本文選題：表皮生長(zhǎng)因子　切入點(diǎn)：Ret指樣蛋白3　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:肺癌是嚴(yán)重危害人類健康的疾病,其死亡率及發(fā)病率均居全球癌癥的首位,肺癌中近80%為非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)。近年來,雖然EGFR和ALK重排的激酶抑制劑、單克隆抗體藥物、新的化療藥物逐漸出現(xiàn),放療技術(shù)大幅進(jìn)步,手術(shù)方式逐步改進(jìn),但大多數(shù)肺癌患者的5年生存率并沒有明顯改善,僅約17%。其中的重要原因有兩方面,第一,缺乏可靠的早期診斷方法的;第二,欠缺明顯改善晚期肺癌患者生存期的治療方法。因此,盡快從基因水平上揭示肺癌發(fā)病機(jī)制、發(fā)現(xiàn)新的治療靶點(diǎn),對(duì)改善肺癌患者預(yù)后意義重大。RET指樣蛋白3(ret finger protein-like 3,RFPL3)在NSCLC細(xì)胞系和腫瘤病理組織中比肺正常細(xì)胞和癌旁組織顯著過表達(dá),RFPL3蛋白主要分布在NSCLC的細(xì)胞核中,與肺癌淋巴結(jié)轉(zhuǎn)移有密切關(guān)系,是新的hTERT啟動(dòng)子的轉(zhuǎn)錄增強(qiáng)因子。RFPL3被CREB結(jié)合蛋白(CREB binding protein,CBP)乙酰化后,兩者緊密結(jié)合于人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase,hTERT)啟動(dòng)子區(qū)域,共同調(diào)控端粒酶活性、活化hTERT啟動(dòng)子,促進(jìn)肺癌細(xì)胞增殖和腫瘤發(fā)展。上述研究提示,RFPL3過表達(dá)及其細(xì)胞核富集分布在肺癌發(fā)生、發(fā)展過程中發(fā)揮重要作用,有望成為肺癌診斷及治療干預(yù)的新靶點(diǎn)。但國(guó)內(nèi)外關(guān)于誘導(dǎo)RFPL3蛋白表達(dá)增加及核富集的因素及其對(duì)細(xì)胞生物學(xué)功能影響的研究很少。本實(shí)驗(yàn)重點(diǎn)研究RFPL3在肺正常細(xì)胞(人胚肺成纖維細(xì)胞(human embryonic lung fibroblast,HLF));人支氣管上皮細(xì)胞(16 human bronchial epithelial cell,16HBE)和肺腺癌細(xì)胞(A549、H1299)的表達(dá)和分布情況,觀察、檢測(cè)表皮生長(zhǎng)因子(epidermal growth factor,EGF)對(duì)肺正常細(xì)胞和NSCLC細(xì)胞RFPL3蛋白的量及分布的影響及轉(zhuǎn)染RFPL3基因引起的RFPL3蛋白量及分布的變化對(duì)凋亡影響的初步研究,以期為后續(xù)深入探索RFPL3與肺癌發(fā)生、發(fā)展的基礎(chǔ)研究及指導(dǎo)肺部惡性腫瘤臨床治療提供新的參考資料。隨著計(jì)算機(jī)斷層掃描應(yīng)用普及,肺磨玻璃結(jié)節(jié)(groundglassnodule,ggn)的發(fā)現(xiàn)日益增多,表現(xiàn)為ggn的早期肺癌的發(fā)現(xiàn)也明顯增加。但目前關(guān)于老年人肺磨玻璃結(jié)節(jié)的報(bào)道很少。因此,本文對(duì)比分析體檢肺ct發(fā)現(xiàn)的老年與中青年單個(gè)ggn影像及病理特點(diǎn),為臨床體檢發(fā)現(xiàn)的老年人磨玻璃密度結(jié)節(jié)診治提供參考。方法:預(yù)實(shí)驗(yàn)中應(yīng)用免疫印跡(westernblot)技術(shù)檢測(cè)不同質(zhì)量濃度(0、5、10、20ng/ml)和不同處理時(shí)間(24h、48h、72h)egf對(duì)正常肺細(xì)胞(hlf、16hbe)及肺腺癌細(xì)胞(a549、h1299)中rfpl3蛋白表達(dá)的影響,篩選影響不同細(xì)胞rfpl3表達(dá)的最適濃度及時(shí)間。將細(xì)胞分為有efg刺激的實(shí)驗(yàn)組和無egf刺激的觀察組,運(yùn)用westernblot和免疫熒光技術(shù)(if;immunofluorescencetechnique)檢測(cè)經(jīng)最適條件egf刺激前后,rfpl3蛋白在正常肺細(xì)胞及肺腺癌細(xì)胞中表達(dá)及亞分布情況變化。運(yùn)用瞬時(shí)轉(zhuǎn)染技術(shù),將人工合成的rfpl3重組質(zhì)粒轉(zhuǎn)染a549細(xì)胞;運(yùn)用熒光顯微鏡檢測(cè)轉(zhuǎn)染效率;運(yùn)用westernblot技術(shù)檢測(cè)各轉(zhuǎn)染組rfpl3總蛋白及核蛋白的表達(dá)水平差異;運(yùn)用流式細(xì)胞術(shù)檢測(cè)a549細(xì)胞不同轉(zhuǎn)染組間凋亡率的差異�；仡櫺苑治鑫以�56例體檢肺ct發(fā)現(xiàn)并取得病理結(jié)果的ggn,對(duì)比老年人及中青年人磨玻璃密度結(jié)節(jié)和結(jié)節(jié)病理的特點(diǎn)。所有數(shù)據(jù)運(yùn)用合適的統(tǒng)計(jì)學(xué)方法,檢測(cè)差異性。結(jié)果:(1)egf質(zhì)量濃度在0-20ng/ml范圍內(nèi),肺腺癌細(xì)胞(a549、h1299)rfpl3蛋白表達(dá)量與egf呈劑量依賴關(guān)系,最佳刺激質(zhì)量濃度為20ng/ml;肺正常細(xì)胞(hlf、16hbe)rfpl3蛋白表達(dá)量與egf濃度呈st-t曲線關(guān)系,egf濃度達(dá)10ng/ml時(shí)rfpl3蛋白表達(dá)達(dá)最大。egf促進(jìn)肺細(xì)胞(hlf、16hbe、a549、h1299)rfpl3蛋白表達(dá)的最佳時(shí)間為48h。(2)(1)觀察組westernblot結(jié)果表明,rfpl3蛋白在肺細(xì)胞(hlf、16hbe、a549、h1299)中均有表達(dá),且肺癌細(xì)胞總蛋白及核蛋白表達(dá)量明顯高于肺正常細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(2)實(shí)驗(yàn)組經(jīng)egf20ng/ml刺激48h后,westernblot結(jié)果表明同一種肺細(xì)胞(hlf、16hbe、a549、h1299)中rfpl3總蛋白、核蛋白較觀察組明顯增多,肺腺癌細(xì)胞中rfpl3總蛋白、核蛋白表達(dá)高于肺正常細(xì)胞,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。(3)觀察組if結(jié)果表明肺腺癌細(xì)胞(a549、h1299)中rfpl3蛋白主要分布在細(xì)胞質(zhì),細(xì)胞核也有少量分布;肺正常細(xì)胞(hlf、16hbe)中rfpl3蛋白全部分布在細(xì)胞質(zhì)中。