【作者】 曾希；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 背景：恶性肿瘤难以治愈的主要原因是早期不容易被发现,一旦出现明显症状就诊,大多数已经到中晚期,伴有较广泛的浸润或转移,临床治疗效果差。目前,最佳抗肿瘤治疗策略仍然有赖于早期发现及密切监测早期复发。寻找方便有效的肿瘤早期诊断分子标志物和方法,一直是肿瘤研究亟待解决的问题。血液天然反映着机体的生理和病理状态,且血液标本较易获取,是寻找疾病分子标志物最理想的材料。鼻咽癌在欧美等西方国家罕见,而在我国高发,鼻咽癌也被形象的称为“中国癌”。鼻咽癌发病隐秘,早期没有明显临床表现,不容易发现,且鼻咽癌淋巴结转移发生早。因此,鼻咽癌早期诊断在我国尤为重要。由于鼻咽癌与EB病毒感染存在一定相关性,目前,临床上针对鼻咽癌的筛查主要是检测血液中EB病毒感染情况,如EBV-DNA、EBV-VCA-IgA等检测。但鼻咽癌与EBV并非完全相关,且许多疾病(如淋巴瘤、胃癌、乳腺癌、咽炎、传染性单核细胞增多症等)都可伴有EBV感染,健康人群也有一定比例感染者,大量的临床实践证明,EBV相关检测诊断鼻咽癌的灵敏度与特异性都不高,达不到作为早期诊断标志物的要求。肿瘤本身就是组织微环境的产物,肿瘤与微环境之间相互影响,组织微环境的多肽成分可以进入血液循环。血清中包含大量的低分子肽,这些肽受到机体生理和病理状态的影响,天然的“记录”着机体的生理和病理信息。因此,血清肽很可能是进行疾病血液学诊断的良好资源,昔日认为是生物垃圾的血清肽如今成了“诊断黄金”。在多种肿瘤中,血清肽的研究也正在兴起。miRNA是一种非编码小RNA,在基因的转录后调控中发挥巨大作用。miRNA的异常表达在许多疾病中扮演重要角色,特别是在肿瘤性疾病中,miRNA的意义尤其重要。近年来的研究发现,血清中存在miRNA,血清(?)niRNA的表达受疾病的影响,且这些miRNA十分稳定,具有成为疾病标志物的天然优势,是十分有应用前景的疾病诊断标志物。目的：本研究拟通过对鼻咽癌患者血清和非癌人群血清的血清肽谱和血清miRNA表达谱进行比较研究,经过筛选、验证,寻找差异表达血清肽(表达模式)和血清miRNA,并建立鼻咽癌血清肽和血清miRNA诊断模型或判别方程,为鼻咽癌早期诊断、病情监测和预后提供实验依据,并可能为治疗提供思路和靶点。方法：1.制定血清采集的标准程序按照标准程序广泛收集鼻咽癌患者(包括治疗前、一个疗程后)血清和非癌人群血清标本,完善基本资料和相关临床资料,建立血清样本库；2.使用EBV-VCA-IgA ELISA检测试剂盒检测所有血清标本中EBV的感染情况；3.运用WCX磁珠固相分离与MALDI-TOF质谱技术相结合的方法,获得鼻咽癌血清肽谱和非癌血清肽谱,对其二组血清肽谱进行比较研究,使用Clinpro Tools软件和CARS (competitive adaptive reweighted sampling)算法建立鼻咽癌血清肽诊断模型(判别方程),同时观察不同临床阶段鼻咽癌血清肽谱和鼻咽癌治疗前后血清肽变化情况；通过随访,分析血清肽表达模式与预后的关系；4.通过TaqMan Low-Density array (TLDA) miRNA芯片获取鼻咽癌患者血清和非癌人群血清的miRNA表达谱,通过比较分析和定量PCR验证筛选鼻咽癌差异表达血清miRNA;建立鼻咽癌血清miRNA诊断模型,并验证诊断模型的诊断效率；随访患者,采用Kaplan-Meier法分析鼻咽癌差异表达血清miRNA与预后的相关性。结果：1.收集了一定量的鼻咽癌血清样本和非癌血清样本,初步建立了资料齐全的血清样本库；2.通过EBV-VCA-IgA检测得到非癌血清样本EBV阳性率为11.4%,鼻咽癌患者血清样本EBV阳性率为66.2%,差异有高度显著性。3.在正式实验前,通过磁珠-质谱实验条件摸索,发现了一系列影响结果的实验条件,制订了合理可靠的磁珠-质谱方法实验流程；4.通过不同年龄段和不同性别血清肽的比较研究,发现年龄对血清肽谱有较大影响,性别对血清肽谱的影响相对较小；5.获得了基于WCX磁珠和MALDI-TOF质谱技术的鼻咽癌血清肽谱和非癌人群血清肽谱。6.使用Clinpro Tools建立鼻咽癌血清肽诊断模型,外部验证其特异性为85.7%、灵敏度为80.7%；7.运用CARS算法建立鼻咽癌的诊断判别方程：Y=0.2884X1+0.2073X2-0.2223 X3+0.3985 X4+0.2446 X5(X1：1741.10Da；X2：2991.11Da；X3：3192.36Da；X4：3890.71Da；X5：5064.67Da),其特异性为84.08%,灵敏度为93.50%,曲线下面积(AUC)为0.9425；8.通过对不同临床阶段鼻咽癌血清肽谱的比较分析发现,血清肽的改变主要发生在正常-早期鼻咽癌过程中,鼻咽癌各个临床阶段之间血清肽表达模式无明显差异；9.鼻咽癌治疗前后血清肽表达模式有较小差异；10.EBV阳性和阴性鼻咽癌血清肽谱无明显差异；11.生存曲线分析提示3890Da血清肽与预后存在相关性；12.使用TLDA芯片获得了鼻咽癌患者和非癌人群血清miRNA表达谱,分别检测到103个和125个miRNA表达；13.对鼻咽癌患者和非癌人群血清miRNA表达谱进行比较分析和验证,发现miR-17、miR-20a、miR-486-3p在鼻咽癌血清中表达升高,miR-29c、miR-223在鼻咽癌血清中表达下降。14.建立了鼻咽癌血清miRNA诊断模型：A=(CtmiR-29c+CtmiR-223)-(CtmiR-17+CtmiR-20a)>-3.30(即A>-3.30诊断为鼻咽癌；A<-3.30诊断为非癌),其灵敏度和特异性在96%以上。15.生存曲线分析提示血清miR-20a与预后存在相关性。结论：1.血清肽受到除疾病之外的多种因素的影响,血清肽组研究必须制定并按照严格标准统一的实验流程进行；2.鼻咽癌患者与非癌人群的血清肽表达模式及血清(?)niRNA表达谱都存在一定差异；3.血清肽表达模式的改变是鼻咽癌的早期事件；4.鼻咽癌血清肽和血清miRNA诊断模型的可靠性显著高于血清EBV-VCA-IgA检测方法,可能应用于临床鼻咽癌血清学诊断,且可能成为评价鼻咽癌预后的参考指标。更多还原

【Abstract】 Introduction:The main reason of the difficulty of curing malignant tumors is that they are not easy to be detected early. Once clinical manifestation appears, the majority has reached the middle or advanced stage, with extensive invasion or metastasis and poor clinical treatment effect. At present, the optimal strategy for anti-cancer depends on early detecting and close monitoring for early recurrence to adjust treatment plan reasonably. Development of minimally invasive biomarker assays for early detection and effective clinical management of pancreatic cancer is urgently needed to reduce high morbidity and mortality associated with this malignancy. There has been widespread consensus that bloodstream is considerably suitable for the detection of noninvasive biomarker in population. However, few serum (or plasma) markers with high specificity and sensitivity have been applied. Nasopharyngeal carcinoma (NPC) is seldom seen in western countries, however, it has a high incidence in China and it is also called’Chinese Cancer’. The morbility of nasopharyngeal carcinoma is very concealed without obvious clinical