北京师范大学 生物化学与分子生物系 基因工程药物与生物技术北京市重点实验室, 北京 100875
收稿日期：2020-04-17；接收日期：2020-07-06；网络出版时间：2020-08-13
基金项目：国家重点研发计划(Nos. 2018YFC0910202, 2016YFC1306300)，中央高校基础研究经费(No. 2020KJZX002)，北京市自然科学基金(No. 7172076), 北京市合作建设项目(No. 110651103), 北京师范大学(No. 11100704)，北京协和医院(No. 2016-2.27)资助
摘要：不同的微生物都可以引起腹腔感染，文中尝试利用尿液来区分不同的微生物感染。通过在大鼠腹腔内分别注射大肠杆菌、金黄色葡萄球菌和白色念球菌建立3种模型，收集感染后0、12、36、72 h的尿液，并使用液相色谱串联质谱技术(LC-MS/MS)对尿蛋白进行分析。与感染前相比，在大肠杆菌腹腔注射模型中共鉴定到69个差异蛋白，在金黄色葡萄球菌腹腔注射模型中共鉴定到31个差异蛋白，在白色念球菌腹腔注射模型中共鉴定到38个差异蛋白。结果表明，腹腔注射不同的微生物时尿蛋白质组不同，提示尿液可能对不同的腹腔感染有鉴别诊断的潜能。
关键词：腹腔感染尿液蛋白组学鉴别诊断
Comparison of urine proteome in rats intraperitoneally infected by different microorganisms
Wenshu Meng, Yameng Zhang, Yuanrui Hua, Yijin Jiang, Jiawen Kong, Youhe Gao
Gene Engineering Drug and Biotechnology Beijing Key Laboratory, Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, China
Received: April 17, 2020; Accepted: July 6, 2020; Published: August 13, 2020
Supported by: National Key Research and Development Program of China (Nos. 2018YFC0910202, 2016YFC1306300), Fundamental Research Funds for the Central Universities (No. 2020KJZX002), Beijing Natural Science Foundation (No. 7172076), Beijing Cooperative Construction Project (No. 110651103), Beijing Normal University (No. 11100704), Peking Union Medical College Hospital (No. 2016-2.27)
Corresponding author: Youhe Gao. Tel: +86-10-58804285; E-mail: gaoyouhe@bnu.edu.cn.
Abstract: Different microorganisms can cause intraperitoneal infection. This study was to distinguish different microbial infections by urine analysis. Rats were intraperitoneally injected with Escherichia coli, Staphylococcus aureus, and Candida albicans, separately. Urine samples were collected from rats at 0, 12, 36 and 72 h after infection. Urinary proteins were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Compared with the control (without infection), a total of 69 differential proteins were identified in rats injected with E. coli. A total of 31 differences proteins were identified in rats injected with S. aureus. A total of 38 differential proteins were identified in rats injected with C. albicans. Urine proteome was different when rats were infected by different microorganisms, suggesting that urine may have the potential for differential diagnosis of different intraperitoneal infections.
