鼻咽癌血清蛋白质分子标志物的iTRAQ定量、验证、功能分析和调控通路构建

For the diagnosis, treatment effect and prognosis evaluation of human diseases, we cannot detect without various biomarkers in the blood, which is the most commonly utilized and suitable bodily liquid. Guangxi is a high-incidence area of nasopharyngeal carcinoma (NPC), but it lacks special and excellent assistant biomarkers in the screening, evaluation at different clinical stages, mechanisms of invasion and metastasis of NPC. In our recent studies, compare the serum of NPC patients and normal peoples using SELDI-TOF-MS and iTRAQ-coupled LC/MS-MS technology, the results showed that there are some differential proteins would be potential biomarkers in NPC serum. In order to have a deeper understanding the differential proteins, based on the found so far, this project will investigate detailed qualitative and quantitative analysis for the differential proteins in the serum of different clinical stage (I, II, III, IV period) and of EBV-VCA-IgA-positive and -negative patients with NPC by iTRAQ-coupled LC-MS/MS technology. For scientific significance differential proteins will be verified and functional analyzed as candidate protein molecular marker. Moreover, construct the regulatory pathways of interaction or regulation between the candidate protein molecular markers and other proteins. Finally, this project would obtain the protein molecular markers of NPC serum, which will make research foundations for the early diagnosis, prevention, treatment, the development of medication target, and to explore the pathogenesis of NPC, and so on.

人类疾病的诊断、疗效和预后评估等都离不开血液中各标志物的检测，血液是最常利用和最合适的体液。广西属鼻咽癌高发区，对鼻咽癌的筛查、分期评估以及浸润转移机制的了解等均缺乏特异性和优秀的辅助性标志物。在我们利用SELDI-TOF-MS和iTRAQ结合LC-MS/MS技术比较鼻咽癌患者与正常人血清的研究中，发现鼻咽癌血清中存在有可能作为标志物的差异蛋白质。为了更深入地了解那些差异蛋白质，本项目将在原有的研究基础上，利用iTRAQ-LC-MS/MS技术对不同临床分期（Ⅰ、Ⅱ、Ⅲ、Ⅳ期）及EBV-VCA-IgA阳性和阴性鼻咽癌患者血清中的差异蛋白质进行详尽的定性和定量分析，对其中具有科学意义的差异蛋白质作为鼻咽癌候选分子标志物进行验证和功能分析，并构建其蛋白质之间相互作用或调控的通路图谱，最后获得鼻咽癌血清蛋白质分子标志物，为鼻咽癌的早期诊断、防治和用药靶标的开发、探寻鼻咽癌的发病机制等研究奠定基础。

广西属鼻咽癌高发区，因此寻找鼻咽癌早诊早治以及侵袭迁移的分子生物标志物具有重要意义。项目通过收集各期鼻咽癌病人及正常人血清样本，经多重亲和免疫去除高丰度蛋白后运用iTRAQ结合2D LC-MS/MS技术进行2次重复定量蛋白组学分析，共鉴定出274个置信蛋白，2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，其中表达上调蛋白中S100A8和S100A9 在2次定量实验中均稳定显著高表达。血浆验证实验中，采用ELISA法检测非角化性低分化初诊鼻咽癌患者（Ⅰ、Ⅱ期36例，Ⅲ期107例，Ⅳ期104例）和健康人（111例）血浆中S100A8、S100A9的表达，结果显示，鼻咽癌患者血浆中S100A8、S100A9含量显著高于正常人，其中Ⅰ、Ⅱ期和Ⅲ期鼻咽癌患者血浆中S100A8、S100A9高于正常人，差异显著，Ⅳ期鼻咽癌患者血浆中S100A9高于正常人，而S100A8的均值虽高于正常人，但无显著差异，且S100A8、S100A9在鼻咽癌Ⅰ、Ⅱ期与Ⅳ期之间、Ⅲ期与Ⅳ期之间具有统计学差异，而Ⅰ、Ⅱ期与Ⅲ期之间无显著差异，同时结果亦显示鼻咽癌患者血浆中S100A8、S100A9的表达呈正相关。组织验证实验中，免疫组化分析显示S100A8、S100A9表达上调于鼻咽癌组织，表达于癌细胞的胞浆，S100A8阳性率70%以上，S100A9阳性率91.7%，均较健康咽炎患者高。细胞实验Western Blot分析鼻咽癌高分化CNE1和低分化CNE2细胞系及正常上皮细胞NP69，结果显示CNE1和CNE2中的S100A8、S100A9表达均高于NP69；使用S100A8 siRNA与S100A9 siRNA分别转染CNE1后进行划痕实验，结果显示CNE1迁移功能减弱，且MMP7基因的表达量亦有不同程度的减弱；使用1µg/ml的S100A8/S100A9浓度培养CNE1和CNE2，侵袭和迁移实验结果显示，S100A8、S100A9可促进鼻咽癌细胞的侵袭和迁移，同时细胞免疫组化结果显示1µg/ml的S100A8/S100A9浓度培养CNE1，β-Catenin的免疫阳性染色细胞增多，MMP7表达上调。本项目还成功构建了携带绿色荧光报告基因的EBV-LMP1真核表达载体与S100A8-S100A9共表达质粒，为S100A8、S100A9的后续研究打下了基础。