本文選題：非小細(xì)胞肺癌　切入點(diǎn)：人肝細(xì)胞生長(zhǎng)因子　出處：《延邊大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：肺癌是常見(jiàn)惡性腫瘤,其發(fā)病率和死亡率逐年上升,近年來(lái)已成為我國(guó)第一大癌癥。按照全國(guó)腫瘤登記機(jī)構(gòu)2014年統(tǒng)計(jì)結(jié)果發(fā)現(xiàn),在新發(fā)惡性腫瘤中,肺癌已占據(jù)病發(fā)率首位,占全部腫瘤的1/5左右,死亡率占惡性腫瘤死亡的24.87%。非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)在肺癌發(fā)病中占首位,約80～85%,但當(dāng)患者發(fā)現(xiàn)病情時(shí),多已處在晚期,已錯(cuò)失手術(shù)良機(jī),所以NSCLC患者一般采取化療,往往使用鉑類藥物為基礎(chǔ)的二藥聯(lián)合應(yīng)用。但臨床上使用的化療藥常受到很大限制,存在生存期短、特異性小、副作用大、預(yù)后差等缺點(diǎn)。而靶向藥物治療為NSCLC患者帶來(lái)了福音。表皮生長(zhǎng)因子受體酪氨酸激酶抑制劑(epidermal growth factor receptor-tyrosine kinase inhibiter,EGFR-TKI)為一種通過(guò)與表皮生長(zhǎng)因子受體(epidermal growth factor receptor-tyrosine,EGFR)酪氨酸激酶區(qū)(Tyr 激酶區(qū))ATP結(jié)合位點(diǎn)有機(jī)結(jié)合的最為普遍應(yīng)用于治療NSCLC的靶向分子藥物(如gefitinib,erlotinib等),該藥物可抑制該酶的活化而相關(guān)信號(hào)傳遞受阻,使癌細(xì)胞增殖受抑制,開啟細(xì)胞凋亡。EGFR高表達(dá)是應(yīng)用EGFR-TKI的基礎(chǔ),TKI的療效與EGFR突變緊密關(guān)聯(lián)。研究發(fā)現(xiàn)對(duì)EGFR-TKI敏感的NSCLC患者一般均存在EGFR21、19、18外顯子基因突變,但此類突變往往在女性、亞裔、非吸煙、腺癌NSCLC患者體內(nèi)發(fā)生,患者瘤細(xì)胞帶有EGFR激活突變時(shí),EGFR-TKI治療率可超過(guò)七成。但EGFR-TKI具有原發(fā)或獲得耐藥,導(dǎo)致藥物的失效,治療的失敗,這種耐藥原理尚未能解釋清楚。EGFR二次突變(T790M)與MET基因擴(kuò)增是當(dāng)前EGFR-TKI獲得性耐藥的兩大分子機(jī)理,其余可能性機(jī)理有蛋白酪氨酸磷酸酶相關(guān)基因缺失、胰島素生長(zhǎng)因子1受體高表達(dá)、肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor,HGF)過(guò)量表達(dá)等。HGF與腫瘤侵襲緊密相關(guān),為成纖維細(xì)胞的一種衍生物,NSCLC患者血清內(nèi)其含量明顯偏高。特異性受體c-Met與HGF結(jié)合而起到作用,異常的HGF/c-Met信號(hào)傳導(dǎo)途徑與腫瘤的生長(zhǎng)、粘附、轉(zhuǎn)移和凋亡等因素相關(guān)。HGF誘導(dǎo)NSCLC對(duì)EGFR-TKI耐藥,可能與其受體c-Met激活有關(guān)。所以本項(xiàng)研究思路首先在體外探討HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞對(duì)厄洛替尼耐藥及耐藥機(jī)制;隨后建立裸鼠移植瘤模型,在體內(nèi)探討HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞對(duì)厄洛替尼耐藥及耐藥機(jī)制。