食管癌血清多肽标志和ABCC4基因调控研究

发布时间：2018-04-21 09:16

本文选题：ESCC + 血清标志物　；参考：《北京协和医学院》2016年博士论文

【摘要】：食管鳞状细胞癌(esophageal squamous cell carcinoma, ESCC)是全球常见恶性肿瘤之一,由于缺乏明显的早期症状和可靠的诊断技术,多数患者就诊时已为中晚期,治疗效果欠佳,5年平均生存率仅为3040%。如能早期诊断,其5年生存率可达90%以上。目前尚无ESCC可靠的血清学标志分子。本研究首先应用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测了ESCC患者、食管癌前病变不典型增生和健康对照个体血清中角化素蛋白片段19(Cyfra21-1)及鳞状细胞癌抗原(squamous cell carcinoma antigen, SCC)水平。结果发现,与健康对照组相比,Cyfra21-1和SCC在食管不典型增生和食管癌患者血清中均高表达(P0.05)。伴有淋巴结转移的ESCC患者血清Cyfra21-1水平显著高于无转移组(P=0.005)。而血清Cyfra21-1和SCC水平与患者年龄、性别、肿瘤部位、分化程度、肿瘤大小和TNM分期等特征无关。化学发光法定量检测Cyfra21-1和SCC表达水平并计算曲线下面积(area under curve, AUC),结果提示联合检测Cyfra21-1和SCC血清标志诊断食管癌效果(AUC=0.777)优于Cyfra21-1(AUC=0.757)或SCC (AUC=0.661)单独检测的效果。化学发光法与ELISA法检测血清Cyfra21-1和SCC表达水平结果一致。接下来,为了发现和筛选新的ESCC循环标志物,应用弱阳离子交换磁珠与基质辅助激光解吸电离飞行时间质谱(magnetic bead-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MB-based MALDI-TOF-MS)联用,利用477例肿瘤和健康对照样本,建立了一个由三个多肽差异峰(1,925.5,2,950.6and 5,900.0 Da)组成的ESCC血清诊断模型,该模型在100例ESCC和98例健康对照样本组成的训练集中的诊断灵敏度和特异度分别达到97.00%(97/100)和95.92%(94/98),101例ESCC和98例健康对照样本组成的验证集的诊断灵敏度和特异度分别达到97.03% (98/101)和100.00% (98/98)。该模型的诊断效果优于SCC和Cyfra 21-1联用,且该模型对于早期ESCC样本的诊断灵敏度达到42/43(97.67%)。通过轨道阱组合式高分辨液质联用系统对差异峰进行了鉴定,三个差异峰分别来源于α2 HS糖蛋白(Alpha2-HS glycoprotein, AHSG),血小板反应素-1(Thrombospondin-1, TSP1)和纤维蛋白原α链(Fibrinogen A alpha-chain, FGA)蛋白片段。此外,TSP1在ESCC组织和血清检测中表达升高且与ESCC进展相关,组织中TSP1的表达是ESCC不良预后的独立风险因素。由于TSP1蛋白N端的一个多肽在ESCC患者血清中明显升高,据此序列制备了特异性识别该多肽片段的抗体,并初步建立了一种特异性检测血清中的该多肽片段的竞争性ELISA方法。测试分析发现,食管癌患者血清中该TSP1N端多肽的表达水平显著高于健康对照组(P0.05)。新制备的抗TSP1 N端多肽片段的抗体,以及基于此抗体的竞争性ELISA方法,可以作为检测和诊断ESCC的新工具。这一工作仍在进行中,还需更多的样本来进一步验证其临床意义。最后,本实验室前期研究发现ABCC4基因的种系DNA拷贝数扩增和蛋白的异常表达促进食管癌的发生发展。ABCC4 (ATP-binding cassette transporter family class C4, ABCC4)是跨膜的ABC转运蛋白C家族成员之一,在转运生理性、内源性和外源性物质中发挥重要作用,也被称为多药抗药性相关蛋白4(Multidrug resistance-associated protein 4,MRP4)。本研究进一步检测了ESCC细胞系中ABCC4表达情况与化疗药顺铂耐药的关系。结果发现与ABCC4低表达细胞系相比,ABCC4高表达细胞系对顺铂更耐药。用siRNA敲降KYSE140细胞中ABCC4表达后,细胞对顺铂的敏感性显著增高。此外,基于前期在ABCC4基因内含子区发现的增强子元件,进一步用报告基因实验获得其核心区段,并通过染色质免疫共沉淀-聚合酶链式反应(Chromatin Immunoprecipitation-Polymerase Chain Reaction, ChIP-PCR)实验发现了与该核心区段结合的转录因子。这些结果提示ABCC4在ESCC化疗敏感性调控中发挥重要作用,而ESCC中ABCC4基因的表达调控还需要进一步深入研究。
[Abstract]:Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in the world. Because of the lack of obvious early symptoms and reliable diagnostic techniques, most patients have been in the middle and late stages of medical treatment. The treatment effect is poor and the average survival rate of 5 years is only 3040%. If early diagnosis, the 5 year survival rate can reach 90%. There is no ESCC reliable serological marker. In this study, enzyme-linked immunosorbent assay (ELISA) was used to detect the serum keratinin fragment 19 (Cyfra21-1) and squamous cell carcinoma antigen (squamous cell carcinoma antigen) in the serum of ESCC patients, atypical hyperplasia of precancerous lesions and healthy controls. The results showed that Cyfra21-1 and SCC were highly expressed in the sera of the atypical hyperplasia of the esophagus and the patients with esophageal cancer compared with the healthy control group (P0.05). The serum Cyfra21-1 level of the ESCC patients with lymph node metastasis was significantly higher than that in the non metastasis group (P=0.005). The serum Cyfra21-1 and SCC levels were associated with the patient's age, sex, tumor location, and differentiation. The extent, tumor size and TNM staging were not related. Chemiluminescence was used to quantify the expression of Cyfra21-1 and SCC and to calculate the area under the curve (area under curve, AUC). The results suggested that the combined detection of Cyfra21-1 and SCC serum markers in the diagnosis of esophageal cancer (AUC=0.777) was better than that of Cyfra21-1 (AUC=0.757) or individual detection. The results of chemiluminescence and ELISA detection were consistent with the levels of serum Cyfra21-1 