本文關(guān)鍵詞：Filamin A影響EGFR T790M突變肺腺癌H1975細(xì)胞對三代EGFR-TKIs敏感性的研究　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 非小細(xì)胞肺癌 細(xì)絲蛋白A 表皮生長因子受體 酪氨酸磷酸化 AZD9291

【摘要】：目的:肺癌是我國腫瘤相關(guān)死亡的主要病因,在男性惡性腫瘤中其發(fā)病率及死亡率最高,女性發(fā)病率居第二位,但其死亡率仍為女性腫瘤死亡的首位。非小細(xì)胞肺癌(NSCLC)在肺癌中最常見,約占85%。針對其治療方法主要有手術(shù)、化療、放療、分子靶向治療以及免疫治療等。近年來化療及手術(shù)治療的水平雖有所提高,但其5年生存率僅達(dá)15%。分子靶向治療開啟了肺癌治療的新紀(jì)元,即以腫瘤原癌基因或其信號通路分子為治療靶點(diǎn),有針對性對腫瘤細(xì)胞發(fā)揮抑制作用,降低對正常細(xì)胞的損傷,為腫瘤患者帶來了福音。表皮生長因子受體(EGFR)是一類具有酪氨酸激酶活性的受體,屬于Erb B受體家族的一員。EGFR胞外區(qū)與配體如表皮生長因子(EGF)結(jié)合形成同源二聚體,在酪氨酸殘基上發(fā)生磷酸化,進(jìn)一步活化下游信號通路,如MAPK/ERK、PI3K/AKT、JAK/STAT等,參與調(diào)控腫瘤細(xì)胞增殖、侵襲、轉(zhuǎn)移及血管生成等。研究證實(shí),EGFR活化突變與NSCLC的進(jìn)展和預(yù)后不良相關(guān)。最常見的EGFR激活突變以19外顯子缺失或21外顯子L858R替代突變的形式發(fā)生,在西方人群中大約10%-15%非小細(xì)胞肺癌的患者攜帶著EGFR激活突變,而在亞洲人群約占40%。針對其分子治療的靶向藥物已成功應(yīng)用于臨床,第一代EGFR酪氨酸激酶抑制劑(EGFR-TKIs),如吉非替尼和厄洛替尼廣泛用于晚期NSCLC患者的一線治療方案,對比以鉑類為主的雙藥化療能夠顯著延長中位無疾病進(jìn)展生存期(PFS),改善生活質(zhì)量。然而,大部分患者在應(yīng)用9-14個月治療后,往往將會面臨EGFR-TKIs耐藥。最常見的獲得性耐藥機(jī)制是T790M突變,增加了與ATP的親和力從而降低抑制劑療效。此外,還包括MET基因擴(kuò)增、小細(xì)胞肺癌轉(zhuǎn)化、EGFR突變基因丟失等。另外,約30%患者對EGFR-TKIs表現(xiàn)為原發(fā)性耐藥。針對這一耐藥問題,后續(xù)又研發(fā)出了第二代阿法替尼及第三代奧希替尼(AZD9291),其中AZD9291是一個新的口服藥,有效的、選擇性的抑制EGFR敏感突變和T790M耐藥突變的不可逆抑制劑,對野生型EGFR效果差些。所以,我們進(jìn)一步來探討NSCLC患者對EGFR-TKIs的敏感性的問題,為后續(xù)治療提供一定的指導(dǎo)作用。細(xì)絲蛋白A(Filamin A,FLNa)是一種非肌性肌動蛋白結(jié)合蛋白,主要存在細(xì)胞漿中。FLNa包括一個位于氨基末端的肌動蛋白結(jié)合結(jié)構(gòu)域和一個由24個串聯(lián)重復(fù)序列組成的棒狀結(jié)構(gòu)域,多層β折疊的結(jié)構(gòu)在這個棒狀結(jié)構(gòu)域?yàn)榈鞍踪|(zhì)之間相互作用提供了界面。FLNa通過調(diào)節(jié)信號傳導(dǎo)分子可以改變致瘤微環(huán)境。目前,Zhu等研究發(fā)現(xiàn)減少FLNa表達(dá)量后能夠促進(jìn)肺癌PC-9細(xì)胞的增殖、遷移和侵襲;減少FLNa表達(dá)量也能導(dǎo)致肺癌PC-9細(xì)胞對第一代EGFR-TKIs敏感性下降。然而,患者經(jīng)過一段時間治療后往往出現(xiàn)耐藥問題,針對其最常見的機(jī)制T790M突變,研發(fā)出了第三代抑制劑AZD9291,其單苯胺基嘧啶化合物在結(jié)構(gòu)和藥理學(xué)上不同與其他幾代TKIs。為此我們了解一下藥物結(jié)構(gòu)不同的TKI與FLNa的相互作用,及細(xì)胞對藥物敏感性的影響。本研究擬用體外培養(yǎng)EGFR第21外顯子敏感突變和20外顯子T790M耐藥突變的NSCLC H1975細(xì)胞株,通過:采用質(zhì)粒轉(zhuǎn)染技術(shù),構(gòu)建沉默F(xiàn)LNa的H1975細(xì)胞株;應(yīng)用MTS法、平板克隆、劃痕修復(fù)試驗(yàn)及侵襲試驗(yàn)檢測穩(wěn)定轉(zhuǎn)染細(xì)胞株增殖、遷移和侵襲生物學(xué)行為的能力;應(yīng)用Western blot印跡檢測AZD9291對穩(wěn)定轉(zhuǎn)染細(xì)胞信號分子水平的影響,從而探討FLNa影響EGFR T790M突變肺腺癌H1975細(xì)胞對三代EGFR-TKIs的敏感性。方法:1 Western bolt檢測穩(wěn)定轉(zhuǎn)染細(xì)胞株中FLNa沉默效果分別將本實(shí)驗(yàn)室保存的肺腺癌H1975、H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于35mm培養(yǎng)皿,完全1640培養(yǎng)基培養(yǎng)至貼壁長滿。收集細(xì)胞并提取蛋白,應(yīng)用Western blot檢測FLNa的水平,以?-actin作為內(nèi)參蛋白。2 MTS比色分析法檢測兩組細(xì)胞增殖能力分別將H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于96孔板,培養(yǎng)24h,棄去上清液,換用含有不同濃度AZD9291(0、2、4、8、16μmol/L)的完全1640培養(yǎng)基繼續(xù)培養(yǎng)48h,棄去上清液加入MTS,測定各孔的吸光度(OD)值,并計(jì)算細(xì)胞生長抑制率和AZD9291的半數(shù)抑制濃度(IC50)。3平板克隆法檢測兩組細(xì)胞貼壁后克隆形成變化分別將H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于35mm培養(yǎng)皿,在有無(1μmol/L)AZD9291作用下培養(yǎng)2周,蘇木素染色,倒置顯微鏡下觀察并計(jì)數(shù)細(xì)胞克隆形成數(shù)量。4劃痕修復(fù)實(shí)驗(yàn)檢測兩組細(xì)胞遷移能力分別將H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于24孔板,培養(yǎng)24h,使用無菌移液器吸頭在單層細(xì)胞上均勻劃痕,兩種細(xì)胞分別用含有或不含有1μmol/L AZD9291的完全1640培養(yǎng)基繼續(xù)培養(yǎng),觀察到各組細(xì)胞遷移變化無明顯差異,故增加藥物濃度至2μmol/L,定時觀察并拍照記錄至劃痕愈合。測量寬度變化并計(jì)算細(xì)胞的遷移率。