吉非替尼和顺铂联合使用在非小细胞肺癌治疗中的拮抗作用及其机制研究
发布时间：2018-07-18 21:47
【摘要】：背景与目的:支气管肺癌是最常见的呼吸系统肿瘤,在全球范围内发病率及死亡率均较高。非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)占支气管肺癌发病的大多数,目前手术仍然是治疗NSCLC的主要手段。但是对于手术患者来说,两年内的复发率约40%。故对于术后复发的NSCLC患者及无手术指征的晚期NSCLC患者,全身性化疗及靶向治疗仍为其主要的治疗手段,但目前常见的NSCLC一线化疗方案疗效仍不理想,有效率仅为30%左右,因此寻找新的有价值治疗手段和策略显得十分必要。靶向治疗与化疗联合能否提高化疗疗效,成为当前国内外研究的重要课题。含铂类的联合化疗仍然是晚期NSCLC的标准一线化疗方案,其中第一代铂类药物顺铂更是NSCLC化疗的基石。然而单一化疗并不能取得理想的治疗效果,随着生物制剂靶向治疗药物的出现,化疗联合靶向治疗是否可以提高疗效是一个值得研究的课题。吉非替尼是第一代表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)酪氨酸激酶抑制剂,该酶通常表达于上皮来源的实体瘤。作为第一代小分子酪氨酸激酶抑制剂,目前相关大型三期多中心临床实验证实在NSCLC的一线治疗中,吉非替尼相对于化疗来说,对于亚裔、腺癌和EGFR突变患者具有明显的无进展生存期(Progress-Free Survival,PFS)优势,然而在能否与化疗联用方面,四个大型多中心临床实验均证实表皮生长因子受体酪氨酸激酶抑制剂(Tyrosine-Kinase Inhibitor TKI),例如吉非替尼和厄罗替尼都与与化疗药物联用时对晚期非小细胞肺癌患者总生存期(Overall Survival,OS)无明显生存获益。提示吉非替尼与包括顺铂在内的化疗药物联用时可能存在拮抗作用。虽然临床试验结果显示顺铂和吉非替尼联合使用存在拮抗作用,但导致这种拮抗作用的机制仍然不是十分清楚。阐明导致拮抗作用的机制可为临床治疗提供新的思路。本课题拟从自噬和外泌体两个角度揭示吉非替尼与顺铂联合使用时如何影响顺铂治疗疗效。研究一吉非替尼通过上调非小细胞肺癌细胞自噬水平诱导影响顺铂疗效的作用及其机制研究方法(1)使用吉非替尼与不同浓度的顺铂联合作用于人NSCLC细胞株PC9,采用MTT法测定不同浓度顺铂对PC9细胞增殖力的影响,同时计算吉非替尼和不同浓度顺铂之间的相互作用指数来分析吉非替尼对不同浓度顺铂的作用。Western-Blot方法分析吉非替尼与顺铂联用与单独使用顺铂作用于PC9细胞后自噬标记蛋白LC3-Ⅱ及P62的变化,电镜观察上述两组自噬小体数量的变化,进而了解吉非替尼对顺铂诱导自噬水平的影响。(2)为进一步评估自噬强度,使用5μg/ml的溶酶体抑制剂氯喹(chloroquine,CQ),即抑制溶酶体降解的自噬抑制剂预处理细胞1小时,透射电子显微镜(Transmission Electron Microscope,TEM)用于确认CQ抑制自噬溶酶体的降解效果。然后加入吉非替尼和/或顺铂,比较无CQ时组间LC3表达的改变。从而判断PC9细胞的自噬水平的改变是吉非替尼和/或顺铂诱导,而不是自噬降解缺陷所致。(3)为观察抑制自噬后对吉非替尼和顺铂间拮抗效应的影响,我们使用MTT法分别检测给予CQ处理后的吉非替尼和顺铂组PC9细胞,根据所测得OD值计算CDI,了解自噬在吉非替尼导致顺铂耐药过程中的作用。(4)采用Annexin V-FITC/PI流式细胞技术检测研究上述处理后各组细胞凋亡率,同时提取PC9细胞相应组别凋亡蛋白Bcl-2和Bax,以了解自噬水平改变后对PC9细胞凋亡的影响。同时我们采用流式细胞学技术(Flow cytometry,FCM)进行细胞周期分析,了解自噬对PC9细胞周期的影响。结果1吉非替尼联合顺铂导致拮抗效应(1)采用MTT法,检测不同浓度吉非替尼和顺铂对PC9细胞的增殖抑制作用,结果显示,吉非替尼和顺铂对NSCLC细胞的抑制作用具有时间和浓度依赖性。相对于顺铂或吉非替尼单药,其他各种联合用药组细胞增殖力并无明显下降。(2)采用药物相互作用系数(Coefficient of Drug Interaction,CDI)评估药物作用性质,给予不同浓度吉非替尼和顺铂后CDI值为1.19±0.1(1.07-1.35),提示存在拮抗作用。2吉非替尼联合顺铂诱导产生自噬(1)采用Western Blot法观察吉非替尼和/或顺铂处理后的PC9细胞自噬标记物LC3及p62的表达情况,结果显示,与对照未处理组相比,吉非替尼和顺铂可诱导上调LC3-II转化,而吉非替尼合并顺铂与单独吉非替尼用药相比,LC3-II转化明显上升,但无统计学差异。同时吉非替尼和/或顺铂均显著下调p62水平。相比单药刺激,吉非替尼合并顺铂导致p62蛋白更加显著的下调,提示吉非替尼合并顺铂相对单药可诱导更高水平自噬。(2)采用透射电子显微镜(Transmission electron microscopy,TEM),观察吉非替尼和/或顺铂处理后的PC9细胞自噬小体变化情况,结果显示与单药处理组相比,吉非替尼联合顺铂相对单药处理组自噬小体数目增多),并且吉非替尼合并顺铂相对单药可诱导更高水平自噬。(3)采用Western Blot法观察CQ预处理后,吉非替尼和/或顺铂处理后的PC9细胞自噬标记物LC3及p62的表达情况。该结果提示吉非替尼和/或顺铂可诱导PC9细胞自噬,而不是溶酶体降解所致。3抑制自噬可抵抗吉非替尼和顺铂间的拮抗效应(1)采用TEM检测CQ预处理后吉非替尼和/或顺铂处理后的PC9细胞自噬溶酶体变化情况,结果显示与吉非替尼和/或顺铂组相比,加用CQ后自噬溶酶体显著增多,提示CQ可抑制自噬过程中自噬溶酶体的降解。(2)采用MTT检测CQ预处理后,吉非替尼和/或顺铂处理后的PC9细胞增殖力,结果提示与无CQ组相比,给予CQ预处理时吉非替尼和顺铂显著降低细胞增殖力。同时CQ预处理后吉非替尼和顺铂的协同作用相对增加。(3)采用Annexin V-FITC/PI凋亡检测研究与无CQ预处理相比,CQ预处理可增加各组PC9凋亡细胞比例。CQ预处理时无论单药还是两药联合使用均可显著增加凋亡细胞比,提示自噬在凋亡过程中扮演重要角色。4抑制自噬可促进凋亡(1)采用流式细胞技术(Flow Cytometry,FCM)进行对各组PC9细胞周期分析,结果提示吉非替尼和顺铂单药均可导致G1期停滞,然而,吉非替尼和顺铂两药联用仅稍微提高G1期PC9细胞比例。为进一步证实自噬与细胞周期停滞的关系,我们给吉非替尼和/或顺铂CQ(5μg/ml)预处理1小时,结果显示CQ抑制自噬不能逆转吉非替尼和/或顺铂诱导的细胞周期停滞。(2)采用Western Blot检测是否CQ预处理可影响各组PC9细胞促凋亡蛋白和抗凋亡蛋白的表达水平。CQ处理后Bcl-2表达水平(经典抗凋亡家族蛋白)和Bax(促凋亡蛋白)表达分别显著降低和升高。然而,与无CQ组相比,吉非替尼和/或顺铂加CQ预处理组的Bax/Bcl-2比例无显著差异(分别为p=0.592,0.09及0.06)。上述结果提示CQ抑制自噬可通过上调Bax和下调Bcl-2表达来促进PC9细胞凋亡。研究二吉非替尼通过外泌体上调自噬拮抗顺铂诱导的非小细胞肺癌细胞凋亡的作用研究方法(1)采用Exo Quick沉淀液分离正常对照组PC9细胞或吉非替尼(1μM)/顺铂(7.5μM)给药组的外泌体。外泌体从吉非替尼或顺铂作用的PC9细胞上清液提取,分为Exo-Con(Exosomes derived from negative Control),Exo-GF(Exosomes derived from gefitinib treated EGFR mutant lung cancer cells),Exo-DDP(Exosomes derived from cisplatin treated EGFR mutant lung cancer cells)。使用BCA蛋白检测试剂盒对不同胞外体蛋白进行标准定量。(2)采用透射电子显微镜(TEM)观察外泌体形成,鉴定是否提取成功。(3)采用CCK-8法检测外泌体预处理后的各组细胞抑制率,我们使用一定浓度外泌体预孵育24小时后加1μM吉非替尼或7.5μM顺铂孵育24小时。使用CCK-8法计算其细胞增殖活力,同时计算其细胞抑制率。并计算两种药物间的药物相互作用系数(Coefficient of Drug Interaction,CDI)。然后使用用外泌体抑制剂GW4869作用于各药物处理组,了解抑制外泌体分泌对吉非替尼或顺铂对于PC9细胞效果的影响。(4)分别使用各组外泌体和药物共同孵育细胞,Western Blot法检测自噬标记蛋白LC3和p62的变化,以了解外泌体和自噬之间的相关性。同时对以上各组使用Annexin V-FITC/PI复染试剂及流式细胞技术(FCM)检测细胞凋亡。并采用Western Blot法检测凋亡蛋白Bcl-2和Bax,检测外泌体对PC9细胞凋亡的影响。