manifestation in earlier period. It is not easy to be detected and the diversion of lymphatic gland happens early. Therefore, early diagnosis of nasopharyngeal carcinoma is very urgent in China. At present, the detecting of the infection status of EBV (eg. EBV-DNA, BV-VCA-IgA, etc) has been commonly used in clinical screening for nasopharyngeal carcinoma. However, nasopharyngeal carcinoma is not completely related with EBV. People with other diseases (eg. lymphoma, gastric carcinoma, breast cancer, pharyngitis, acute lymphadenosis, etc) have EBV infection, and a proportion of healthy individual also has EBV infection. A large number of clinical practice demonstrate that the sensitivity and specificity of EBV-related detection for diagnosing nasopharyngeal carcinoma are poor, which can not meet the requirements of early diagnosing.It had been demonstrated that cancer is a product of the tissue microenvironment. Serum contains a large number of low molecular weight (LMW) peptides which are "records" for the cellular and extracellular events that take place at the level of the cancer-tissue microenvironment. They could be a rich source of cancer-specific diagnostic information. The blood peptidomic were a higher dimension of information content for cancer biomarker discovery. There is a growing interest in the research of using serum peptidome as markers in a series of tumor.MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level by degrading or blocking translation of messenger RNA (mRNA) targets. MiRNAs play important regulatory roles in a variety of cellular functions as well as in several diseases, including cancer. MiRNA-specific expression profiles have been reported for several pathological conditions. Recently, the discovery of miRNAs in serum opens up the possibility of using miRNAs as biomarkers of disease. The potential of microRNAs (miRNAs) as novel tumor markers has been the focus of recent scrutiny because of their tissue specificity, stability, and association with clinicopathological parameters. Therefore, circulating microRNAs (serum miRNAs/plasma miRNAs) were looked as stable blood-based markers for cancer detection and circulating microRNAs have been reported as potential biomarkers for the noninvasive diagnosis of cancer.Objectives:The aim of this study was to compare serum peptide profiling and serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma; to screen for serum molecular biomarkers(serum peptide pattern or serum miRNA) and establish diagnosis model to improve early diagnosis, disease monitoring and prognosis evaluation; and to provide possible target for treatment of nasopharyngeal cancer.Methods:1. Formulate standard procedure for serum sampling, extensive serum specimen of tumor free population and patients with nasopharyngeal carcinoma were gathered (including prior treatment, after one course of treatment), basic and clinical data were consummated, and the storeroom of serum specimen was established; 2. EBV-VCA-IgA ELISA was used to detect the infection of EBV in all serum specimen.3. WCX Magnetic bead combined with MALDI-TOF mass spectrometry were performed to obtain serum peptide profiling of tumor free population and patients with nasopharyngeal carcinoma, and these two groups of serum peptide profiling were compared. Clinpro Tools and CARS were used to establish diagnosis model of serum peptide pattern of nasopharyngeal carcinoma, the variation in different clinical stages was observed, and the relation between the prognosis and serum peptide profiling was analyzed.4. TaqMan Low-Density array (TLDA) was used to obtain serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma, quantity real-time PCR was used to verify and screen for serum miRNAs with differential expression, the diagnosis model