生物标志物是与指生理以及病理过程相关的可监测的变化。机体因为受内环境稳态机制的严格调控，生理及病理因素产生的变化会被及时清除，使得血液维持稳定。而尿液是接收血液代谢废物的地方，可以早期且敏感地反映疾病进展，是理想的生物标志物来源。本文采用液相色谱串联质谱技术，对walker-256肿瘤骨种植大鼠模型、肿瘤腘窝淋巴结转移大鼠模型和哮喘小鼠模型进行尿液蛋白质组学的研究。本论文的前言着重介绍了蛋白质组、尿液筛选标志物的优势，以及蛋白质组学技术。 实验探究了walker-256肿瘤骨种植大鼠模型尿液蛋白质组的变化。骨骼是恶性肿瘤常见的发生及转移部位。我们将walker-256细胞种植到大鼠左腿胫骨处的肿瘤骨种植大鼠模型来模拟人类肿瘤骨转移患者，用非标记定量的方法鉴定建模后第3、5、7、13天和对照组大鼠尿液蛋白质。通过LC-MS/MS分析，将实验组鉴定到的蛋白质与对照组相比，发现69个具有人同源蛋白的差异蛋白。在种植肿瘤细胞后的第3天，鉴定到25个差异蛋白，其中6个差异蛋白在前人的研究中被报道与骨转移相关。GO分析表明，第3天的差异蛋白参与多种反应，包括急性期反应、适应性免疫反应和先天免疫反应。第7天的差异蛋白参与了矿物吸收途径。第13天的差异蛋白参与维生素D结合和钙离子结合。这些过程可能与骨转移有关。尿液能够敏感地反映移植骨癌早期的变化，这为今后研究尿肿瘤骨转移的生物标志物提供了有价值的线索。 我们还探究了Walker-256移植瘤腘窝淋巴结转移大鼠模型早期的尿液蛋白质组变化。该部分内容是承接硕士毕业黄师姐的实验内容，前期已经完成了Walker-256肿瘤细胞大鼠腘窝淋巴结转移模型的构建及评估，收集尿液样本。前期的研究结果发现，在形态学方面：注射Walker-256肿瘤细胞27天后，与对照组相比，实验组部分大鼠腘窝淋巴结明显肿大，部分未肿大。本人对收集的第3、8、14天尿液样本进行了酶切处理和LC-MS/MS分析。经质谱鉴定后，肿大组筛选得到155个与人同源的差异蛋白。未肿大组筛选得到148个差异蛋白。对肿大组差异蛋白进行GO分析发现，第14天有2个差异蛋白（Adenosine deaminase，Carcinoembryonic antigen-related cell adhesion molecule 1）参与了Peyer's patch development这一与淋巴结相关的生物过程，但在未肿大组中只有Adenosine deaminase在差异蛋白中出现。 我们研究了哮喘小鼠的尿液蛋白质组变化。哮喘（asthma）是常见的气道慢性炎症疾病。目前还没有针对该疾病的精确的诊断测试。哮喘的鉴别诊断也存在困难。哮喘预后通常是很不错的，尤其对病情较轻的儿童来说，因此需要早发现早治疗。我们将卵白蛋白（OVA）（佐剂：氢氧化铝）采用腹腔注射（D1、D7）和滴鼻（OVA：D14、D15、D16）结合的方式对C56BL/6小鼠给药，来建立小鼠哮喘模型。在多个时间点对小鼠的尿液进行收集，并对对照组、D2、D7、D14和D17的小鼠尿液样本进行前处理和质谱分析。在D7、D14各取一只小鼠肺组织做病理。D18处死余下小鼠做病理，且取肺泡灌洗液进行流式分析。采用数据非依赖性定量法来分析尿液蛋白质组。共鉴定到331个蛋白质，对蛋白进行筛选后得到53个差异蛋白，在第2天共有26个差异蛋白，其中21个上调，5个下调；第7天有24个差异蛋白，其中15个上调，9个下调；第14天有14个差异蛋白，其中6个上调，8个下调；第17天有20个差异蛋白，其中7个上调，13个下调。

Biomarkers are the measurable changes associated with a physiological or pathophysiological process. The body is strictly regulated by the homeostasis mechanism of the internal environment. The changes caused by physiological and pathological factors will be eliminated in time, so that the blood remains stable. Urine is an ideal source of biomarkers because it receives metabolic wastes from blood and can reflect disease progress early and sensitively. Using Liquid chromatography-tandem mass spectrometry((LC-MS/MS), the urine proteome of bone implantation rat model, lymph node metastasis rat model constructed by Walker-256 tumor cells and asthmatic mouse model were studied. The foreword of this paper focuses on the proteomic, advantages of urine biomarkers, and proteomics technology. The experiment explored the changes of urine proteome in early stage of Walker-256 tumor cell bone implantation rat model. Bone is a common site of growth and metastasis of malignant tumors. Walker-256 cells were implanted into the tibia of the left leg of rats to simulate human bone metastasis. Urine samples were then collected on days 3, 5, 7, and 13 and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It was found that 69 differentially proteins with human homologous proteins were identified in the experimental group as compared with the proteins in the control group. Twenty-five differentially proteins were identified on the third day after implantation of tumor cells. Six of the proteins were reported to be associated with bone metastasis in previous studies. GO analysis shows that the differential proteins on day 3 were involved in several responses, including the acute phase response, the adaptive immune response and the innate immune response. The