结果1吉非替尼合并顺铂治疗产生拮抗效应采用CCK-8法,检测吉非替尼和/或顺铂对PC9细胞增殖力的影响,结果显示相比各浓度单药吉非替尼(0.4-2μΜ)治疗,联合顺铂治疗的吉非替尼抗增殖效应仅稍微增加。同时计算两药在PC9及A549细胞中的CDI值。结果提示在吉非替尼和顺铂联用于EGFR突变及野生型NSCLC PC9细胞株时均存在相互拮抗作用。2 Exo-GF降低顺铂的抗肿瘤活性研究吉非替尼和顺铂间的拮抗效应由Exo-GF还是Exo-DDP介导。收集外泌体,加入一定浓度顺铂或吉非替尼共同作用于PC9细胞。CCK-8结果显示Exo-GF可剂量依赖性对抗顺铂的抗肿瘤效应,然而Exo-Con对顺铂诱导的增殖抑制无显著影响,同时Exo-DDP对吉非替尼几乎无影响。采用CCK-8法检测外泌体抑制剂GW4869预处理后,吉非替尼和/或顺铂作用于PC9细胞的抑制率,结果显示加入GW4869后,单药吉非替尼和顺铂对PC9细胞抑制率仅有轻微增加。但吉非替尼和顺铂联合组使用GW4869预处理后抑制率显著上升,同时两药CDI值在加入GW4869后显著下降,提示抑制Exo-GF分泌可逆转吉非替尼和顺铂间的拮抗作用。3 Exo-GF上调顺铂诱导的自噬水平采用Western blot检测Exo-Con,Exo-GF及Exo-DDP作用于PC9细胞的LC3及p62表达。与对照未处理PC9细胞相比,Exo-Con,Exo-GF及Exo-DDP均可显著上调自噬活性。相比单药顺铂组,Exo-GF预处理顺铂组的自噬显著增强。但Exo-DDP预处理几乎对吉非替尼诱导的自噬无影响。4 Exo-GF下调顺铂诱导的细胞凋亡采用流式细胞技术(Flow Cytometry,FCM)检测Exo-GF对于顺铂诱导的PC9细胞凋亡的影响,结果显示相比使用Exo-Con预处理的顺铂组及顺铂单药组,Exo-GF预处理加顺铂单药组的凋亡细胞显著减少。同CCK-8实验结果一致,Exo-DDP对吉非替尼诱导凋亡无明显影响。进一步采用Western Blot检测上述处理各组PC9细胞Bcl-2及Bax的蛋白表达水平。与顺铂组或Exo-Con预处理加顺铂组相比,Exo-GF预处理加顺铂组PC9细胞Bcl-2表达显著上调伴Bax下调。相比单纯顺铂组,Exo-GF预处理加顺铂组Bcl-2/Bax比值显著增高。但与单纯吉非替尼组相比,Exo-DDP预处理加吉非替尼组PC9细胞凋亡水平无显著差异。Exo-GF可显著下调顺铂诱导的细胞凋亡。结论1.吉非替尼联合顺铂作用于NSCLC细胞可产生拮抗效应;2.吉非替尼通过外泌体上调自噬拮抗顺铂诱导的NSCLC细胞凋亡;3.抑制外泌体分泌可通过抑制自噬逆转吉非替尼和顺铂间的拮抗效应。
[Abstract]:Background and purpose: bronchial lung cancer is the most common respiratory system tumor, with high morbidity and mortality worldwide. Non small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for most of the incidence of bronchial lung cancer, and the operation is still the main means to treat NSCLC. The rate of hair is about 40%., so for patients with recurrent NSCLC and late NSCLC with no indication of operation, systemic chemotherapy and targeted therapy are still the main treatment methods. However, the common NSCLC first-line chemotherapy is still not ideal and the effective rate is only about 30%. Therefore, it is necessary to find new methods and Strategies of valuable treatment. The combination of targeted therapy and chemotherapy has become an important topic at home and abroad. Platinum group chemotherapy is still the standard first-line chemotherapy for advanced NSCLC. The first generation platinum group cisplatin is the cornerstone of NSCLC chemotherapy. However, single chemotherapy can not achieve the ideal therapeutic effect with the biological system. Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor, which is the first representative of epidermal growth factor receptor (EGFR), is the first representative of the tyrosine kinase inhibitor, which is the first representative of the small molecule of casein. Acid kinase inhibitors, which are currently related to large three phase multicenter clinical validation of NSCLC first-line treatment, gefitinib has a distinct progression free survival (PFS) advantage for Asian, adenocarcinoma and EGFR mutations in patients with Asian, adenocarcinoma and EGFR mutations. However, in connection with chemotherapy, four large multicenter patients can be used in combination with chemotherapy. The bed test showed that the epidermal growth factor receptor tyrosine kinase inhibitor (Tyrosine-Kinase Inhibitor TKI), such as gefitinib and erlotinib, had no significant survival benefit for the total survival period of patients with advanced non-small cell lung cancer (Overall Survival, OS) when combined with chemotherapeutic drugs. There may be antagonism when combined with drugs. Although clinical trials have shown that the combination of cisplatin and gefitinib is antagonistic, the mechanism that causes this antagonism is still not very clear. Clarifying the mechanism that leads to antagonism can provide new ideas for clinical treatment. This topic is intended to be from two angles of autophagy and exocytosis. The effect of gefitinib and cisplatin on the therapeutic effect of cisplatin was revealed. The study of the effect and mechanism of gefitinib on the effect of autophagy induced autophagy in non small cell lung cancer cells (1) using gefitinib