因此,本研究主要分為以下兩個(gè)部分:第一部分人肝細(xì)胞生長(zhǎng)因子誘導(dǎo)非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼耐藥及機(jī)制的體外研究目的探討肝細(xì)胞生長(zhǎng)因子在體外是否誘導(dǎo)不同EGFR基因型非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼的耐藥及是否c-Met及下游信號(hào)通道蛋白參與體外HGF誘導(dǎo)不同EGFR基因型非小細(xì)胞肺癌細(xì)胞株對(duì)于厄洛替尼的耐藥。方法用HGF、厄洛替尼單獨(dú)或聯(lián)合處理人NSCLC細(xì)胞株EGFR突變型PC9、PC9/R和EGFR野生型H292及A549,將實(shí)驗(yàn)分成四組:不加藥對(duì)照組(C組)、HGF干預(yù)組(H組)、厄洛替尼干預(yù)組(E組)、HGF聯(lián)合厄洛替尼干預(yù)組(HE組)。采用MTT法檢測(cè)其對(duì)細(xì)胞增殖的影響,流式細(xì)胞術(shù)檢測(cè)其對(duì)細(xì)胞周期和凋亡的影響,利用Western blotting檢測(cè)其對(duì)細(xì)胞中c-Met、EGFR、ErbB3及其磷酸化蛋白表達(dá)的影響,利用慢病毒包裝干擾RNA感染不同NSCLC細(xì)胞,收集感染的肺癌細(xì)胞,裂解細(xì)胞并提取總RNA,利用RT-PCR檢測(cè)干擾后c-Met-mRNA及蛋白的表達(dá)。利用MTT法檢測(cè)干擾c-Met后厄洛替尼對(duì)細(xì)胞增殖的影響,Western blotting方法檢測(cè)厄洛替尼或/和HGF處理對(duì)c-Met-shRNA感染的NSCLC細(xì)胞中c-Met及其下游通道蛋白表達(dá)的影響。結(jié)果在正常情況下,PC9和A549細(xì)胞的c-Met及其磷酸化蛋白呈低表達(dá)或不表達(dá),H292和PC9/R細(xì)胞的c-Met及其磷酸化蛋白均呈高表達(dá)。隨厄洛替尼濃度增高,H292、PC9及A549細(xì)胞的生長(zhǎng)抑制也增強(qiáng),通過(guò)HGF誘導(dǎo)后,厄洛替尼抑制細(xì)胞的生長(zhǎng)曲線顯著往右移。H292、PC9及A549細(xì)胞的HE組凋亡率均顯著低于E組(均P0.05)。當(dāng)厄洛替尼作用于四種細(xì)胞時(shí),相比于C組,E組G0/G1期比例顯著上升,有統(tǒng)計(jì)學(xué)差異(P0.05)。相比于E組,HE組PC9、PC9/R細(xì)胞的G0/G1期比例無(wú)統(tǒng)計(jì)學(xué)上的差異(P0.05)。H組A549細(xì)胞的G0/G1期比例與C組比較顯著降低,S期比例顯著上升,差異存在統(tǒng)計(jì)學(xué)上的意義(P0.05);相比于E組,HE組A549細(xì)胞的G0/G1期比例顯著降低,S期比例顯著上升,差異存在統(tǒng)計(jì)學(xué)上的意義(P0.05)。相比于E組,HE組H292的G0/G1期比例顯著降低,S期及G2/M期比例顯著上升,差異存在統(tǒng)計(jì)學(xué)上的意義(P0.05)。HGF能激活PC9、H292、PC9/R、A549細(xì)胞中c-Met及其下游信號(hào)通道蛋白。相比于E組,HE組H292、PC9與A549細(xì)胞的p-Met、p-Akt、p-Stat3、p-Erk1/2蛋白表達(dá)量明顯上升,在PC9/R細(xì)胞中無(wú)明顯增高。病毒感染的PC9和H292細(xì)胞中c-Met-mRNA和c-Met蛋白的表達(dá)受到了抑制。HGF誘導(dǎo)病毒感染的PC9和H292細(xì)胞,其厄洛替尼藥物濃度-細(xì)胞存活率曲線可恢復(fù)到原來(lái)水平。病毒感染的PC9和H292細(xì)胞,HGF刺激后,未見(jiàn)細(xì)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組與E組相比,Stat3、Akt、Erk1/2磷酸化蛋白均明顯受到抑制。結(jié)論1.在體外HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞株對(duì)厄洛替尼耐藥。2.