【作者】 童永清；

【导师】 李官成；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 鼻咽癌（Nasopharyngeal carcinoma,NPC）是一种上皮源性的高发于我国南方及东南亚一带的头颈部恶性肿瘤,在我国广东、广西、福建、湖南、江西尤为突出,据估计全世界鼻咽癌病例中80%发生于我国,年发病率为10-25/10万。由于鼻咽癌起病隐匿,部位特殊,早期症状缺乏特异性,导致早期诊断十分困难,通常在鼻咽癌确诊时,70%以上的患者局部已经高度恶性或扩散到淋巴结,已属于临床中晚期。因此,早期诊断鼻咽癌将极大提高鼻咽癌的治愈率和提高患者预后。许多蛋白质在肿瘤组织中高表达,这些蛋白质能刺激机体的免疫系统产生抗体,这些自身抗体比肿瘤蛋白质更容易检测,因此将常规检测这些高表达的蛋白质改为检测肿瘤患者体内针对这些肿瘤相关抗原的自身抗体是一种更有效的策略。相当部分的自身抗体作为肿瘤的诊断和预后判断的指标有着潜在的价值,如抗BRD2和α-甲基-辅酶A消旋酶（alpha-methylacyl-CoA racemase,AMACR）自身抗体用于前列腺癌的诊断,抗Annexin XI-A和Grb2相关蛋白自身抗体用于乳腺癌的诊断,抗表皮细胞粘附分子（Epithelial cell adhesion molecule,Ep-CAM）和肌酸激酶B（creatine kinase B,CKB）自身抗体用于卵巢癌的诊断等。目前在鼻咽癌中也发现了一些高表达的蛋白质,如fibronectin、Mac-2BP、plasminogen、和zinc-alpha（2）-glycoprotein（ZAG）等,提示一系列的针对这些高表达蛋白质的自身抗体有可能用于鼻咽癌的诊断和监测,具有很高的应用价值。因此研究针对鼻咽癌特异性或者相关性肿瘤抗原的自身抗体将大大提高鼻咽癌的诊断效率,这些自身抗体有望作为鼻咽癌的肿瘤标志物。首先,探讨了鼻咽癌患者血清中是否存在抗鼻咽癌自身抗原的抗体,为寻找鼻咽癌新的肿瘤标志物提供依据。以Epstein-Barr病毒阴性的鼻咽癌细胞株CNE1细胞为固相抗原板,酶联免疫吸附实验（ELISA）分析其与32例鼻咽癌患者血清和54例正常血清反应的差异性;提取CNE1总蛋白,Western印迹分析其与鼻咽癌患者血清反应是否存在特异性的蛋白质。ELISA分析结果显示鼻咽癌患者血清平均OD₄₀₅值（0.904±0.032）明显高于正常人血清平均OD₄₀₅值（0.736±0.028）（P＜0.01）;Western印迹分析发现鼻咽癌患者血清与正常人血清相比,部分阳性条带位置一致,但条带阳性强度增加,还发现出现了部分新的阳性条带,这些条带可能属于鼻咽癌相关抗原或鼻咽癌特异性抗原。说明鼻咽癌患者血清中存在抗鼻咽癌自身抗原的抗体,这些抗体并非针对Epstein-Barr病毒,为寻找鼻咽癌血清肿瘤标志物提供了依据。其次,用8例病理确诊的鼻咽癌组织活检标本构建鼻咽癌T7噬菌体cDNA文库,然后再利用来源于鼻咽癌临床分期在Ⅱ-Ⅳ期患者的血清和正常人血清进行生物淘洗,并将生物淘洗获得的22个噬菌体重组蛋白进行免疫印迹和ELISA实验鉴定。随后通过ELISA分析鼻咽癌患者和正常人血清中的5个抗噬菌体重组蛋白自身抗体的水平,ROC分析这些自身抗体作为鼻咽癌诊断标志物的特异性和敏感性。结果成功构建了鼻咽癌混合组织T7噬菌体cDNA文库,原始库容量为8.0×10⁷pfu/ml,放大后的库容量为3.47×10¹⁰pfu/ml,为生物淘洗奠定了基础。利用鼻咽癌患者血清和正常人血清经生物淘洗分离了31个肿瘤相关蛋白质,DNA序列分析显示这31个蛋白质中有22个为独立的与已知或者假定的蛋白质相匹配。说明通过生物淘洗能有效富集与鼻咽癌患者血清发生反应的蛋白质,而这些蛋白质不与正常人血清发生反应,为进一步实验提供了依据。RT-PCR检测发现BMI-1基因在7株不同的鼻咽癌细胞株中均有不同程度的表达,而在正常鼻咽上皮细胞株NP69中不表达。随后以BMI-1噬菌体重组蛋白与鼻咽癌患者血清进行免疫印迹分析,结果显示在不同的鼻咽癌患者血清中存在不同滴度的抗BMI-1自身抗体,且抗BMI-1自身抗体与BMI-1具有良好的亲合力。进一步ELISA检测鼻咽癌血清中抗BMI-1自身抗体的水平,发现在鼻咽癌患者血清中,抗BMI-1自身抗体平均OD₄₀₅值为0.791±0.025,而在正常人血清中平均OD₄₀₅值为0.488±0.042,统计学分析二者之间具有显著性差异（P＜0.01）。ROC分析曲线下面积为0.8044,说明抗BMI-1自身抗体作为鼻咽癌诊断的标志物,具有良好的特异性和敏感性。以鼻息肉患者血清为对照,进一步分析抗BMI-1自身抗体与鼻咽癌临床资料之间的关系,发现抗BMI-1自身抗体与鼻咽癌的转移有密切关系,在原位鼻咽癌患者血清中的水平与鼻息肉患者血清水平相比,具有显著性差异（P＜0.01）,提示抗BMI-1自身抗体有可能作为鼻咽癌的早期诊断标志物。在探讨抗BMI-1自身抗体作为鼻咽癌标志物的基础上,又进一步检测了抗CD44、MAGE、fibronectin、HSP70和EBNA-1的自身抗体。CD44、MAGE、fibronectin和HSP70均能与不同的鼻咽癌患者血清发生反应,这种反应强度与正常人血清比较,具有明显差异（P＜0.05）。ELISA分析显示抗CD44、MAGE、fibronectin和HSP70的自身抗体具有与4个人噬菌体蛋白良好的亲合力,在鼻咽癌患者血清中的水平明显高于正常人血清和鼻息肉患者血清中的水平（P＜0.05）,而在正常人血清和鼻息肉患者血清中的水平无明显差异（P＞0.05）,进一步为以抗CD44、MAGE、fibronectin和HSP70的自身抗体作为鼻咽癌的标志物提供了依据。ROC分析抗CD44、MAGE、fibronectin和HSP70自身抗体作为鼻咽癌标志物的ROC曲线下面积分别为0.7857、0.8922、0.8091和0.8435,而抗EBNA-1自身抗体作为鼻咽癌诊断标志物的ROC分析曲线下面积为0.6110,说明抗CD44、MAGE、fibronectin和HSP70的自身抗体作为鼻咽癌诊断的标志物,其特异性和敏感性均高于抗EBNA-1自身抗体作为鼻咽癌诊断的标志物。进一步分析抗CD44、MAGE、fibronectin和HSP70自身抗体是否可以作为鼻咽癌早期诊断的标志物,显示鼻咽癌患者血清中抗MAGE自身抗体水平与鼻咽癌的临床分期紧密相关,抗CD44、fibronectin和HSP70的自身抗体的水平也与鼻咽癌的临床分期相关,提示抗MAGE自身抗体可能作为鼻咽癌早期诊断的标志物,这些鼻咽癌患者血清自身抗体的水平可以用来预测鼻咽癌的临床分期。总之,通过本研究,证实了在鼻咽癌患者血清中存在针对鼻咽癌组织的自身抗体,这些自身抗体有可能作为鼻咽癌的肿瘤标志物,用于鼻咽癌的早期诊断或者预后判断,但是由于这些自身抗体也存在于其它肿瘤患者血清中,所以难以单独用于鼻咽癌的诊断,而将多个自身抗体联合起来用于鼻咽癌的早期诊断是有可能的。