5侵襲試驗(yàn)檢測兩組細(xì)胞侵襲能力分別將H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于鋪有基質(zhì)膠的Transwell小室的上室,兩種細(xì)胞分別于含有或不含有1μmol/L AZD9291的無胎牛血清1640培養(yǎng)基中培養(yǎng),下室放入含10%胎牛血清的1640培養(yǎng)基,繼續(xù)培養(yǎng)24h后,固定,染色,光鏡拍照并計(jì)算穿膜細(xì)胞數(shù)。6 Western blot檢測兩組細(xì)胞信號通路分子活性變化分別將H1975/FLNa(KD)及H1975/ctrl細(xì)胞接種于35mm培養(yǎng)皿,完全1640培養(yǎng)基培養(yǎng)至貼壁長滿。予以1μmol/L AZD9291進(jìn)行干預(yù),分別于0h、2h、4h、8h后收細(xì)胞,提取總蛋白,Western blot檢測p-EGFR、EGFR、p-ERK、ERK、FLNa的蛋白水平,?-actin作為內(nèi)參蛋白。結(jié)果:1穩(wěn)定轉(zhuǎn)染H1975細(xì)胞中FLNa蛋白表達(dá)水平的變化Western blot檢測兩組細(xì)胞FLNa蛋白水平,掃描蛋白條帶并統(tǒng)計(jì)分析,結(jié)果:H1975/FLNa(KD)細(xì)胞中FLNa的表達(dá)量(0.34±0.01)明顯低于H1975/ctrl細(xì)胞(0.91±0.02),差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。2穩(wěn)定轉(zhuǎn)染H1975細(xì)胞AZD9291的IC50MTS法檢測兩組細(xì)胞的增殖能力,結(jié)果:不同濃度的AZD9291作用下,其對H1975/FLNa(KD)細(xì)胞的生長抑制率均高于相應(yīng)濃度的H1975/ctrl細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義(P0.05);H1975/FLNa(KD)組AZD9291的IC50值(2.17±0.19)明顯低于H1975/ctrl組細(xì)胞(6.09±0.11),差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。H1975/FLNa(KD)組對AZD9291的敏感性高于H1975/ctrl組。3穩(wěn)定轉(zhuǎn)染H1975細(xì)胞貼壁克隆生長能力的比較平板克隆法檢測兩組細(xì)胞克隆生長能力,結(jié)果:在無AZD9291作用2周后,H1975/FLNa(KD)組細(xì)胞克隆形成率(91.18±3.27%)明顯低于H1975/ctrl組(143.26±6.49%),差異均有統(tǒng)計(jì)學(xué)意義(P0.01);在含1μmol/L AZD9291作用2周后,H1975/FLNa(KD)組細(xì)胞克隆形成率(7.95±2.02%)明顯低于H1975/ctrl組(21.07±3.21%),差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。4穩(wěn)定轉(zhuǎn)染H1975細(xì)胞遷移能力的比較劃痕修復(fù)實(shí)驗(yàn)檢測兩組轉(zhuǎn)染細(xì)胞遷移能力,結(jié)果:在無AZD9291作用下,H1975/FLNa(KD)組細(xì)胞在6h、12h、18h的遷移率(5.21±0.85%,12.30±0.32%,19.09±0.66%)均明顯低于H1975/ctrl組(13.79±1.88%,33.49±1.92%,50.00±0.00%),差異均有統(tǒng)計(jì)學(xué)意義(P0.01);在2μmol/L AZD9291作用下,H1975/FLNa(KD)組細(xì)胞6h、12h、18h的遷移率(4.07±0.66%,9.26±1.48%,13.07±1.35%)明顯低于H1975/ctrl組細(xì)胞(7.93±1.05%,21.90±1.50%,33.90±1.27%),差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。5穩(wěn)定轉(zhuǎn)染H1975細(xì)胞侵襲能力的比較Transwell法檢測兩組轉(zhuǎn)染細(xì)胞侵襲能力,結(jié)果:在無AZD9291作用下,H1975/FLNa(KD)組穿膜的細(xì)胞數(shù)量(127.80±4.49)少于H1975/ctrl組(221.60±5.86),在1μmol/L AZD9291作用下,H1975/FLNa(KD)組穿膜的細(xì)胞數(shù)量(29.00±2.24)少于H1975/ctrl組(70.20±2.39),差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。6穩(wěn)定轉(zhuǎn)染H1975細(xì)胞EGFR活化水平及其下游信號分子活性的比較Western blot檢測1μmol/L AZD9291作用于兩組細(xì)胞0h、2h、4h、8h后各蛋白的表達(dá)水平,結(jié)果:H1975/FLNa(KD)組p-EGFR的水平(0.30±0.02;0.08±0.02;0.10±0.02;0.01±0.00)均明顯低于H1975/ctrl組(0.36±0.02;0.44±0.01;0.16±0.01;0.06±0.01),差異具有統(tǒng)計(jì)學(xué)意義(P0.05);H1975/FLNa(KD)組p-ERK的水平(0.90±0.04;0.31±0.00;0.28±0.01;0.18±0.06)均低于H1975/ctrl組(1.11±0.09;1.42±0.02;1.12±0.12;1.01±0.11),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1下調(diào)FLNa表達(dá)可加強(qiáng)AZD9291對T790M突變肺腺癌H1975細(xì)胞增殖、遷移和侵襲的抑制作用。2下調(diào)FLNa通過降低EGFR磷酸化水平,抑制MAPK/ERK信號通路活化,提高H1975細(xì)胞對三代EGFR-TKI AZD9291的敏感性。
