人肝细胞生长因子诱导非小细胞肺癌细胞对厄洛替尼耐药及机制研究

发布时间：2018-03-03 04:38

  本文选题：非小细胞肺癌　切入点：人肝细胞生长因子　出处：《延边大学》2017年博士论文　论文类型：学位论文

【摘要】：肺癌是常见恶性肿瘤,其发病率和死亡率逐年上升,近年来已成为我国第一大癌症。按照全国肿瘤登记机构2014年统计结果发现,在新发恶性肿瘤中,肺癌已占据病发率首位,占全部肿瘤的1/5左右,死亡率占恶性肿瘤死亡的24.87%。非小细胞肺癌(non-small cell lung cancer,NSCLC)在肺癌发病中占首位,约80～85%,但当患者发现病情时,多已处在晚期,已错失手术良机,所以NSCLC患者一般采取化疗,往往使用铂类药物为基础的二药联合应用。但临床上使用的化疗药常受到很大限制,存在生存期短、特异性小、副作用大、预后差等缺点。而靶向药物治疗为NSCLC患者带来了福音。表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibiter,EGFR-TKI)为一种通过与表皮生长因子受体(epidermal growth factor receptor-tyrosine,EGFR)酪氨酸激酶区(Tyr 激酶区)ATP结合位点有机结合的最为普遍应用于治疗NSCLC的靶向分子药物(如gefitinib,erlotinib等),该药物可抑制该酶的活化而相关信号传递受阻,使癌细胞增殖受抑制,开启细胞凋亡。EGFR高表达是应用EGFR-TKI的基础,TKI的疗效与EGFR突变紧密关联。研究发现对EGFR-TKI敏感的NSCLC患者一般均存在EGFR21、19、18外显子基因突变,但此类突变往往在女性、亚裔、非吸烟、腺癌NSCLC患者体内发生,患者瘤细胞带有EGFR激活突变时,EGFR-TKI治疗率可超过七成。但EGFR-TKI具有原发或获得耐药,导致药物的失效,治疗的失败,这种耐药原理尚未能解释清楚。EGFR二次突变(T790M)与MET基因扩增是当前EGFR-TKI获得性耐药的两大分子机理,其余可能性机理有蛋白酪氨酸磷酸酶相关基因缺失、胰岛素生长因子1受体高表达、肝细胞生长因子(hepatocyte growth factor,HGF)过量表达等。HGF与肿瘤侵袭紧密相关,为成纤维细胞的一种衍生物,NSCLC患者血清内其含量明显偏高。特异性受体c-Met与HGF结合而起到作用,异常的HGF/c-Met信号传导途径与肿瘤的生长、粘附、转移和凋亡等因素相关。HGF诱导NSCLC对EGFR-TKI耐药,可能与其受体c-Met激活有关。所以本项研究思路首先在体外探讨HGF诱导不同EGFR基因型NSCLC细胞对厄洛替尼耐药及耐药机制;随后建立裸鼠移植瘤模型,在体内探讨HGF诱导不同EGFR基因型NSCLC细胞对厄洛替尼耐药及耐药机制。因此,本研究主要分为以下两个部分:第一部分人肝细胞生长因子诱导非小细胞肺癌细胞对厄洛替尼耐药及机制的体外研究目的探讨肝细胞生长因子在体外是否诱导不同EGFR基因型非小细胞肺癌细胞对厄洛替尼的耐药及是否c-Met及下游信号通道蛋白参与体外HGF诱导不同EGFR基因型非小细胞肺癌细胞株对于厄洛替尼的耐药。方法用HGF、厄洛替尼单独或联合处理人NSCLC细胞株EGFR突变型PC9、PC9/R和EGFR野生型H292及A549,将实验分成四组:不加药对照组(C组)、HGF干预组(H组)、厄洛替尼干预组(E组)、HGF联合厄洛替尼干预组(HE组)。采用MTT法检测其对细胞增殖的影响,流式细胞术检测其对细胞周期和凋亡的影响,利用Western blotting检测其对细胞中c-Met、EGFR、ErbB3及其磷酸化蛋白表达的影响,利用慢病毒包装干扰RNA感染不同NSCLC细胞,收集感染的肺癌细胞,裂解细胞并提取总RNA,利用RT-PCR检测干扰后c-Met-mRNA及蛋白的表达。利用MTT法检测干扰c-Met后厄洛替尼对细胞增殖的影响,Western blotting方法检测厄洛替尼或/和HGF处理对c-Met-shRNA感染的NSCLC细胞中c-Met及其下游通道蛋白表达的影响。结果在正常情况下,PC9和A549细胞的c-Met及其磷酸化蛋白呈低表达或不表达,H292和PC9/R细胞的c-Met及其磷酸化蛋白均呈高表达。随厄洛替尼浓度增高,H292、PC9及A549细胞的生长抑制也增强,通过HGF诱导后,厄洛替尼抑制细胞的生长曲线显著往右移。H292、PC9及A549细胞的HE组凋亡率均显著低于E组(均P0.05)。当厄洛替尼作用于四种细胞时,相比于C组,E组G0/G1期比例显著上升,有统计学差异(P0.05)。相比于E组,HE组PC9、PC9/R细胞的G0/G1期比例无统计学上的差异(P0.05)。H组A549细胞的G0/G1期比例与C组比较显著降低,S期比例显著上升,差异存在统计学上的意义(P0.05);相比于E组,HE组A549细胞的G0/G1期比例显著降低,S期比例显著上升,差异存在统计学上的意义(P0.05)。相比于E组,HE组H292的G0/G1期比例显著降低,S期及G2/M期比例显著上升,差异存在统计学上的意义(P0.05)。HGF能激活PC9、H292、PC9/R、A549细胞中c-Met及其下游信号通道蛋白。相比于E组,HE组H292、PC9与A549细胞的p-Met、p-Akt、p-Stat3、p-Erk1/2蛋白表达量明显上升,在PC9/R细胞中无明显增高。病毒感染的PC9和H292细胞中c-Met-mRNA和c-Met蛋白的表达受到了抑制。HGF诱导病毒感染的PC9和H292细胞,其厄洛替尼药物浓度-细胞存活率曲线可恢复到原来水平。病毒感染的PC9和H292细胞,HGF刺激后,未见细胞中c-Met、Stat3、Akt、Erk1/2磷酸化蛋白的激活。病毒感染的PC9和H292中HE组与E组相比,Stat3、Akt、Erk1/2磷酸化蛋白均明显受到抑制。结论1.在体外HGF诱导不同EGFR基因型NSCLC细胞株对厄洛替尼耐药。2.