combined with different concentrations of cisplatin in human NSCLC cell line PC9, the MTT assay was used to determine the effect of gefitinib The effect of different concentrations of cisplatin on the proliferation of PC9 cells and the interaction index between gefitinib and cisplatin at different concentrations to analyze the effect of gefitinib on different concentrations of cisplatin by.Western-Blot method analysis gefitinib and cisplatin combined with cisplatin and the action of cisplatin alone on PC9 cell autophagic protein LC3- II and P62 Change and electron microscopy to observe the changes in the number of autophagic corpuscles in these two groups, and then understand the effect of gefitinib on the autophagy induced autophagy. (2) to further evaluate autophagy, the use of chloroquine (CQ), a lysosome inhibitor (CQ), which is a lysosome inhibitor, is used to pretreat cells for 1 hours and transmission electron microscopy. Transmission Electron Microscope (TEM) was used to confirm the degradation effect of CQ on autophagic lysosomes. Then we added gefitinib and / or cisplatin to compare the changes in LC3 expression between groups without CQ. Thus, the changes in the autophagy level of PC9 cells were determined by gefitinib and / or cisplatin, rather than autophagic degradation defects. (3) observation of inhibition (3) The effect of autophagy on the antagonism of gefitinib and cisplatin, we used MTT to detect PC9 cells in gefitinib and cisplatin group after CQ treatment, and calculated the CDI according to the measured OD value. (4) the study of Annexin V-FITC/PI flow cytometry was used to investigate the role of autophagy in the process of cisplatin resistance. The apoptosis rate of each cell after treatment was described, and the apoptosis protein Bcl-2 and Bax of the corresponding group of PC9 cells were extracted to understand the effect of the autophagy level on the apoptosis of PC9 cells. At the same time, we used the flow cytometry (Flow cytometry, FCM) to carry out the cell cycle analysis to understand the effect of autophagy on the cycle of PC9 cells. Results 1 combined with gefitinib. The antagonistic effect of cisplatin (1) was used to detect the inhibitory effect of gefitinib and cisplatin on the proliferation of PC9 cells by MTT. The results showed that the inhibition of gefitinib and cisplatin on NSCLC cells was time and concentration dependent. (2) the drug interaction coefficient (Coefficient of Drug Interaction, CDI) was used to evaluate the properties of the drug. The CDI value of gefitinib and cisplatin at different concentrations was 1.19 + 0.1 (1.07-1.35), suggesting the existence of the antagonistic effect of.2 gefitinib and cisplatin induced autophagy (1) using Western Blot to observe gefitinib and / or cisplatin. The expression of autophagic markers LC3 and p62 after treatment showed that gefitinib and cisplatin could induce the up-regulation of LC3-II transformation compared with the control group, while gefitinib and cisplatin were significantly increased in LC3-II transformation compared with the single gefitinib, but no statistical difference was found. Both gefitinib and / or cisplatin were significantly different. Lower p62 level. Compared with single drug stimulation, gefitinib and cisplatin resulted in a more significant downregulation of p62 protein, suggesting that gefitinib and cisplatin could induce higher level autophagy relative to the single drug. (2) a transmission electron microscope (Transmission electron microscopy, TEM) was used to observe the autophagic corpuscle of PC9 cells treated with gefitinib and / or cisplatin. The results showed that the number of autophagic corpuscles in gefitinib combined with cisplatin was increased in comparison with the single drug treatment group, and a higher level of autophagy could be induced by gefitinib and cisplatin relative to the single drug. (3) the Western Blot method was used to observe the autophagic marker LC of PC9 cells treated with gefitinib and / or cisplatin. 