[Abstract]:Objective: lung cancer is a major cause of cancer death in China, its incidence and mortality is highest in male malignant tumors, the incidence of women ranked second, but the mortality is still female cancer death first. Non small cell lung cancer (NSCLC) is the most common in lung cancer, accounting for about 85%. for the main treatment method there are surgery, radiotherapy, chemotherapy, molecular targeted therapy and immunotherapy. In recent years, chemotherapy and surgical treatment level has improved, but the 5 year survival rate is only 15%. molecular targeted therapy to open a new era in the treatment of lung cancer with tumor oncogene or its signal pathway as a therapeutic target targeted, exert inhibitory effects on tumor cells, reduce the damage of normal cells, to bring the gospel. For patients with tumors of the epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor, B receptor belongs to the Erb family a Member of the extracellular domain of.EGFR and ligands such as epidermal growth factor (EGF) combined with two homodimers, phosphorylated at tyrosine residues, further activation of downstream signaling pathways, such as MAPK/ERK, PI3K/AKT, JAK/STAT etc., involved in the regulation of tumor cell proliferation, invasion, metastasis and blood of Guan Shengcheng et al. Study confirmed that the EGFR mutation and activation the progress and prognosis of NSCLC. The most common adverse related activation of EGFR mutations in exon 19 and exon 21 L858R substitution mutation occurs in the form of the population in the west, approximately 10%-15% of patients with non-small cell lung cancer with EGFR activating mutations, and in the Asian population accounts for about 40%. in molecular therapy target the drug has been successfully used in clinic, the first generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib are widely used for first-line treatment of patients with advanced NSCLC, double chemotherapy compared with platinum based Could significantly prolong the median progression free survival (PFS), to improve the quality of life. However, most of the patients in 9-14 months after treatment, often will face EGFR-TKIs resistance. The most common mechanisms of acquired drug resistance is T790M mutation, increased the affinity to ATP so as to reduce the effect of inhibitors. In addition, also includes MET gene amplification, small cell lung cancer, mutations in the EGFR gene loss. In addition, about 30% of patients with EGFR-TKIs showed primary resistance. The resistance, follow-up and developed the second generation and the three generation of afatinib OHI imatinib (AZD9291), where AZD9291 is a new oral drug, effective, selective inhibition of EGFR sensitive mutant irreversible inhibitor of T790M mutation and resistance, the effect of wild type EGFR. Therefore, we further discuss the sensitivity of NSCLC patients to EGFR-TKIs problems, provide for the subsequent treatment A guiding role of filamin A (Filamin A FLNa) is a nonmuscle actin binding protein, mainly exist in the cytoplasm of.FLNa cells including one located at the amino terminus of actin binding domain and one consisting of 24 tandem repeats of the rod domain structure of multilayer beta sheet in this bar domains for protein-protein interactions provides.FLNa interface through the regulation of signal transduction molecules can change the tumorigenic microenvironment. At present, Zhu found that the reduced FLNa expression could promote lung cancer PC-9 cell proliferation, migration and invasion; reduce the expression of FLNa can also lead to lung cancer PC-9 cells decreased to the first