活化的c-Met及其下游信号通道蛋白可能是HGF诱导不同EGFR基因型NSCLC细胞株对厄洛替尼耐药的一种重要机制。3.HGF/c-Met系统可作为治疗EGFR-TKI耐药的NSCLC的靶向位点,为临床提供EGFR-TKI联合c-Met抑制剂或HGF拮抗剂有效克服不同EGFR基因型NSCLC对EGFR-TKI耐药的依据。第二部分人肝细胞生长因子诱导非小细胞肺癌细胞对厄洛替尼耐药及机制的体内研究目的探讨肝细胞生长因子是否在体内诱导不同EGFR基因型非小细胞肺癌细胞对厄洛替尼的耐药及是否c-Met及下游通道蛋白在体内参与HGF诱导不同EGFR基因型非小细胞肺癌细胞株对厄洛替尼耐药。方法选取人NSCLC细胞株EGFR突变型PC9、EGFR野生型H292与MRC-5(人胚肺成纤维细胞),使用ELISA法检测H292、PC9和MRC-5细胞上清液中HGF浓度。用MRC-5细胞培养上清液诱导PC9、H292细胞,采用Western blotting法检测c-Met及下游通道蛋白表达情况。将56只雌性(SPF级BALB/c)裸鼠随机分为8组,每组7只。在PC9细胞诱导模型中,对照组(C组)和厄洛替尼处理组(E组)裸鼠皮下接种PC9细胞悬液,MRC-5诱导组(H组)、MRC-5和厄洛替尼处理组(HE组)裸鼠皮下接种PC9+MRC-5细胞悬液;当移植瘤直径达到4mm时,C组和H组用0.9%氯化钠溶液灌胃,E组和HE组用厄洛替尼灌胃,每周给药5天,连续4周。在H292细胞诱导模型分为C组、E组、H组和HE组,C组和E组裸鼠皮下接种H292细胞悬液,H组和HE组裸鼠皮下接种H292+MRC-5细胞悬液;模型建立后灌胃方式亦同上。每3～4天测量移植瘤体积,给药四周结束后处死裸鼠,比较PC9、H292细胞诱导模型中各组移植瘤重量,计算其相应抑瘤率。使用免疫组化法对实验裸鼠移植瘤中c-Met及下游通道蛋白的表达状况进行观察。结果在H292与PC9细胞培养上清液中均没有发现HGF,在MRC-5培养上清液中HGF浓度是(1262.07± 89.78)pg/mL。Western blotting检测结果示MRC-5细胞培养上清液中HGF能刺激PC9、H292中p-Met、p-Akt、p-Stat3、p-Erk1/2的表达活性。PC9细胞诱导模型中:4组第4、7天移植瘤体积比较,差异无统计学意义(P0.05);4组第11、14、18、21、25天移植瘤体积比较,差异有统计学意义(P0.05);其中E组第11、14、18、21、25天移植瘤体积均小于C组,差异有统计学意义(P0.05);HE组第18、21、25天移植瘤体积小于H组,差异有统计学意义(P0.05);HE组第11、14、18、21、25天移植瘤体积均大于E组,差异有统计学意义(P0.05)。H292细胞诱导模型中:4组第4、7天移植瘤体积比较,差异无统计学意义(P0.05);4组第11、14、18、21、25天移植瘤体积比较,差异有统计学意义(P0.05);其中E组第11、14、18、21、25天移植瘤体积均小于C组,差异有统计学意义(P0.05);HE组第11、14、18、21、25天移植瘤体积小于H组,差异有统计学意义(P0.05);HE组第11、14、18、21、25天移植瘤体积均大于E组,差异有统计学意义(P0.05)。PC9细胞诱导模型中:C组、H组、E组、HE 组移植瘤重量分别为(680.31±373.38)、(804.84±492.36)、(108.89±83.76)、(361.19±172.08)mg。H292 细胞诱导模型中:C 组、H 组、E 组、HE 组移植瘤重量分别为(920.43±121.72)、(979.14±175.65)、(389.29±109.40)、(564.00±176.08)mg。厄洛替尼对PC9细胞E组、HE组抑瘤率分别为83.99%和46.91%,对H292细胞E组、HE组抑瘤率分别为57.71%和38.72%。PC9细胞诱导模型中:E组移植瘤重量小于C组(P0.05);HE组移植瘤重量小于H组,大于E组(P0.05)。H292细胞诱导模型中:E组移植瘤重量小于C组(P0.05);HE组移植瘤重量小于H组,大于E组(P0.05)。免疫组化结果示c-Met、p-Met分别定位于细胞膜和细胞质。在PC9、H292细胞诱导模型中:C组、H组、E组、HE组c-Met表达水平比较,差异都不存在统计学上的意义(P0.05);H组、HE组p-Met表达水平均比C、E组升高(P0.05)。Stat3定位于细胞质,p-Stat3定位于细胞核。在PC9、H292细胞诱导模型中:C组、H组、E组、HE组Stat3表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Stat3表达水平分别高于C组、E组(P0.05)。Akt、p-Akt均定位于细胞质。在PC9、H292细胞诱导模型中:C组、H组、E组、HE组Akt表达水平比较,差异都不存在统计学上的意义(P0.05);H组、HE组p-Akt表达水平均比C组、E组高(P0.05)。Erk1/2定位于细胞质,p-Erk1/2定位于细胞核。在PC9、H292细胞诱导模型中:C组、H组、E组、HE组Erk1/2表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Erk1/2表达水平分别高于C组、E组(P0.05)。结论1.在体内HGF诱导不同EGFR基因型NSCLC细胞株对厄洛替尼产生耐药。2.活化的c-Met及其下游信号通道蛋白可能是在体内HGF诱导不同EGFR基因型NSCLC细胞株对厄洛替尼耐药的一种重要机制。3.HGF/c-Met系统是NSCLC对EGFR-TKI耐药的另一个重要途径,为临床提供EGFR-TKI联合c-Met抑制剂或HGF拮抗剂有效克服不同EGFR基因型NSCLC对EGFR-TKI耐药的依据。