活化的c-Met及其下游信號(hào)通道蛋白可能是HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞株對(duì)厄洛替尼耐藥的一種重要機(jī)制。3.HGF/c-Met系統(tǒng)可作為治療EGFR-TKI耐藥的NSCLC的靶向位點(diǎn),為臨床提供EGFR-TKI聯(lián)合c-Met抑制劑或HGF拮抗劑有效克服不同EGFR基因型NSCLC對(duì)EGFR-TKI耐藥的依據(jù)。第二部分人肝細(xì)胞生長(zhǎng)因子誘導(dǎo)非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼耐藥及機(jī)制的體內(nèi)研究目的探討肝細(xì)胞生長(zhǎng)因子是否在體內(nèi)誘導(dǎo)不同EGFR基因型非小細(xì)胞肺癌細(xì)胞對(duì)厄洛替尼的耐藥及是否c-Met及下游通道蛋白在體內(nèi)參與HGF誘導(dǎo)不同EGFR基因型非小細(xì)胞肺癌細(xì)胞株對(duì)厄洛替尼耐藥。方法選取人NSCLC細(xì)胞株EGFR突變型PC9、EGFR野生型H292與MRC-5(人胚肺成纖維細(xì)胞),使用ELISA法檢測(cè)H292、PC9和MRC-5細(xì)胞上清液中HGF濃度。用MRC-5細(xì)胞培養(yǎng)上清液誘導(dǎo)PC9、H292細(xì)胞,采用Western blotting法檢測(cè)c-Met及下游通道蛋白表達(dá)情況。將56只雌性(SPF級(jí)BALB/c)裸鼠隨機(jī)分為8組,每組7只。在PC9細(xì)胞誘導(dǎo)模型中,對(duì)照組(C組)和厄洛替尼處理組(E組)裸鼠皮下接種PC9細(xì)胞懸液,MRC-5誘導(dǎo)組(H組)、MRC-5和厄洛替尼處理組(HE組)裸鼠皮下接種PC9+MRC-5細(xì)胞懸液;當(dāng)移植瘤直徑達(dá)到4mm時(shí),C組和H組用0.9%氯化鈉溶液灌胃,E組和HE組用厄洛替尼灌胃,每周給藥5天,連續(xù)4周。在H292細(xì)胞誘導(dǎo)模型分為C組、E組、H組和HE組,C組和E組裸鼠皮下接種H292細(xì)胞懸液,H組和HE組裸鼠皮下接種H292+MRC-5細(xì)胞懸液;模型建立后灌胃方式亦同上。每3～4天測(cè)量移植瘤體積,給藥四周結(jié)束后處死裸鼠,比較PC9、H292細(xì)胞誘導(dǎo)模型中各組移植瘤重量,計(jì)算其相應(yīng)抑瘤率。使用免疫組化法對(duì)實(shí)驗(yàn)裸鼠移植瘤中c-Met及下游通道蛋白的表達(dá)狀況進(jìn)行觀察。結(jié)果在H292與PC9細(xì)胞培養(yǎng)上清液中均沒(méi)有發(fā)現(xiàn)HGF,在MRC-5培養(yǎng)上清液中HGF濃度是(1262.07± 89.78)pg/mL。Western blotting檢測(cè)結(jié)果示MRC-5細(xì)胞培養(yǎng)上清液中HGF能刺激PC9、H292中p-Met、p-Akt、p-Stat3、p-Erk1/2的表達(dá)活性。PC9細(xì)胞誘導(dǎo)模型中:4組第4、7天移植瘤體積比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);4組第11、14、18、21、25天移植瘤體積比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);其中E組第11、14、18、21、25天移植瘤體積均小于C組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);HE組第18、21、25天移植瘤體積小于H組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);HE組第11、14、18、21、25天移植瘤體積均大于E組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。