膀胱癌患者尿液exosome中潜在肿瘤标志物研究

Bladder cancer remains a highly prevalent and lethal malignancy in china. Currently, no tools exist for effective detection and monitoring of this cancer. Human urinary exosomes are microvesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary track and may serve as a suitable non-invasive starting material for biomarker discovery relevant to a variety of renal disease. Some bladder cancer specific proteins are found to be present in the urinary exosomes of individuals with bladder cancer in previous researches, and it's of great researching potential and application value to mining bladder cancer related biomarkers from these vesicles. In the present research, we aim to extensively analyze the protein profile of the urinary exosomes of individuals with bladder cancer, and to identify and validate bladder cancer related biomarkers by proteomic methods. We'll further explore the relevance between candidate biomarkers and bladder cancer by analyzing the expression and location of the candidate biomarkers on the urinary exosomes, bladder cancer tissue and cells. The result of this research may develop a new method of objective and non-invasive diagnosis and monitoring of bladder cancer in urine and lay foundation for the treatment and prevention of bladder cancer.

膀胱癌是我国泌尿系统最常见的恶性肿瘤，目前对其诊断、监测尚缺乏有效可靠的生物学指标，发现新的膀胱癌标志物对于其初筛、早诊及预后预测都具有十分重要的意义。尿液exosome是由泌尿道上皮细胞所分泌的小囊泡，已有研究结果显示，膀胱癌患者尿液exosome中存在膀胱癌特异性的蛋白质，从中筛选相关的肿瘤标志物具有较大研究潜力和应用价值。本课题在前期成功分离尿液exosome和相关蛋白质组学研究的基础上全面分析膀胱癌尿液exosome中蛋白质的表达情况，拟运用比较蛋白质组学方法从中筛选膀胱癌相关的差异表达蛋白质并对其进行临床验证，进一步筛选出具有一定特异性和敏感性的膀胱癌潜在标志物，分析其在尿液exosome、膀胱癌组织和细胞中的表达、定位情况，并结合相关的功能研究探讨其与膀胱癌的相关性及在膀胱癌发生发展中的作用，为发展新的准确无创的膀胱肿瘤诊断、监测方法奠定基础，为膀胱癌的防治提供新思路与新靶点。

摘要：通过比较正常对照者和膀胱癌患者尿中exosomes蛋白质组，筛选可能存在于尿exosomes中的与膀胱癌相关的特异性蛋白质。方法：以超速离心法分离正常人和膀胱癌患者尿中exosomes，通过电镜对exosomes进行鉴定，运用1D SDS-PAGE对exosomes蛋白质组进行分离，分别从凝胶上切取正常对照组和疾病组45KD～35KD条带，胰蛋白酶酶解后，进行HPLC-CHIP-MS/MS分析，串联质谱数据进入UniProtKB/SWISS-PORT数据库搜索，找出差异蛋白。结合western blot技术和免疫组化技术分析鉴定搜索得到的差异蛋白——锌-α2糖蛋白（AZGP1）。结果：通过UniProtKB/SWISS-PORT数据库搜索，从尿中exosomes蛋白条带中共鉴定出42种蛋白质，其中16种仅从膀胱癌患者组中鉴定出。通过实验发现AZGP1蛋白在膀胱癌患者尿液exosomes和癌组织中均有较高表达。结论：膀胱癌患者尿液exosomes中的AZGP1可能来源于肿瘤组织，AZGP1可能是一种膀胱癌相关肿瘤标志物的候选分子。