膀胱癌是严重威胁人类健康的疾病之一，具备难以筛查易于复发的特点。现有的临床检测手段具有侵入性及灵敏度较低等问题，往往不适用于早期筛查和预后的长期检测。在尿液检查中，血尿的鉴定与尿脱落细胞的研究对于膀胱癌的诊断有很重要的意义，且已有多项研究表明泌尿外泌体与膀胱癌疾病的发展高度相关，是潜在的膀胱癌症检测生物标志物，但是目前还缺乏便捷高效集成式的尿液细胞与外泌体多标志物床旁诊断手段。因此，本文基于微流控系统、免疫荧光技术和蛋白质组学技术，以构建用于膀胱癌筛查和预后的全集成便捷分析芯片为目标，具体开展工作如下：（1）设计与构建对尿脱落细胞和血细胞进行捕获和分析的细胞分析模块，研究微流控芯片上尿液样本中尿脱落细胞和血细胞捕获与分析技术。（2）设计与实现对尿液中总外泌体和膀胱癌相关外泌体进行捕获和分析的外泌体分析模块，研究微流控芯片上尿液样本中总外泌体与膀胱癌特异性外泌体捕获与分析技术。（3）在尿液外泌体捕获与分析的基础上，通过二硫键设计实现非破坏性外泌体完整洗脱，从尿液中分离回收膀胱癌相关外泌体以进行后续分析，有助于未来进一步丰富基于尿液的膀胱癌筛查和预后检测项目。总之，本论文围绕构建临床驱动的尿液中细胞、外泌体床旁诊断技术而展开，为膀胱癌的诊断和研究探索新的路径。
Bladder cancer is a major threat to human health globally, which is usually found at late stages and prone to recurrence. Current clinical detection methods are not suitable for early screening and prognosis due to their invasiveness and low sensitivity. The identification of hematuria in urine and the study of epithelial cells in urine are of great significance for the diagnosis of bladder cancer. Currently, a number of studies have shown that urinary exosomes are highly correlated with the development of bladder cancer, and are potential biomarkers for bladder cancer detection. Therefore, based on the custom-made microfluidic system, immunofluorescence technology and protein detection, this paper aims to construct a fully-integrated analysis chip for point-of-care bladder cancer screening and prognosis. Specific works are as follows: (1) Design and construction of a microfluidic cell analysis module, and study of the trapping and detection technology for blood cells and epithelial cells in urine.(2) Devisal and implementation of an exosome analysis module, and study of the capture and examination techniques for total exosomes and bladder cancer-specific exosomes in urine samples in the microfluidic chip.(3) On the basis of microfluidic urinary exosome capture and analysis, disulfide bonds were introduced to achieve non-destructive recovery of whole exosomes for downstream analysis of surface antigens and internal molecules, facilitating multiplex analysis of urinary exosomes for screening and prognosis of bladder cancer in the future. In summary, this thesis focuses on the development of clinic-driven point-of-care detection techniques for the capture and analysis of cells and exosomes in urine, enabling exploration of new approaches for the diagnosis and research of bladder cancer.