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common cancers in the Chinese and other Asian populations. It is one of the serious health problems in Southern China where an annual incidence of more than 20 cases per 100,000 was reported. Metastasis was found in 7% of the patients at the time of initial diagnosis and 20% or more developed metastasis after treatment. However, NPC is often diagnosed late due to its deep location and vague symptoms. Therefore, a diagnostic assay that can detect NPC early would greatly improve the treatment outcome of NPC.Many proteins are overexpressed in tumor tissues, which could activate the immune system to produce their antibodies. Instead of measuring the levels of these over expressed proteins, an alternative approach is to measure the circulating antibodies against tumor-associate -d proteins in the sera of patients. Some autoantibodies could have diagnostic and prognostic value, such as antibodies against BRD2 and alpha-methylacyl-CoA racemase (AMACR) for prostate cancer, antibodies against annexin XI-A and Grb2-associated protein 2 for breast cancer, and antibodies against the epithelial cell adhesion molecule (Ep-CAM) and creatine kinase B for ovarian cancer. There were also several overexpressed proteins that could be used as potential tumor markers for NPC, such as fibronectin, Mac-2 binding protein, plasminogen, inter-α-trypsin inhibitor precursor and zinc-alpha(2)-glycoprotein (ZAG). These observations strongly suggested that a list of potential autoantibody markers will greatly facilitate the development of an assay with high predictive value. Studies of the autoantibodies in NPC patients towards these NPC specific antigens could provide an effective diagnostic assay for NPC. Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma.At first, in this study, to investigate whether there are autoantibodies to NPC in the patients sera in order to find new nasopharyngeal carcinoma(NPC) biomarker, we prepared the cell plate of Epstein Barr-virus negative NPC cell line CNE1, and analyzed the differential reactions between 32 NPC patient serum and 54 normal serum by ELISA. Extracted the total protein of CNE1, and analyzed whether had the specific proteins to react with NPC serum by Western blot.The results of ELISA showed that the average of NPC patients sera antibody absorbance values(0.904±0.032) were significantly elevated above the mean of normal sera antibody absorbance values (0.736±0.028) (P<0.01). The analysis with Western blot showed there were positive bands ,and some of these were unanimously bands, but the intensity increased, and some of these were new bands compared with normal serum. These positive bands may be NPC tumor-associated antigens or NPC tumor-specific antigens. There are autoantibodies reacted with NPC in the sera of patients with NPC, and which not reacted with Epstein-Barr virus. It provided the basis to seek the tumor biomarkers in NPC serum.Secondly, to construct a T7 phage cDNA library from mixed nasopharyngeal carcinoma tissue samples of 8 patients using Novagen’s OrientExpress cDNA Synthesis and Cloning Systems, and biopan the T7 phage cDNA library with sera pooled from ten NPC patients (stages II-IV) and from normal healthy donors. Twenty two phage expressed immunogenic proteins were identified through immunochemical detection and ELISA. Then we measured the levels of autoantibodies against five identified proteins in the sera of NPC patients and in the sera of healthy donors by ELISA to evaluate the sensitivity and specificity of these autoantibodies for NPC detection. Logistic regression was used to evaluate the probability of a sample to be the serum of a patient or the serum of a health donor.We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor-associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage-displayed