3 and p62 expression. The results suggest that gefitinib and / or cisplatin can induce autophagy in PC9 cells, instead of lysosomal degradation induced by.3 inhibition of autophagy against gefitinib and cisplatin (1) changes in the autophagic lysosomes of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning. The results show that the changes in the autophagy of the autophagic lysosomes after the treatment of gefitinib and / or cisplatin Compared with gefitinib and / or cisplatin group, autophagic lysosomes increased significantly after adding CQ, suggesting that CQ could inhibit the degradation of autophagic lysosomes during autophagy. (2) the proliferation of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning was detected by MTT. The results suggested that gefitinib and cisplatin were given to CQ pretreatment than in the CQ group. The synergism of gefitinib and cisplatin was increased by CQ pretreatment. (3) compared with Annexin V-FITC/PI apoptosis, CQ preconditioning could increase the proportion of apoptotic cells in each group of PC9 cells to increase the ratio of apoptotic cells, regardless of the combination of single or two drugs. Autophagy plays an important role in the process of apoptosis,.4 inhibits autophagy and promotes apoptosis (1) using flow cytometry (Flow Cytometry, FCM) to analyze the cycle of PC9 cells in each group. The results suggest that gefitinib and cisplatin can lead to the stagnation of G1 phase, however, the combined use of gefitinib and cisplatin only slightly increases the proportion of PC9 cells in G1 phase. To further confirm the relationship between autophagy and cell cycle stagnation, we pretreated gefitinib and / or cisplatin CQ (5 g/ml) for 1 hours. The results showed that the inhibition of autophagy by CQ could not reverse the cell cycle stagnation induced by gefitinib and / or cisplatin. (2) the use of Western Blot to detect CQ preconditioning could affect the apoptotic protein and resistance of PC9 cells in each group. The expression level of Bcl-2 (classic anti apoptotic family protein) and Bax (pro apoptotic protein) was significantly reduced and elevated after.CQ treatment. However, there was no significant difference in the proportion of Bax/Bcl-2 in gefitinib and / or cisplatin plus CQ preconditioning group (p=0.592,0.09 and 0.06 respectively). The results suggested CQ inhibition. Autophagy can promote the apoptosis of PC9 cells by up regulation of Bax and down regulation of Bcl-2. Study the effect of two gefitinib on the inhibition of autophagy against cisplatin induced non small cell lung cancer cell apoptosis (1) the separation of PC9 cells from normal control group or gefitinib (1 M) / cisplatin (7.5 mu M) by Exo Quick precipitate PC9 cell supernatant from gefitinib or cisplatin is extracted from the supernatant of PC9 cells, which is divided into Exo-Con (Exosomes derived from negative Control). The white detection kit carried out a standard quantitative determination of different extracellular body proteins. (2) a transmission electron microscope (TEM) was used to observe the formation of exosbodies and whether the extraction was successful. (3) the cell inhibition rate was detected by CCK-8 method. We used a certain concentration of exote for 24 hours to add 1 mu M to gefitinib or 7.5 M cisplatin. The cell proliferation activity was calculated by CCK-8 method and the cell inhibition rate was calculated by CCK-8 method. The drug interaction coefficient (Coefficient of Drug Interaction, CDI) between the two drugs was calculated. Then the exocrine inhibitor GW4869 was used in the drug treatment group to understand the inhibition of exocrine secretion to gefitinib or cisplatin. The influence of PC9 