鼻咽癌相关血清标志物的筛选与验证

发布时间：2018-06-20 12:17

本文选题：鼻咽癌 + 血清标志物　；参考：《广西医科大学》2013年硕士论文

【摘要】：鼻咽癌(nasopharyngeal carcinoma, NPC)是源于鼻咽部上皮组织的一种恶性肿瘤,95%以上临床病例属低分化癌和未分化癌,恶性程度高。目前确诊鼻咽癌主要依靠病理诊断,由于鼻咽癌早期不易察觉,大部分病人确诊时已处于中晚期,远处转移机会多,治疗效果差,患者生存期较短。因此,寻找鼻咽癌相关标志物对于NPC的筛选、治疗靶标、探究肿瘤发生发展机理重要意义。本研究旨在运用定量蛋白质组学方法筛选能作为筛选与诊断的鼻咽癌血清标志物,并对候选标志物进行验证和初步的研究。第一部分iTRAQ结合2D LC-MS/MS筛选鼻咽癌相关血清标志物目的：运用基于二维液相色谱/质谱联用的稳定同位素标记方法(iTRAQ结合2D LC-MS/MS)对鼻咽癌患者血清进行定量蛋白组学分析,获得差异蛋白谱,从中筛选潜在的肿瘤标志物。方法：正常组、鼻咽癌组(Ⅱ期,Ⅲ期,Ⅳ期)共4组血清样本(每组6例)经多重亲和免疫去除14种高丰度蛋白后运用iTRAQ结合2D LC-MS/MS技术进行定量蛋白组学分析,重复实验一次,汇总2次实验结果,获得差异蛋白谱并通过生物信息学方法从中挖掘潜在的生物标志物。结果：两次质谱实验共鉴定出274个置信蛋白,两次实验鉴定蛋白重复率为70.8%,在2次实验中均被鉴定和定量到的差异蛋白有15个,其中5个相对于正常组表达上调(1.4倍),10个表达下调(0.7倍)。S100A8和S100A9在2次定量实验中均稳定显著高表达,而S100A8/A9两者作为炎症相关因子的功能已被广泛认可,近来越来越多的研究表明其不仅可作为肿瘤标志物,在肿瘤发展和转移过程中也发挥重要作用。第二部分S100A8/A9,有潜力的鼻咽癌标志物目的：对质谱实验得到的差异蛋白S100A8/A9进行验证,确认它们的差异表达并初步研究其与鼻咽癌的关系。方法：①ELISA技术检测S100A8/A9在血清中的表达水平；②IHC技术检测S100A8/A9在鼻咽癌组织和鼻咽炎组织中的表达和分布；③ICC和Western Blot技术检测S100A8/A9在永生化正常人鼻咽细胞系NP69、高分化鼻咽癌细胞系CNE1、低分化鼻咽癌细胞系CNE2中表达与分布；④RT-PCR技术检测S100A8/A9/A12在3个细胞系中mRNA的表达水平；⑤通过不同浓度的Zn2+作用细胞,CCK8法检测3个细胞系的活性与增值并细胞计数,⑥RT-PCR检测随Zn2+浓度改变S100A8/A9/A12的mRNA表达水平变化。结果：①血清和病理组织中S100A8/A9含量水平变化趋势符合质谱结果,癌症组高于正常组。②S100A8/A9在鼻咽癌细胞系、其他头颈部高分化鳞癌细胞、高分化的表皮细胞中强表达而在低分化的腺体细胞、基底细胞中不表达提示S100A8/A9可能与分化有关。③S100A8/A9/A12的mRNA水平随培养时间的延长在癌细胞系CNE1、CNE2中表达降低,正常细胞NP69中表达升高。④Zn2+50μM时可以促进3个细胞系的增殖但浓度超过70gM后则迅速表现出细胞毒性,20μM时绝大部分细胞已死亡,CNE2表现出更强的耐受性。另外值得一提的是当细胞生长发生堆叠后特别是癌细胞系,对Zn2+的耐受性明显增强,110μM时大部分细胞仍存活。⑤随着Zn2+浓度增加,当S100A8/A9/A12的mRNA表达降低时细胞增殖与活性加强,表明S100A8/A9/A12可能参与细胞的生长进程。结论：iTRAQ结合2DLC-MS/MS是强有力的定量蛋白组学手段,其高通量、高重复性与准确性有助于筛选生物标志物。本研究首次采用iTRAQ技术筛选鼻咽癌血清相关标志物并获得一系列差异蛋白,从中挖掘分析将能获得潜在的标志物。对差异蛋白S100A8/A9的验证和研究表明是有潜力的鼻咽癌标志物,与鼻咽癌的发展密切相关。对这些潜在标志物的进一步研究将有助于鼻咽癌的筛选和诊断,发现新的治疗靶标和探究癌症发生发展的机制。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the epithelial tissue of the nasopharynx. More than 95% of the clinical cases are low differentiated and undifferentiated, and the malignant degree is high. At present, the diagnosis of nasopharyngeal carcinoma is mainly based on the pathological diagnosis. Because the early diagnosis of nasopharyngeal carcinoma is not easy to detect, most patients are in the middle and late stage, and the distant metastasis machine has been diagnosed. There are many, poor therapeutic effects and shorter survival time. Therefore, it is important to search for the related markers of nasopharyngeal carcinoma for the screening of NPC, the target of treatment, and the significance of the mechanism of the development of the tumor. A preliminary study.
Part 1 screening of nasopharyngeal carcinoma related serum markers by iTRAQ combined with 2D LC-MS/MS
Objective: to use the stable isotope labeling method based on two-dimensional liquid chromatography / mass spectrometry (iTRAQ combined with 2D LC-MS/MS) to analyze the serum of patients with nasopharyngeal carcinoma by quantitative proteomic analysis, to obtain the differential protein spectrum and to screen the potential tumor markers.
Methods: the normal group and the nasopharyngeal carcinoma group (stage II, stage III, IV) had 4 groups of serum samples (6 cases in each group) after multiple affinity immunization to remove 14 kinds of high abundance proteins, and then used iTRAQ combined with 2D LC-MS/MS to carry out quantitative proteomic analysis. Excavate potential biomarkers.