proteins, 22 have sequence identity with known or putative tumor-associated proteins. The results of immunochemical reactivity of patients’ sera with phage expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors.The phage expressing BMI-1 protein was isolated by screening of a mixture of nasopharyngeal carcinoma (NPC) cDNA T7 phage library and found that the antibody against BMI-1 was elevated in the sera from NPC patients. BMI-1 mRNA was over-expressed at different levels in seven NPC cell lines compared with normal nasopharyngeal epithelial cell line NP69. Histochemistry showed that patient sera were more reactive with BMI-1 than normal sera. Antibody affinity assay using sera from 40 NPC patients and 54 controls showed that BMI-1 antibody was significantly greater in patient sera than in normal controls (patient 0.791±0.025 and normal 0.488±0.042; P<0.001) and the BMI-1 autoantibody be significantly related with the progress of NPC (Benign versus LNPC P=0.001; LNPC versus MNPC P=0.047). Analysis of the results with logistic regression and receiver operating characteristics (ROC) curves showed that BMI-1 antibody was a modest marker for NPC (sensitivity 0.74 and specificity 0.73;AUC=0.8044). The showed that BMI-1 antibody as a potential marker of NPC may be rational, and could have diagnostic and prognostic value.We measured the levels of antibodies against another four phage expressed proteins and the phage expressed protein EBNA-1. The c statistic (i.e., area under the curve) of EBNA-1, which is the positive control, was 0.6110 (P=0.0120). MAGE was the most significant with a c statistic of 0.8922 (P<0.0001). The c statistic of HSP70 was 0.8435 (P<0.0001), the c statistic of CD44 was 0.7857 (P<0.0001), and the c statistic of fibronectin was 0.8091 (P<0.0001). These results showed that phage expressed proteins, HSP70, CD44, fibronectin and MAGE, as markers had greater sensitivity and specificity to predict the NPC disease than EBNA-1.There was a significant correlation between the levels of HSP70, CD44, fibronectin or MAGE antibodies with the clinical stage (P < 0.001) in the NPC groups. The autoantibody level of MAGE positively correlated with T classification: higher T classification correlated with higher autoantibody levels of MAGE(P<0.05). And the antibody levels against other three proteins partially correlated with the clinical stages of NPC. The levels of antibodies can be used to predict the clinical stages of NPC.Our studies suggested that the autoantibodies against tumor-associ -ated antigens in the sera of NPC patients could be used as a screening test for NPC and these autoantibodies can be used for the early detection of NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy. However, analysis of more serum samples is needed to improve the statistical power and to validate this method as a clinically reliable assay. Efficient alternatives detected by ELISA could facilitate development of a blood test for NPC prediction. Testing a full panel of antibodies specific for NPC will be an important part of assay validation and eventually defines the clinical applicability.In summary, we identified immunogenic proteins and their autoantibodies in peripheral blood and generated a panel of antibody markers, which could have significant diagnostic, therapeutic and scientific values.更多还原