[Abstract]:Lung cancer is a common malignant tumor, its incidence and mortality increased year by year, in recent years has become the first major cancer in China. According to the National Cancer Registry statistics agency on 2014, in the new primary malignant tumors, lung cancer incidence has occupied the first place, the total tumor 1/5, mortality rate of cancer deaths 24.87%. small cell lung cancer (non-small cell lung cancer, NSCLC) occupied the first place in the pathogenesis of lung cancer, about 80 ~ 85%, but when the patient found the disease, has been in the late stage, already miss the opportunity to surgery, so most of the NSCLC patients with chemotherapy, often using platinum based chemotherapy. But the combination of the two drugs in clinic use often is very limited, there are short survival, specific small, side effects, disadvantages of poor prognosis. And targeted therapy for NSCLC patients to bring the gospel. The epidermal growth factor receptor tyrosine kinase inhibitor (epidermal growth factor receptor-tyrosine kinase agent inhibiter, EGFR-TKI) is a kind of with epidermal growth factor receptor (epidermal growth factor receptor-tyrosine, EGFR) tyrosine kinase (Tyr kinase) ATP organic binding sites are most common in the treatment of NSCLC targeting molecules (such as gefitinib, erlotinib etc.), activation the related signal transduction blocked drugs can inhibit the enzyme, the proliferation of cancer cells by inhibiting apoptosis, open the high expression of.EGFR is the basis of the application of EGFR-TKI effect and EGFR TKI mutation is closely related to EGFR-TKI. The study found that patients with NSCLC are sensitive to the exon of EGFR21,19,18 gene mutations, but these mutations often in women, Asian, non smoking, adenocarcinoma in patients with NSCLC, patients with EGFR activating mutations in tumor cells, EGFR-TKI treatment rate can be more than 70%. But EGFR-TKI Has a primary or acquired resistance, leading to the drug failure, treatment failure, this principle is not resistant to explain.EGFR two mutation (T790M) and MET gene amplification is two EGFR-TKI the molecular mechanism of acquired resistance, the possible mechanism of deletion gene related protein tyrosine phosphatase, insulin-like growth factor expression 1 receptor, hepatocyte growth factor (hepatocyte growth, factor, HGF).HGF overexpression and tumor invasion are closely related, as a derivative of fiber cells, serum NSCLC content was significantly higher. Its specific receptor c-Met binding with HGF and play the role of HGF/c-Met signal transduction pathway and tumor abnormal growth.HGF, adhesion, metastasis and apoptosis related