cell effect. (4) the changes of autophagic marker protein LC3 and p62 were detected by Western Blot method, and the correlation between autophagy and autophagy was detected by using both exocrine and drug incubating cells. The apoptosis of the cells was detected by Annexin V-FITC/PI reagents and flow cytometry (FCM). The effects of Bcl-2 and Bax on apoptosis of PC9 cells were detected by Western Blot. Results the antagonistic effect of 1 gefitinib combined with cisplatin was detected by CCK-8, and the effects of gefitinib and / or cisplatin on the proliferation of PC9 cells were detected. The results showed that compared with the concentration of the single drug gefitinib (0.4-2 mu), the combination of cisplatin and cisplatin was compared. The antiproliferative effect of gefitinib was only slightly increased. The CDI values of the two drugs in PC9 and A549 cells were calculated. The results suggest that the antagonistic effect of.2 Exo-GF on the antitumor activity of cisplatin and the antagonism of gefitinib and cisplatin in the combination of gefitinib and cisplatin in the EGFR mutation and the wild type NSCLC PC9 cell lines It should be mediated by Exo-GF or Exo-DDP. Collecting exosecrete, adding a certain concentration of cisplatin or gefitinib to the PC9 cell.CCK-8 results showed that Exo-GF was dose-dependent against the antitumor effect of cisplatin. However, Exo-Con had no significant effect on cisplatin induced proliferation inhibition, while Exo-DDP had little effect on gefitinib. CCK-8 The inhibition rates of gefitinib and / or cisplatin on PC9 cells were detected by GW4869 pretreatment with exo secreting inhibitors. The results showed that the inhibition rate of PC9 cells was only slightly increased after the addition of GW4869 to GW4869, but the inhibition rate of GW4869 in the combination group of gefitinib and cisplatin increased significantly, and the CDI value of the two drugs was at the same time. After adding GW4869, the inhibition of Exo-GF secretion could reverse the antagonism of gefitinib and cisplatin..3 Exo-GF up-regulated the level of autophagy induced by cisplatin. Western blot was used to detect Exo-Con, Exo-GF and Exo-DDP were used in LC3 and p62 expressions in PC9 cells. Compared with cisplatin group, Exo-GF pretreated cisplatin group significantly increased autophagy, but Exo-DDP pretreatment almost caused gefitinib induced autophagy.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2132875
[Abstract]:Background and purpose: bronchial lung cancer is the most common respiratory system tumor, with high morbidity and mortality worldwide. Non small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for most of the incidence of bronchial lung cancer, and the operation is still the main means to treat NSCLC. The rate of hair is about 40%., so for patients with recurrent NSCLC and late NSCLC with no indication of operation, systemic chemotherapy and targeted therapy are still the main treatment methods. However, the common NSCLC first-line chemotherapy is still not ideal and the effective rate is only about 30%. Therefore, it is necessary to find new methods and Strategies of valuable treatment. The combination of targeted therapy and chemotherapy has become an important topic at home and abroad. Platinum group chemotherapy is still the standard first-line chemotherapy for advanced NSCLC. The first generation platinum group cisplatin is the cornerstone of NSCLC chemotherapy. However, single chemotherapy can not achieve the ideal therapeutic effect with the biological system. Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor, which is the first representative of epidermal growth factor receptor (EGFR), is the first representative of the tyrosine kinase inhibitor, which is the first representative of the small molecule of casein. Acid kinase inhibitors, which are currently related to large three phase multicenter clinical validation of NSCLC first-line treatment, gefitinib has a distinct progression free survival (PFS) advantage for Asian, adenocarcinoma and EGFR mutations in patients with Asian, adenocarcinoma and EGFR mutations. However, in connection with chemotherapy, four large multicenter patients can be used in combination with chemotherapy. The bed test showed that the epidermal growth factor receptor tyrosine kinase inhibitor (Tyrosine-Kinase Inhibitor TKI), such as gefitinib and erlotinib, had no significant survival benefit for the total survival period of patients with advanced non-small cell lung cancer (Overall Survival, OS) when combined with chemotherapeutic drugs. There may be antagonism when combined with drugs. Although clinical trials have shown that the combination of cisplatin and gefitinib is antagonistic, the mechanism that causes this antagonism is still not very clear. Clarifying the mechanism that leads to antagonism can provide new ideas for clinical treatment. This topic is intended to be from two angles of autophagy and exocytosis. The effect of gefitinib and cisplatin on the therapeutic effect of cisplatin was revealed. The study of the effect and mechanism of gefitinib on the effect of autophagy induced autophagy in non small cell lung cancer cells (1) using gefitinib combined with different concentrations of cisplatin in human NSCLC cell line PC9, the MTT assay was used to determine the effect of gefitinib The effect of different concentrations of cisplatin on the proliferation of PC9 cells and the interaction index between gefitinib and cisplatin at different concentrations to analyze the effect of gefitinib on different concentrations of cisplatin by.Western-Blot method analysis gefitinib and cisplatin combined with cisplatin and the action of cisplatin alone on PC9 cell autophagic protein LC3- II and P62 Change and electron microscopy to observe the changes in the number of autophagic corpuscles in these two groups, and then understand the effect of gefitinib on the autophagy induced autophagy. (2) to further evaluate autophagy, the use of chloroquine (CQ), a lysosome inhibitor (CQ), which is a lysosome inhibitor, is used to pretreat cells for 1 hours and transmission electron microscopy. Transmission Electron Microscope (TEM) was used to confirm the degradation effect of CQ on autophagic lysosomes. Then we added gefitinib and / or cisplatin to compare the changes in LC3 expression between groups without CQ. Thus, the changes in the autophagy level of PC9 cells were determined by gefitinib and / or cisplatin, rather than autophagic degradation defects. (3) observation of inhibition (3) The effect of autophagy on the antagonism of gefitinib and cisplatin, we used MTT to detect PC9 cells in gefitinib and cisplatin group after CQ treatment, and calculated the CDI according to the measured OD value. (4) the study of Annexin V-FITC/PI flow cytometry was used to investigate the role of autophagy in the process of cisplatin resistance. The apoptosis rate of each cell after treatment was described, and the apoptosis protein Bcl-2 and Bax of the corresponding group of PC9 cells were extracted to understand the effect of the autophagy level on the apoptosis of PC9 cells. At the same time, we used the flow cytometry (Flow cytometry, FCM) to carry out the cell cycle analysis to understand the effect of autophagy on the cycle of PC9 cells. Results 1 combined with gefitinib. The antagonistic effect of cisplatin (1) was used to detect the inhibitory effect of gefitinib and cisplatin on the proliferation of PC9 cells by MTT. The results showed that the inhibition of gefitinib and cisplatin on NSCLC cells was time and concentration dependent. (2) the drug interaction coefficient (Coefficient of Drug Interaction, CDI) was used to evaluate the properties of the drug. The