(4)實(shí)驗(yàn)組if結(jié)果提示,肺細(xì)胞(HLF、16HBE、A549、H1299)經(jīng)EGF刺激后RFPL3蛋白主要分布于細(xì)胞核中。(3)熒光顯微鏡結(jié)果表明轉(zhuǎn)染效率為90%以上。western blot結(jié)果表明轉(zhuǎn)染組RFPL3總蛋白、核蛋白表達(dá)較空白對(duì)照組及陰性對(duì)照組明顯增加,差異有統(tǒng)計(jì)學(xué)差異(P0.05),空白對(duì)照組及陰性對(duì)照組間表達(dá)無明顯差異(P0.05)。流式細(xì)胞術(shù)檢測(cè)結(jié)果表明,轉(zhuǎn)染組凋亡率明顯低于空白對(duì)照組及陰性對(duì)照組,轉(zhuǎn)染組與空白對(duì)照及陰性對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),空白對(duì)照組與陰性對(duì)照組間比較差異無統(tǒng)計(jì)學(xué)意義(P0.05)。(4)老年人與中青年人磨玻璃結(jié)節(jié)在大小、位置、邊緣(分葉、毛刺、光滑)、空泡征、臨近結(jié)構(gòu)改變(血管集束征、胸膜凹陷征)等方面差異無統(tǒng)計(jì)學(xué)意義。老年人GGN以mGGN多見,形態(tài)多不規(guī)則,邊界常模糊,多為實(shí)性。中青年人GGN多為pGGN,形態(tài)常為圓形或者類圓形,邊界多清晰,內(nèi)部常含支氣管充氣征。老年人GGN惡變率較中青年人高,差異有統(tǒng)計(jì)學(xué)意義。結(jié)論:(1)EGF顯著上調(diào)體外肺正常及NSCLC細(xì)胞RFPL3蛋白表達(dá),誘導(dǎo)RFPL3蛋白由細(xì)胞質(zhì)穿梭入核,EGF與NSCLC細(xì)胞RFPL3表達(dá)上調(diào)和核濃聚關(guān)系密切。過表達(dá)及核富集分布的RFPL3蛋白,抑制肺腺癌A549細(xì)胞早期凋亡。RFPL3有望成為NSCLC基因治療的新靶點(diǎn)和早期診斷新指標(biāo)。(2)老年人GGN惡變率較中青年人更高,加強(qiáng)老年人肺CT的體檢篩查及管理,警惕老年人GGN有益于早期發(fā)現(xiàn)肺癌,改善預(yù)后。
[Abstract]:Background and objective: lung cancer is a serious disease, the mortality and incidence of cancer in the world were first, nearly 80% of lung cancer in non small cell lung cancer (non-small cell lung cancer, NSCLC). In recent years, although the kinase inhibitor EGFR and ALK rearrangement, monoclonal antibody drugs, new drugs gradually appeared, radiotherapy technology greatly improved surgical methods improved gradually, but the 5 year survival rate of most lung cancer patients were not significantly improved, an important reason is only about 17%. which has two aspects, first, the lack of reliable early diagnosis method; second, the lack of treatment significantly improved survival in patients with advanced lung cancer. Therefore, as soon as possible to reveal the pathogenesis of lung cancer at gene level, to find new therapeutic targets, to improve the prognosis in patients with lung cancer is significant.RET like protein 3 (RET finger 3 protein-like, RFPL3) in NSCLC cell line and swollen Tumor tissues than in normal lung tissue adjacent to cancer cells and significant overexpression of RFPL3 protein mainly distributed in the nuclei of NSCLC, lung cancer and lymph node metastasis is closely related, is the new hTERT transcription promoter enhancer factor.RFPL3 binding protein by CREB (CREB binding protein, CBP) after acetylation, the combination of in the human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) promoter region, CO regulation of telomerase activity, activation of the hTERT promoter, promote cell proliferation and tumor development of lung cancer. These results suggested that overexpression of RFPL3 and its nuclear enrichment distribution in lung cancer, play an important role in the process of development, is expected to become a new target for diagnosis and treatment of lung cancer. But few studies at home and abroad on the induction of RFPL3 protein expression increase and nuclear enrichment factors on cell biology function of this experiment. Focus on the study of RFPL3 in normal lung cells (human embryonic lung fibroblast cells (human embryonic lung fibroblast, HLF)); human bronchial epithelial cells (16 human bronchial epithelial cell, 16HBE) and lung adenocarcinoma cells (A549, H1299) to