factors NSCLC induced resistance to EGFR-TKI, may be related to the activation of c-Met receptor. So this study ideas first in vitro study by HGF The EGFR gene of NSCLC cells to erlotinib resistance and resistance mechanisms; followed by the establishment of nude mice model in vivo, to investigate the HGF of erlotinib resistance and resistance mechanisms induced by different EGFR genotypes. NSCLC cells therefore, this research is mainly divided into the following two parts: the first part of human hepatocyte growth factor induced by non in vitro study of small cell lung cancer cells to erlotinib resistance and mechanism of hepatocyte growth factor in vitro induced by different EGFR genotypes of non small cell lung cancer cells to erlotinib resistance and whether c-Met and its downstream signal channel proteins induced by HGF in vitro in different genotypes of EGFR in non-small cell lung cancer cell lines for erlotinib resistance to imatinib. Methods HGF, erlotinib treatment alone or in combination with human NSCLC cell line EGFR mutant PC9, PC9/R and EGFR of wild type H292 and A549, were divided into four groups: without medicine The control group (C group), HGF group (group H), erlotinib intervention group (E group), HGF combined with erlotinib in the intervention group (HE group). To detect the effect on cell proliferation by MTT method to detect the effect on cell cycle and apoptosis by flow cytometry and its detection EGFR on c-Met cells by Western, blotting, ErbB3 and its effects on phosphorylation of protein expression by using lentiviral packaging interference RNA infection of different NSCLC cells collected from infected lung cancer cells, cell lysis and extraction of total RNA, using the c-Met-mRNA and protein expression of RT-PCR and c-Met. After detection of interference interference detection by MTT method after erlotinib for Nigeria effect on cell proliferation, Western blotting method for detection of erlotinib and / or HGF treatment effect on the expression of c-Met and its downstream channel c-Met-shRNA infected cells NSCLC protein. Results in the normal condition, c-Met and phosphorylation of protein PC9 and A549 cells Low or no expression, high c-Met expression and phosphorylation of H292 protein and PC9/R cells were H292. With erlotinib concentration, PC9, and A549 cell growth inhibition was enhanced by HGF after induced by erlotinib inhibits the cell growth curve was shifted to the right of.H292, PC9 and A549 cells HE the apoptosis rate of group were significantly lower than E group (P0.05). When erlotinib in four