EGF促进肺细胞RFPL3表达及入核抑制肺腺癌A549细胞凋亡并老年肺GGN特点分析

发布时间：2018-03-20 18:40

本文选题：表皮生长因子　切入点：Ret指样蛋白3　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:肺癌是严重危害人类健康的疾病,其死亡率及发病率均居全球癌症的首位,肺癌中近80%为非小细胞肺癌(non-small cell lung cancer,NSCLC)。近年来,虽然EGFR和ALK重排的激酶抑制剂、单克隆抗体药物、新的化疗药物逐渐出现,放疗技术大幅进步,手术方式逐步改进,但大多数肺癌患者的5年生存率并没有明显改善,仅约17%。其中的重要原因有两方面,第一,缺乏可靠的早期诊断方法的;第二,欠缺明显改善晚期肺癌患者生存期的治疗方法。因此,尽快从基因水平上揭示肺癌发病机制、发现新的治疗靶点,对改善肺癌患者预后意义重大。RET指样蛋白3(ret finger protein-like 3,RFPL3)在NSCLC细胞系和肿瘤病理组织中比肺正常细胞和癌旁组织显著过表达,RFPL3蛋白主要分布在NSCLC的细胞核中,与肺癌淋巴结转移有密切关系,是新的hTERT启动子的转录增强因子。RFPL3被CREB结合蛋白(CREB binding protein,CBP)乙酰化后,两者紧密结合于人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子区域,共同调控端粒酶活性、活化hTERT启动子,促进肺癌细胞增殖和肿瘤发展。上述研究提示,RFPL3过表达及其细胞核富集分布在肺癌发生、发展过程中发挥重要作用,有望成为肺癌诊断及治疗干预的新靶点。但国内外关于诱导RFPL3蛋白表达增加及核富集的因素及其对细胞生物学功能影响的研究很少。本实验重点研究RFPL3在肺正常细胞(人胚肺成纤维细胞(human embryonic lung fibroblast,HLF));人支气管上皮细胞(16 human bronchial epithelial cell,16HBE)和肺腺癌细胞(A549、H1299)的表达和分布情况,观察、检测表皮生长因子(epidermal growth factor,EGF)对肺正常细胞和NSCLC细胞RFPL3蛋白的量及分布的影响及转染RFPL3基因引起的RFPL3蛋白量及分布的变化对凋亡影响的初步研究,以期为后续深入探索RFPL3与肺癌发生、发展的基础研究及指导肺部恶性肿瘤临床治疗提供新的参考资料。随着计算机断层扫描应用普及,肺磨玻璃结节(groundglassnodule,ggn)的发现日益增多,表现为ggn的早期肺癌的发现也明显增加。但目前关于老年人肺磨玻璃结节的报道很少。因此,本文对比分析体检肺ct发现的老年与中青年单个ggn影像及病理特点,为临床体检发现的老年人磨玻璃密度结节诊治提供参考。方法:预实验中应用免疫印迹(westernblot)技术检测不同质量浓度(0、5、10、20ng/ml)和不同处理时间(24h、48h、72h)egf对正常肺细胞(hlf、16hbe)及肺腺癌细胞(a549、h1299)中rfpl3蛋白表达的影响,筛选影响不同细胞rfpl3表达的最适浓度及时间。将细胞分为有efg刺激的实验组和无egf刺激的观察组,运用westernblot和免疫荧光技术(if;immunofluorescencetechnique)检测经最适条件egf刺激前后,rfpl3蛋白在正常肺细胞及肺腺癌细胞中表达及亚分布情况变化。运用瞬时转染技术,将人工合成的rfpl3重组质粒转染a549细胞;运用荧光显微镜检测转染效率;运用westernblot技术检测各转染组rfpl3总蛋白及核蛋白的表达水平差异;运用流式细胞术检测a549细胞不同转染组间凋亡率的差异。回顾性分析我院56例体检肺ct发现并取得病理结果的ggn,对比老年人及中青年人磨玻璃密度结节和结节病理的特点。所有数据运用合适的统计学方法,检测差异性。结果:(1)egf质量浓度在0-20ng/ml范围内,肺腺癌细胞(a549、h1299)rfpl3蛋白表达量与egf呈剂量依赖关系,最佳刺激质量浓度为20ng/ml;肺正常细胞(hlf、16hbe)rfpl3蛋白表达量与egf浓度呈st-t曲线关系,egf浓度达10ng/ml时rfpl3蛋白表达达最大。egf促进肺细胞(hlf、16hbe、a549、h1299)rfpl3蛋白表达的最佳时间为48h。(2)(1)观察组westernblot结果表明,rfpl3蛋白在肺细胞(hlf、16hbe、a549、h1299)中均有表达,且肺癌细胞总蛋白及核蛋白表达量明显高于肺正常细胞,差异有统计学意义(p0.05)。(2)实验组经egf20ng/ml刺激48h后,westernblot结果表明同一种肺细胞(hlf、16hbe、a549、h1299)中rfpl3总蛋白、核蛋白较观察组明显增多,肺腺癌细胞中rfpl3总蛋白、核蛋白表达高于肺正常细胞,差异有统计学意义(p0.05)。(3)观察组if结果表明肺腺癌细胞(a549、h1299)中rfpl3蛋白主要分布在细胞质,细胞核也有少量分布;肺正常细胞(hlf、16hbe)中rfpl3蛋白全部分布在细胞质中。(4)实验组if结果提示,肺细胞(HLF、16HBE、A549、H1299)经EGF刺激后RFPL3蛋白主要分布于细胞核中。(3)荧光显微镜结果表明转染效率为90%以上。western blot结果表明转染组RFPL3总蛋白、核蛋白表达较空白对照组及阴性对照组明显增加,差异有统计学差异(P0.05),空白对照组及阴性对照组间表达无明显差异(P0.05)。流式细胞术检测结果表明,转染组凋亡率明显低于空白对照组及阴性对照组,转染组与空白对照及阴性对照组比较差异有统计学意义(P0.05),空白对照组与阴性对照组间比较差异无统计学意义(P0.05)。(4)老年人与中青年人磨玻璃结节在大小、位置、边缘(分叶、毛刺、光滑)、空泡征、临近结构改变(血管集束征、胸膜凹陷征)等方面差异无统计学意义。老年人GGN以mGGN多见,形态多不规则,边界常模糊,多为实性。中青年人GGN多为pGGN,形态常为圆形或者类圆形,边界多清晰,内部常含支气管充气征。老年人GGN恶变率较中青年人高,差异有统计学意义。结论:(1)EGF显著上调体外肺正常及NSCLC细胞RFPL3蛋白表达,诱导RFPL3蛋白由细胞质穿梭入核,EGF与NSCLC细胞RFPL3表达上调和核浓聚关系密切。过表达及核富集分布的RFPL3蛋白,抑制肺腺癌A549细胞早期凋亡。RFPL3有望成为NSCLC基因治疗的新靶点和早期诊断新指标。(2)老年人GGN恶变率较中青年人更高,加强老年人肺CT的体检筛查及管理,警惕老年人GGN有益于早期发现肺癌,改善预后。
[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.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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