observe the expression and distribution of detection of epidermal growth factor (epidermal, growth factor. EGF) a preliminary study on the effect of the change of RFPL3 protein quantity and distribution of normal lung cells and NSCLC cells RFPL3 protein quantity and distribution and the effects of RFPL3 gene transfection on apoptosis induced by, in order to further explore the RFPL3 and lung cancer, to provide a new reference basis for the development of the research and guide the clinical treatment of pulmonary malignant tumor with the popularization of computer tomography applications, pulmonary ground glass nodules (groundglassnodule, GGN) found increasing performance for early lung cancer found GGN also increased significantly. But on the elderly Human pulmonary ground glass nodules were rarely reported. Therefore, the comparison and analysis of physical examination of lung CT found in the old and young single GGN imaging and pathological features, clinical examination found for the elderly ground glass opacity and provide reference. Methods: Western blot pre experiment (Westernblot) technique to detect different concentrations (0,5,10,20ng/ml) and different treatment time (24h, 48h, 72h) EGF on normal lung cells (HLF, 16HBE) and lung adenocarcinoma cells (A549, H1299) on the expression of rfpl3 protein and rfpl3 expression of different cell screening of the optimal concentration and time. The cells were divided into experimental group and observation group EFG stimulation without EGF stimulation, using Westernblot and immunofluorescence technique (if; immunofluorescencetechnique) was measured before and after the optimum conditions of EGF stimulation, and the distribution of rfpl3 protein expression in normal lung cells and lung adenocarcinoma cells by change. Transient transfection, the synthetic rfpl3 recombinant plasmids were transfected into A549 cells by fluorescence microscope detection; transfection efficiency; using Westernblot technology to detect the expression level of each transfection group rfpl3 total protein and nuclear protein were examined by A549 cells; flow cytometry different transfection the apoptosis rate between groups. A retrospective analysis of 56 cases of lung examination CT found in our hospital and the pathological results of GGN, comparing to the characteristics of the elderly and young people in the ground glass opacity and nodules pathology. All data using statistical methods appropriate, detecting differences. Results: (1) EGF concentration in the range of 0-20ng/ml, lung adenocarcinoma cells (A549, H1299) rfpl3 the expression of EGF in a dose dependent manner, the best stimulation concentration was 20ng/ml; normal lung cell (HLF, 16HBE) rfpl3 protein expression was ST-T curve relationship between concentration and EGF concentration, EGF concentration of 10ng/ml rfpl3 protein The expression reached the maximum.Egf promote lung cells (HLF, 16HBE, A549, H1299) the best time for the expression of rfpl3 protein in 48h. (2) (1) Westernblot in the observation group. The results showed that rfpl3 protein in lung cells (HLF, 16HBE, A549, H1299) expressed in lung cancer cells, and total protein and nuclear protein expression the lung was significantly higher than that