Results: 274 confidence proteins were identified in the two mass spectrometry, and the rate of protein repetition in two tests was 70.8%. There were 15 differential proteins identified and quantified in the 2 experiments. 5 of them were up to up (1.4 times) relative to the normal group (1.4 times), and 10 expressions down down (0.7 times).S100A8 and S100A9 were all stable and significant in the 2 quantitative experiments. S100A8/A9, as a function of inflammation related factors, has been widely recognized. Recently, more and more studies have shown that it not only can be used as a tumor marker, but also plays an important role in the process of tumor development and metastasis.
The second part is S100A8/A9, a potential marker for nasopharyngeal carcinoma.
Objective: to verify the differential protein S100A8/A9 obtained by mass spectrometry, confirm their differential expression and preliminarily study their relationship with nasopharyngeal carcinoma.
Methods: (1) ELISA technique was used to detect the expression level of S100A8/A9 in serum; (2) IHC technique was used to detect the expression and distribution of S100A8/A9 in nasopharyngeal carcinoma tissues and nasopharyngitis tissues; (3) ICC and Western Blot techniques were used to detect NP69 in nasopharyngeal cell line in immortalized normal human, CNE1 of highly differentiated nasopharyngeal carcinoma cell line, and CN of low differentiated nasopharyngeal carcinoma cell line. Expression and distribution in E2; (4) RT-PCR technique was used to detect the expression level of mRNA in S100A8/A9/A12 in 3 cell lines; (5) the activity and increment of 3 cell lines were detected by CCK8 method by different concentrations of Zn2+ acting cells, and RT-PCR was used to detect the change of mRNA expression level of S100A8/A9/A12 with Zn2+ concentration.
Results: (1) the change trend of S100A8/A9 content in serum and pathological tissue conforms to the results of mass spectrometry, and the cancer group is higher than the normal group. (2) S100A8/A9 is in the nasopharyngeal carcinoma cell line, the other high differentiated squamous cell carcinoma cells in the head and neck, the highly differentiated epidermal cells are strongly expressed in the low differentiated glandular cell, and the non expression of S100A8/A9 in the basal cells may be suggested as possible. The mRNA level of S100A8/A9/A12 was increased with the prolongation of culture time in the cancer cell line CNE1, CNE2, and the expression of normal cells increased in NP69. (4) Zn2+50 mu M could promote the proliferation of 3 cell lines, but the cytotoxicity was quickly displayed after the concentration of more than 70gM, and most of the cells were dead at 20 u M and CNE2 showed stronger. In addition, it is worth mentioning that when the cell growth is stacked, especially the cancer cell lines, the tolerance to Zn2+ is obviously enhanced, and most of the cells still survive at 110 M. 5. As the concentration of Zn2+ increases, the proliferation and activity of the cell is strengthened when the mRNA expression of S100A8/A9/A12 decreases. It shows that S100A8/A9/A12 may be involved in the cell growth process.
Conclusion: iTRAQ combined with 2DLC-MS/MS is a powerful quantitative proteomic method. Its high flux, high repeatability and accuracy are helpful for screening biomarkers. This study is the first to use iTRAQ technology to screen serum related markers of nasopharyngeal carcinoma and obtain a series of differential proteins. The validation and research of white S100A8/A9 indicate that it is a potential marker for nasopharyngeal carcinoma and is closely related to the development of nasopharyngeal carcinoma. Further research on these potential markers will help the screening and diagnosis of nasopharyngeal carcinoma, discover new therapeutic targets and explore the mechanism of cancer development.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

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