differentially proteins on day 7 were involved in the mineral absorption pathway. The differentially proteins on day 13 were involved in vitamin D binding and calcium ion binding. These processes may be associated with bone metastasis. Urine could sensitively reflect the changes in the early stage of implanted bone cancer. This study provides valuable clues for future studies of urine biomarkers for tumor bone metastasis. We explored the urinary early proteomic changes of Walker-256 transplanted rat model with popliteal lymph node metastasis. This part is to undertake the experimental content of graduate HuangHe. She has completed the construction and evaluation of Walker-256 tumor cell popliteal lymph node metastasis rat model. Urine samples were collected. Previous studies found that: Morphologically, After 27 days’ injection of Walker-256 tumor cells, compared with the control group, the popliteal lymph nodes of some rats in the experimental group were significantly enlarged, and some were not enlarged. The urine samples collected on the 3rd, 8th and 14th days were digested by enzyme and analyzed by LC-MS/MS. 155 differential proteins were screened to be homologous to human in the enlarged group. 148 differential proteins were identified in the non-enlarged group. GO analysis of the enlarged group differentially proteins revealed that two differentially proteins (Adenosine deaminase, Carcinoembryonic antigen-related cell adhesion molecule 1) were involved in the biological process of Peyer's patch development on 14th day. But only Adenosine deaminase appeared in differentially expressed proteins in the non-enlarged group on 14th day. We studied the changes of urine proteome in asthmatic mice. Asthma is a common chronic airway inflammatory disease. There is no accurate diagnostic test for the disease. Differential diagnosis of asthma is also difficult. The prognosis of asthma is usually good, especially for children with mild illness, so early detection and treatment are needed. The mice were given intraperitoneal injection of ovalbumin (OVA)(adjuvant: aluminium hydroxide) on the first and seventh days. On the 14th, 15th and 16th day, mice were given intranasal OVA. The asthma model of C56BL/6 mice was established by the above methods. The urine of mice was collected at several time points. Urine samples from control group, D2, D7, D14 and D17 mice were pretreated and analyzed by mass spectrometry. On the 7th and 14th day, lung tissues were taken from each mouse for pathological examination. On the 17th day, the mice were executed for pathological examination, and the alveolar lavage fluid was taken for flow cytometry. We used data-independent quantitative analysis to analyze urinary proteome. A total of 331 proteins were identified, and 53 differentially proteins were screened. On the 2nd day, there were 26 differentially proteins, 21 of which were up-regulated and 5 down-regulated. On the 7th day, there were 24 differential proteins, of which 15 were up-regulated and 9 were down-regulated. On the 14th day, there were 14 differentially proteins, of which 6 were up-regulated and 8 were down-regulated. On the 17th day, there were 20 differentially proteins, of which 7 were up-regulated and 13 were down-regulated.