H292細(xì)胞誘導(dǎo)模型中:4組第4、7天移植瘤體積比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);4組第11、14、18、21、25天移植瘤體積比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);其中E組第11、14、18、21、25天移植瘤體積均小于C組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);HE組第11、14、18、21、25天移植瘤體積小于H組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);HE組第11、14、18、21、25天移植瘤體積均大于E組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。PC9細(xì)胞誘導(dǎo)模型中:C組、H組、E組、HE 組移植瘤重量分別為(680.31±373.38)、(804.84±492.36)、(108.89±83.76)、(361.19±172.08)mg。H292 細(xì)胞誘導(dǎo)模型中:C 組、H 組、E 組、HE 組移植瘤重量分別為(920.43±121.72)、(979.14±175.65)、(389.29±109.40)、(564.00±176.08)mg。厄洛替尼對(duì)PC9細(xì)胞E組、HE組抑瘤率分別為83.99%和46.91%,對(duì)H292細(xì)胞E組、HE組抑瘤率分別為57.71%和38.72%。PC9細(xì)胞誘導(dǎo)模型中:E組移植瘤重量小于C組(P0.05);HE組移植瘤重量小于H組,大于E組(P0.05)。H292細(xì)胞誘導(dǎo)模型中:E組移植瘤重量小于C組(P0.05);HE組移植瘤重量小于H組,大于E組(P0.05)。免疫組化結(jié)果示c-Met、p-Met分別定位于細(xì)胞膜和細(xì)胞質(zhì)。在PC9、H292細(xì)胞誘導(dǎo)模型中:C組、H組、E組、HE組c-Met表達(dá)水平比較,差異都不存在統(tǒng)計(jì)學(xué)上的意義(P0.05);H組、HE組p-Met表達(dá)水平均比C、E組升高(P0.05)。Stat3定位于細(xì)胞質(zhì),p-Stat3定位于細(xì)胞核。在PC9、H292細(xì)胞誘導(dǎo)模型中:C組、H組、E組、HE組Stat3表達(dá)水平比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);H組、HE組p-Stat3表達(dá)水平分別高于C組、E組(P0.05)。Akt、p-Akt均定位于細(xì)胞質(zhì)。在PC9、H292細(xì)胞誘導(dǎo)模型中:C組、H組、E組、HE組Akt表達(dá)水平比較,差異都不存在統(tǒng)計(jì)學(xué)上的意義(P0.05);H組、HE組p-Akt表達(dá)水平均比C組、E組高(P0.05)。Erk1/2定位于細(xì)胞質(zhì),p-Erk1/2定位于細(xì)胞核。在PC9、H292細(xì)胞誘導(dǎo)模型中:C組、H組、E組、HE組Erk1/2表達(dá)水平比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);H組、HE組p-Erk1/2表達(dá)水平分別高于C組、E組(P0.05)。結(jié)論1.在體內(nèi)HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞株對(duì)厄洛替尼產(chǎn)生耐藥。2.活化的c-Met及其下游信號(hào)通道蛋白可能是在體內(nèi)HGF誘導(dǎo)不同EGFR基因型NSCLC細(xì)胞株對(duì)厄洛替尼耐藥的一種重要機(jī)制。3.HGF/c-Met系統(tǒng)是NSCLC對(duì)EGFR-TKI耐藥的另一個(gè)重要途徑,為臨床提供EGFR-TKI聯(lián)合c-Met抑制劑或HGF拮抗劑有效克服不同EGFR基因型NSCLC對(duì)EGFR-TKI耐藥的依據(jù)。
[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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