cells when compared to the C group, E group significantly increased the proportion of G0/G1, there were significant differences (P0.05). Compared with E group, HE PC9 group, no significant differences in the PC9/R G0/G1 phase cell percentage C G0/G1 (P0.05) and the proportion of group.H group of A549 cells decreased significantly, significantly increased S ratio, the differences were statistically significant (P0.05); compared with E group, HE group, A549 cell ratio of G0/G1 phase decreased significantly, significantly increased the proportion of S phase difference, statistically significance compared to E (P0.05). Group HE, group H292 G0/G1 ratio decreased significantly, S increased significantly and G2/M phase ratio, difference statistically significant (P0.05).HGF can activate PC9, H292, PC9/R, A549 cells c-Met and its downstream signaling pathway protein. Compared with E group, HE group, H292, PC9 and A549 cells p-Met p-Akt, p-Stat3, p-Erk1/2 protein expression was increased in PC9/R cells was not increased. The expression of c-Met-mRNA and c-Met proteins of the virus infected PC9 and H292 cells inhibited.HGF induced by viral infection of PC9 and H292 cells, the erlotinib drug concentration cell survival curve can be restored to its original level. The virus infected PC9 and H292 cells after HGF stimulation, c-Met, no Stat3 in cells, Akt, activation of the phosphorylation of Erk1/2 protein. The PC9 virus infection and H292 in HE group compared with E group, Stat3, Akt, Erk1/2 phosphorylation was significantly inhibited. Conclusion HG 1. in vitro F induced by different EGFR genotypes of NSCLC cell line c-Met and its downstream signal pathway of erlotinib resistant.2. activation may be induced by HGF in different genotypes of EGFR NSCLC cells to erlotinib for an important mechanism of.3.HGF/c-Met system and drug resistance can be used as the treatment of EGFR-TKI resistant NSCLC targets, EGFR-TKI combined with c-Met inhibitor or HGF antagonists overcome different EGFR genotype NSCLC of EGFR-TKI resistant basis for clinical. The second part of human hepatocyte growth factor induced by in vivo to non small cell lung cancer cells to erlotinib resistance and the mechanism of the growth factor in vivo induced by different EGFR genotypes of non small cell lung cancer cells to erlotinib resistance and whether the c-Met and downstream channel proteins participate in HGF induced by different EGFR genotypes in non-small cell lung cancer cell line of erlotinib for acquired liver cells 鑽，

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