generation of EGFR-TKIs sensitivity. However, after a period of time after treatment, patients often appear problems of drug resistance, the most common mechanism of T790M mutation, developed the third generation of AZD9291 inhibitors, the single anilino pyrimidine. Complexes differ in structure and pharmacological and other generations of TKIs. so we know about the interaction between TKI and FLNa drug structure, and the influence on the drug sensitivity of cells. In this study we used in vitro culture EGFR twenty-first exon and exon 20 NSCLC mutation sensitive cell line H1975, mutants resistant to T790M by using plasmid transfection, H1975 cell line construction of FLNa silencing; using MTS method, plate clone, scratch test and repair the invasion test stable transfected cell line proliferation, migration and invasion ability of behavioral biology; effect of AZD9291 was detected by Western blot blotting of stably transfected cell signal molecular level, so as to explore the effect of FLNa EGFR T790M the mutation sensitivity of lung adenocarcinoma H1975 cells of the three generation of EGFR-TKIs. Methods: 1 FLNa Western bolt were detected in lo2-peg10 cells silence effects respectively preserved in our laboratory Lung adenocarcinoma H1975, H1975/FLNa (KD) and H1975/ctrl cells were inoculated in 35mm culture dish, 1640 complete medium to full adherence. Cells were collected and protein extraction, application of Western blot detection of the level of FLNa, with -actin as the reference protein?.2 MTS assay detected two groups of cell proliferation were H1975/FLNa (KD) and H1975/ctrl cells were seeded in 96 well plates, cultured 24h, the supernatant was discarded and replaced by containing different concentration of AZD9291 (0,2,4,8,16 mol/L) complete 1640 culture medium to culture 48h, the supernatant was discarded into MTS, absorption spectrophotometric determination of each hole (OD) value, and calculate the half inhibition rate of cell growth and AZD9291 inhibitory concentration (IC50) of two groups of.3 cells was determined by plate cloning method after adherent colony formation were H1975/FLNa (KD) and H1975/ctrl cells were inoculated in 35mm culture dish, with and without AZD9291 (1 mol/L) under the action of training 2 weeks, hematoxylin staining, inverted Under a microscope to observe and count the number of.4 cell colony formation migration of wound healing assay was two groups of cells were H1975/FLNa (KD) and H1975/ctrl cells were seeded in 24 well plates, cultured 24h, using a sterile pipette tip evenly in the monolayer of scratches on the two kinds of cells were used, containing or not containing 1 mol/L AZD9291 complete 1640 culture medium to culture, observed cell migration changes had no significant difference, so the increase of drug concentration to 2 mol/L, the timing of wound healing was observed and photographed. To measure the width change and calculate the rate of cell migration.5 invasion capability test two groups of cells were H1975/FLNa (KD) and H1975/ctrl cells with a Transwell cell seeded on Matrigel in the upper chamber, two kinds of cells were in containing or not containing 1 mol/L AZD9291 no 1640 fetal bovine serum culture medium containing 10% fetal ventricular into 1640 bovine serum culture medium, cultured 24h after fixation, staining, light microscope photographs and calculate the change of the two groups of cell signal pathway transmembrane cell number.6 Western blot H1975/FLNa (KD) were detected and H1975/ctrl cells were inoculated in 35mm culture dish, totally 1640 cultured adherent to be covered. 