CDI value of gefitinib and cisplatin at different concentrations was 1.19 + 0.1 (1.07-1.35), suggesting the existence of the antagonistic effect of.2 gefitinib and cisplatin induced autophagy (1) using Western Blot to observe gefitinib and / or cisplatin. The expression of autophagic markers LC3 and p62 after treatment showed that gefitinib and cisplatin could induce the up-regulation of LC3-II transformation compared with the control group, while gefitinib and cisplatin were significantly increased in LC3-II transformation compared with the single gefitinib, but no statistical difference was found. Both gefitinib and / or cisplatin were significantly different. Lower p62 level. Compared with single drug stimulation, gefitinib and cisplatin resulted in a more significant downregulation of p62 protein, suggesting that gefitinib and cisplatin could induce higher level autophagy relative to the single drug. (2) a transmission electron microscope (Transmission electron microscopy, TEM) was used to observe the autophagic corpuscle of PC9 cells treated with gefitinib and / or cisplatin. The results showed that the number of autophagic corpuscles in gefitinib combined with cisplatin was increased in comparison with the single drug treatment group, and a higher level of autophagy could be induced by gefitinib and cisplatin relative to the single drug. (3) the Western Blot method was used to observe the autophagic marker LC of PC9 cells treated with gefitinib and / or cisplatin. 3 and p62 expression. The results suggest that gefitinib and / or cisplatin can induce autophagy in PC9 cells, instead of lysosomal degradation induced by.3 inhibition of autophagy against gefitinib and cisplatin (1) changes in the autophagic lysosomes of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning. The results show that the changes in the autophagy of the autophagic lysosomes after the treatment of gefitinib and / or cisplatin Compared with gefitinib and / or cisplatin group, autophagic lysosomes increased significantly after adding CQ, suggesting that CQ could inhibit the degradation of autophagic lysosomes during autophagy. (2) the proliferation of PC9 cells treated with gefitinib and / or cisplatin after CQ preconditioning was detected by MTT. The results suggested that gefitinib and cisplatin were given to CQ pretreatment than in the CQ group. The synergism of gefitinib and cisplatin was increased by CQ pretreatment. (3) compared with Annexin V-FITC/PI apoptosis, CQ preconditioning could increase the proportion of apoptotic cells in each group of PC9 cells to increase the ratio of apoptotic cells, regardless of the combination of single or two drugs. Autophagy plays an important role in the process of apoptosis,.4 inhibits autophagy and promotes apoptosis (1) using flow cytometry (Flow Cytometry, FCM) to analyze the cycle of PC9 cells in each group. The results suggest that gefitinib and cisplatin can lead to the stagnation of G1 phase, however, the combined use of gefitinib and cisplatin only slightly increases the proportion of PC9 cells in G1 phase. To further confirm the relationship between autophagy and cell cycle stagnation, we pretreated gefitinib and / or cisplatin CQ (5 g/ml) for 1 hours. The results showed that the inhibition of autophagy by CQ could not reverse the cell cycle stagnation induced by gefitinib and / or cisplatin. (2) the use of Western Blot to detect CQ preconditioning could affect the apoptotic protein and resistance of PC9 cells in each group. The expression level of Bcl-2 (classic anti apoptotic family protein) and Bax (pro apoptotic protein) was significantly reduced and elevated after.CQ treatment. However, there was no significant difference in the proportion of Bax/Bcl-2 in gefitinib and / or cisplatin plus CQ preconditioning group (p=0.592,0.09 and 0.06 respectively). The results suggested