of normal cells, the difference was statistically significant (P0.05). (2) the experimental group stimulated by egf20ng/ml 48h after Westernblot, the results show that the same kind of lung cells (HLF, 16HBE, A549, H1299) rfpl3 total protein, nuclear protein compared with the observation group increased significantly, the total protein of rfpl3 in lung cancer cells. The nuclear expression was higher than that in normal lung cells, the difference was statistically significant (P0.05). (3) observation group if showed lung adenocarcinoma cells (A549, H1299) rfpl3 protein mainly distributed in the cytoplasm, the nucleus has a small distribution; normal lung cells (HLF, 16HBE) rfpl3 protein located in the cytoplasm. (4). The experimental group if showed lung cells (HLF, 16HBE, A549, H1299) after EGF stimulation by RFPL3 protein mainly distributed in the nucleus. (3) fluorescence microscopy results indicated that the transfection efficiency is more than 90%.Western blot results showed that the transfection group RFPL3 total protein, nuclear protein expression compared with the blank control group and negative control group obviously increased, there was significant difference (P0.05), blank control group and negative control group showed no significant difference (P0.05). The results of flow cytometry showed that the apoptosis rate of transfected group was significantly lower than the blank control group and negative control group, transfection group and blank control group and the negative control group had significant difference (P0.05), blank control group and negative control group no significant differences (P0.05). (4) the elderly ground glass nodules in the size, location and youth, edge (leaf, burr, smooth), vacuole sign, vascular structural changes (near set Beam sign, pleural indentation) no significant differences among the elderly. GGN is mostly mGGN, irregular in shape, the boundary is fuzzy, the solid. The young people GGN pGGN, often round or oval shape, with clear border, often with internal air bronchogram in elderly. GGN more young people in the canceration rate is high, the difference was statistically significant. Conclusion: (1) EGF was significantly up-regulated in vitro normal lung NSCLC cells and the expression of RFPL3 protein, RFPL3 protein induced by cytoplasmic shuttle into nucleus, EGF upregulation and nuclear accumulation is closely related with NSCLC cell RFPL3 expression. The expression and distribution of RFPL3 nuclear enrichment protein, inhibition of lung adenocarcinoma A549 cells in early apoptosis of.RFPL3 NSCLC gene is expected to become a new target for treatment and early diagnosis index. (2) GGN in the elderly malignant rate of young people more than high, strengthen medical screening and management of elderly patients with lung CT, alert elderly GGN is helpful to the early Lung cancer is found in the period, and the prognosis is improved.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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6 張林;何艷玲;李清釗;郝小惠;袁騰;黃娜;郭菲菲;袁聚祥;白玉萍;劉楠;陳剛;袁揚(yáng);姚三巧;;MARCO介導(dǎo)的染矽塵大鼠肺細(xì)胞線粒體凋亡信號(hào)通路研究[A];中國(guó)職業(yè)安全健康協(xié)會(huì)2013年學(xué)術(shù)年會(huì)論文集[C];2013年

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