1 mol/L AZD9291 intervention, respectively in 0h, 2h, 4h, 8h after the resumption of cell total protein extraction, Western detection of blot p-EGFR EGFR, p-ERK, ERK, and protein level of FLNa? -actin as the reference protein. Results: FLNa protein expression changes of Western blot levels in two groups were detected the protein level of FLNa 1 cells stable transfection of H1975 cells, scanning protein bands and statistical analysis, results: H1975/FLNa (KD) expression in FLNa cells (0.34 + 0.01) was significantly lower than that of H1975/ctrl cells (0.91 + 0.02), the differences were statistically significant (P0.01).2 AZD9291 I stably transfected into H1975 cells The proliferation ability, C50MTS assay results: two groups of cells at different AZD9291 concentrations of H1975/FLNa (KD) cell growth inhibition rate was higher than the corresponding concentration of H1975/ctrl cells, the difference was statistically significant (P0.05); H1975/FLNa (KD) AZD9291 IC50 value (2.17 + 0.19) was significantly lower than that of H1975/ctrl group cells (6.09 + 0.11), the difference was statistically significant (P0.01).H1975/FLNa (KD) detection plate cloning method sensitivity group AZD9291 was higher than that of H1975/ctrl group.3 transfected H1975 cells adherent growth of clonal growth ability of two groups of cell clones, results in no AZD9291 effect after 2 weeks, H1975/FLNa (KD) cell clone formation rate (91.18 + 3.27%) was significantly lower than that of H1975/ctrl group (143.26 + 6.49%), the differences were statistically significant (P0.01); in containing 1 mol/L AZD9291 for 2 weeks, H1975/ FLNa group (KD) cell clone formation rate (7.95 + 2.02%) significantly Lower than that of group H1975/ctrl (21.07 + 3.21%), the differences were statistically significant (P0.01) compare the wound healing assay was stably transfected.4 H1975 cells migration of transfected cells in two groups of migration, results: in the absence of AZD9291, H1975/FLNa (KD) cells in 6h, 12h, 18h (5.21 + 0.85%, the rate of migration 12.30 + 0.32%, 19.09 + 0.66%) was significantly lower than that of group H1975/ctrl (13.79 + 1.88%, 33.49 + 1.92%, 50 + 0%), the differences were statistically significant (P0.01); in 2 mol/L AZD9291, H1975/FLNa (KD) 6h 12h, 18h group of cells, the migration rate (4.07 + 9.26 + 0.66%. 1.48%, 13.07 + 1.35%) was significantly lower than that of cells in H1975/ctrl group (7.93 + 1.05%, 21.90 + 1.50%, 33.90 + 1.27%), the differences were statistically significant (P0.01).5 stable transfected H1975 cell invasion detection Transwell method compared two groups of transfected cell invasion. Results: in the presence of AZD9291 H1975/FLNa (KD) group 絀胯啘鐨勭粏鑳?yōu)鏁伴嚕?

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