellsObjective:To evaluate the effect of proliferation and apoptosis of metformin on PC9and PC9/GR cellsMethods:Various concentrations of metformin were given to both PC9and PC9/GR cells. Tetrazolium blue (MTT) assay was used to evaluate proliferation inhibition rate. Flow cytometry was used to analyze the apoptosis rate.Results:1. MTT assay showed that metformin had proliferation inhibition effect on both PC9and PC9/GR manner in a dose and time-dependent manner, and the difference showed statistical significance. And the anti-proliferation effect of metformin showed no difference between PC9and PC9/GR.2. Flow cytometry assay showed that the apoptosis rate of both PC9and PC9/GR increased after48h exposure to metformin. And the apoptosis rate increased accompanied with the increase of metformin concentration.Conclusion:Metformin has anti-proliferation and apoptosis-inducing effect on both PC9and PC9/GR cells. Part ⅡEGFR-TKI sensitization effect of metformin on PC9/GR cell and its possible mechanismObjective:To study the EGFR-TKI sensitization effect of metformin on PC9/GR cell and its possible mechanismMethods:MTT assay was used to evaluate the proliferation inhibition rate of PC9/GR cell after treated with5μmol/L gefitinib combined with various concentration of metformin for48h. Flow cytometry was used to analyze the apoptosis rate of PC9/GR cell and western blot was used to evaluate the expression of AMPK、p-AMPK、mTOR and p-mTOR after treated with2mmol/L metformin,5μmol/L gefitinib and2mmol/L metformin+5μmol/L gefitinib for48h.Results:1. MTT assay showed that gefitinib combined with metformin, compared with gefitinib alone, exerted stronger proliferation inhibition effect on PC9/GR cell. And the difference is statistical significant.2. Flow cytometry assay showed that the apoptosis rate is higher after the treatment of2mmol/L metformin+5μmol/L gefitinib for48h, compared with5μmol/L gefitinib alone.3.Wstern Blot showed that metformin can increase the expression of AMPK、p-AMPK、mTOR, and down-regulate the expression of p-mTOR. While gefitinib could down-regulate the expression of AMPK、p-AMPK、 mTOR, and increase the expression of p-mTOR. The combination of metformin and gefitinib induced a even higher expression of AMPK、 P-AMPK、mTOR, and lower expression of p-mTOR compared with gefitinib alone.Conclusion:Metformin may sensitize PC9/GR cell to EGFR-TKI via affecting the AMPK/mTOR pathway and synergetic apoptosis-inducing effect.更多还原