and SCC. Next, in order to find and screen new ESCC cycle markers, the weak cation exchange magnetic beads and matrix assisted laser desorption ionization time of flight mass spectrometry (magnetic bead-based matrix-assisted laser desorption/ionization time-of-flight MAS) S spectrometry, MB-based MALDI-TOF-MS) combined with 477 cases of tumor and healthy control samples, a ESCC serum diagnostic model consisting of three polypeptide difference peaks (1925.5,2950.6and 5900 Da) was established. The diagnostic sensitivity and specificity of the model were 9 in the training concentration of 100 cases of ESCC and 98 healthy control samples. The diagnostic sensitivity and specificity of 7% (97/100) and 95.92% (94/98), 101 cases of ESCC and 98 healthy control samples are 97.03% (98/101) and 100% (98/98) respectively. The diagnostic effect of this model is better than SCC and Cyfra 21-1, and the diagnostic sensitivity of the model to early ESCC samples is 42/43 (97.67%). Through orbits The well combined high resolution liquid chromatography-mass spectrometry system was used to identify the difference peaks. Three different peaks were derived from alpha 2 HS glycoprotein (Alpha2-HS glycoprotein, AHSG), thrombocytopenin -1 (Thrombospondin-1, TSP1) and fibrinogen alpha chain (Fibrinogen A alpha-chain, FGA) protein fragments. The expression of TSP1 in the tissue is an independent risk factor for the poor prognosis of ESCC. As a polypeptide in the N terminal of the TSP1 protein is obviously elevated in the serum of the patients with the N, the specific antibody to identify the polypeptide fragment is prepared, and a preliminary establishment of a specific detection of the polypeptide fragment in the serum of the ESCC has been established. Contention ELISA method. Test analysis found that the expression level of the TSP1N terminal polypeptide in the sera of the patients with esophageal cancer was significantly higher than that of the healthy control group (P0.05). The new antibody against the TSP1 N peptide fragment and the competitive ELISA method based on this antibody could be used as a new tool for detecting and diagnosing ESCC. This work is still in progress and needs to be done. More samples were used to further verify its clinical significance. Finally, the previous study found that the amplification of the DNA copy number of the ABCC4 gene and the abnormal expression of the protein promoted the development of.ABCC4 (ATP-binding cassette transporter family class C4, ABCC4), one of the member of the ABC transporter C family in the transmembrane. Physiological, endogenous and exogenous substances play an important role, also known as drug resistance related protein 4 (Multidrug resistance-associated protein 4, MRP4). This study further detected the relationship between the expression of ABCC4 in the ESCC cell line and the resistance of chemotherapy to cisplatin. The results showed that the high expression of ABCC4 was compared with the ABCC4 low expression cell line. The cell line was more resistant to cisplatin. The sensitivity of the cells to cisplatin was significantly increased after the expression of ABCC4 in the KYSE140 cells of siRNA knockdown. Furthermore, based on the enhancers found in the intron of the ABCC4 gene, the core segments were obtained by the reporter gene experiment, and the chromatin immunoprecipitation polymerase chain reaction (Chrom) was obtained. Atin Immunoprecipitation-Polymerase Chain Reaction, ChIP-PCR) found the transcription factors associated with the core section. These results suggest that ABCC4 plays an important role in the regulation of chemosensitivity in ESCC, and the regulation of the expression of ABCC4 gene in ESCC needs further study.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1

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