CQ inhibition. Autophagy can promote the apoptosis of PC9 cells by up regulation of Bax and down regulation of Bcl-2. Study the effect of two gefitinib on the inhibition of autophagy against cisplatin induced non small cell lung cancer cell apoptosis (1) the separation of PC9 cells from normal control group or gefitinib (1 M) / cisplatin (7.5 mu M) by Exo Quick precipitate PC9 cell supernatant from gefitinib or cisplatin is extracted from the supernatant of PC9 cells, which is divided into Exo-Con (Exosomes derived from negative Control). The white detection kit carried out a standard quantitative determination of different extracellular body proteins. (2) a transmission electron microscope (TEM) was used to observe the formation of exosbodies and whether the extraction was successful. (3) the cell inhibition rate was detected by CCK-8 method. We used a certain concentration of exote for 24 hours to add 1 mu M to gefitinib or 7.5 M cisplatin. The cell proliferation activity was calculated by CCK-8 method and the cell inhibition rate was calculated by CCK-8 method. The drug interaction coefficient (Coefficient of Drug Interaction, CDI) between the two drugs was calculated. Then the exocrine inhibitor GW4869 was used in the drug treatment group to understand the inhibition of exocrine secretion to gefitinib or cisplatin. The influence of PC9 cell effect. (4) the changes of autophagic marker protein LC3 and p62 were detected by Western Blot method, and the correlation between autophagy and autophagy was detected by using both exocrine and drug incubating cells. The apoptosis of the cells was detected by Annexin V-FITC/PI reagents and flow cytometry (FCM). The effects of Bcl-2 and Bax on apoptosis of PC9 cells were detected by Western Blot. Results the antagonistic effect of 1 gefitinib combined with cisplatin was detected by CCK-8, and the effects of gefitinib and / or cisplatin on the proliferation of PC9 cells were detected. The results showed that compared with the concentration of the single drug gefitinib (0.4-2 mu), the combination of cisplatin and cisplatin was compared. The antiproliferative effect of gefitinib was only slightly increased. The CDI values of the two drugs in PC9 and A549 cells were calculated. The results suggest that the antagonistic effect of.2 Exo-GF on the antitumor activity of cisplatin and the antagonism of gefitinib and cisplatin in the combination of gefitinib and cisplatin in the EGFR mutation and the wild type NSCLC PC9 cell lines It should be mediated by Exo-GF or Exo-DDP. Collecting exosecrete, adding a certain concentration of cisplatin or gefitinib to the PC9 cell.CCK-8 results showed that Exo-GF was dose-dependent against the antitumor effect of cisplatin. However, Exo-Con had no significant effect on cisplatin induced proliferation inhibition, while Exo-DDP had little effect on gefitinib. CCK-8 The inhibition rates of gefitinib and / or cisplatin on PC9 cells were detected by GW4869 pretreatment with exo secreting inhibitors. The results showed that the inhibition rate of PC9 cells was only slightly increased after the addition of GW4869 to GW4869, but the inhibition rate of GW4869 in the combination group of gefitinib and cisplatin increased significantly, and the CDI value of the two drugs was at the same time. After adding GW4869, the inhibition of Exo-GF secretion could reverse the antagonism of gefitinib and cisplatin..3 Exo-GF up-regulated the level of autophagy induced by cisplatin. Western blot was used to detect Exo-Con, Exo-GF and Exo-DDP were used in LC3 and p62 expressions in PC9 cells. Compared with cisplatin group, Exo-GF pretreated cisplatin group significantly increased autophagy, but Exo-DDP pretreatment almost caused gefitinib induced autophagy.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
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