of serum miRNAs of nasopharyngeal carcinoma was established, and its diagnostic efficiency was verified, and the relation between the prognosis and serum miRNAs with differential expression was analyzed using Kaplan-Meier.Results:1. Serum specimen of tumor free population and patients with nasopharyngeal carcinoma were gathered, and the storeroom of serum specimen with complete information was primarily established.2. The positive rate of serum EBV-VCA-IgA was 11.4% in tumor free population, and 66.2% in patients with nasopharyngeal carcinoma. The latter was significantly higher than the former.3. A series of experiment condition affecting results were discovered and a reasonable and stable experiment flow-sheet was formulated by exporing the experiment condition (parameter) of the magnetic bead combined with MALDI-TOF mass spectrometry.4. The result showed that age had great influence on the serum peptidome profiling, but gender had little influence compared with age.5. Serum peptide profiling of tumor free population and patients with nasopharyngeal carcinoma were obtained based on WCX magnetic bead and MALDI-TOF mass spectrometry. 6. The diagnosis model of serum peptide profiling of nasopharyngeal carcinoma was established by using Clinpro Tools. The specificity was 85.7% and the sensitivity was 80.7%.7. The discrimination equations of diagnosis of nasopharyngeal carcinoma was established by using CARS algorithm:Y=0.2884X1+0.2073X2-0.2223 X3+0.3985 X4+0.2446 X5 (X1:1741.10Da; X2:2991.11Da; X3:3192.36Da; X4:3890.71Da; X5: 5064.67Da)The sensitivity was 93.50% and the specificity was 84.08%, AUC was 0.9425.8. The variation of serum peptide profiling happened in the early stage of nasopharyngeal carcinoma, but there are not evident variation among different clinical stages of nasopharyngeal carcinoma, by observing the variation of serum peptide profiling in different stages.9. The variation of serum peptide profiling was a litttle between pre-treatment and post-treatment of nasopharyngeal carcinoma.10. The difference of serum peptide profiling was not obvious in nasopharyngeal carcinoma patients with positive and negative EBV.11. The survival curve suggested that the 3890Da serum peptide was related with prognosis.12. The expression of the 103 and 125 miRNAs were detected by using TaqMan Low-Density array(TLDA) to obtain serum miRNAs expression profiling of nasopharyngeal carcinoma patients and tumor free population.13. The expression of miR-17、miR-20a、miR-486-3p increased, and the expression of miR-29c、miR-223 decrease in serum of nasopharyngeal carcinoma by analyzing and verifying the serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma.14. The diagnosis model of serum miRNAs of nasopharyngeal carcinoma was established A=(CtmiR-29c+CtmiR-223)-(CtmiR-17+CtmiR-20a)>-3.30 (A>-3.30:nasopharyngeal carcinoma; A<-3.30:tumor free), and its sensitivity and specificity were more than 96%. 15. The survival curve showed serum miR-20a was related with prognosis of nasopharyngeal carcinoma.Conclusion:1. Serum peptide was affected by disease and many other factors, so the experiment condition in research of serum peptidome should be standardized strictly.2. There were difference in serum peptide profiling and serum miRNAs expression profiling of tumor free population and patients with nasopharyngeal carcinoma.3. The variation of serum peptide pattern was an early incident of nasopharyngeal carcinoma.4. The reliability of the diagnosis model of serum peptide profiling and serum miRNAs of nasopharyngeal carcinoma was significantly higher than the EBV-VCA-IgA detection. The diagnosis model could be applied in clinical diagnosis of nasopharyngeal carcinoma, and could provide reference for evaluating the prognosis of nasopharyngeal carcinoma.更多还原