Keywords: intraperitoneal infectionurineproteomedifferential diagnosis
不同的微生物都可以引起腹腔感染，当前对腹腔感染的鉴别诊断仍然存在困难。尿液作为研究疾病标志物的良好来源，具有早期和敏感的特点。首先，尿液不受稳态机制的调控，能够富集机体产生的早期变化[1]。已经在多个疾病动物模型中发现，在病理变化出现之前，尿蛋白组就发生了变化[2]。比如，在阿尔兹海默症转基因小鼠的脑部淀粉斑块沉积出现之前，尿液中有29个蛋白发生变化，其中有24个蛋白曾被报道过与阿尔兹海默症有关或作为标志物[3]。其次，尿液能够极其敏感地反映机体的状态。比如，在大鼠皮下注射极少量的肿瘤细胞时，尿蛋白就能反映出与癌症相关的变化[4]。此外，当Walker-256肿瘤细胞被注射到皮下[5]、肝脏[6]、骨[7]、肺[8]和脑部[9]不同的器官生长时，尿蛋白的变化不同，这表明尿液具有区分相同肿瘤细胞在不同器官生长的潜能，提示尿液可能具有良好的鉴别诊断的能力。那么，当不同的微生物注射到腹腔时，尿液能表现出差异吗？
大肠杆菌Escherichia coli、金黄色葡萄球菌Staphylococcus aureus和白色念球菌Candida albicans是常见的腹腔感染的微生物。本研究通过将一定浓度的大肠杆菌、金黄色葡萄球菌和白色念球菌菌液分别注射到大鼠的腹腔建立3种腹腔感染模型，对注射后0、12、36、72 h的尿液进行非标记定量蛋白组学分析，探究3种模型尿液蛋白组的变化情况并进行比较。
1 材料与方法1.1 实验动物模型的建立1.1.1 大肠杆菌腹腔注射模型的建立雄性Wistar大鼠8只，体重140–160 g，购自北京维通利华实验动物技术有限公司，所有动物在标准环境中(室温(22±1) ℃，湿度65%–70%)饲养。动物实验遵循北京师范大学生命科学学院伦理委员会的审查和批准。建立方法如下：实验组和对照组各4只大鼠，实验组大鼠腹腔内注射2 mL大肠杆菌菌液(1×108 CFU/mL)，对照组注射同等体积的生理盐水。
1.1.2 金黄色葡萄球菌腹腔注射模型的建立雄性SD大鼠6只，体重180–200 g。建立方法如下：实验组和对照组各3只大鼠，实验组大鼠腹腔内注射2 mL金黄色葡萄球菌菌液(1×107 CFU/mL)，对照组注射同等体积的生理盐水。
1.1.3 白色念球菌腹腔注射模型的建立雄性SD大鼠6只，体重180–200 g。建立方法如下：实验组和对照组各3只大鼠，实验组大鼠腹腔内注射2 mL白色念球菌菌液(1×107 CFU/mL)，对照组注射同等体积的生理盐水。
1.2 尿液的收集在腹腔注射菌液后的0、12、36、72 h统一将大鼠置于代谢笼中收集尿液，并放入?80 ℃冰箱保存。
1.3 尿液蛋白的提取和蛋白酶解将每个模型中实验组大鼠在4个时间点收集的尿液用12 000×g离心40 min，取上清用3倍体积的乙醇沉淀过夜，然后12 000×g离心30 min。将蛋白沉淀重悬于裂解液中(8 mol/L尿素，2 mol/L硫脲，25 mmol/L二硫苏糖醇，50 mmol/L Tris)。用Bradford法测量蛋白浓度。本研究共提取40个尿蛋白样品，其中大肠杆菌模型16个，金黄色葡萄球菌模型12个，白色念球菌模型12个。
使用滤器辅助样品制备(FASP)方法进行尿蛋白酶解[10]。将尿蛋白加载到10 kDa超滤管的滤膜(Pall，Port Washington，NY，USA)上，用UA溶液(8 mol/L尿素，0.1 mol/L Tris-HCl，pH 8.5)和25 mmol/L NH4HCO3溶液洗涤两次；加入20 mmol/L二硫苏糖醇在37 ℃中变性1 h，再与50 mmol/L碘乙酰胺在黑暗中烷基化30 min，用UA溶液和NH4HCO3洗涤两次，以1︰50比例加入蛋白胰酶在37 ℃水浴中消化14 h。将多肽混合物通过HLB柱(Waters，Milford，MA)除盐后用真空干燥仪进行抽干。
1.4 LC-MS/MS串联质谱分析酶解后的样品用0.1%甲酸复溶，肽段浓度稀释为0.5 μg/μL，每个样品取1 μg使用EASY-nLC 1200色谱系统(Thermo Fisher Scientific，USA)和Orbitrap Fusion Lumos Tribrid质谱仪(Thermo Fisher Scientific，USA)进行质谱采集和分析。
1.5 数据分析将质谱采集的数据用Mascot软件(版本2.4.1)进行数据库检索，设置参数：数据库采用SwissProt大鼠数据库，胰酶酶切，最多允许两个漏切位点，母离子质量容差10 ppm，碎片离子质量容差0.05 Da。半胱氨酸的氨酰甲基化修饰为固定修饰，蛋氨酸的氧化为可变修饰。Mascot检索后的文件导入Scaffold软件(版本4.7.5)，参数设置：多肽FDR设为1%，蛋白水平的FDR设为最大(1%)，每个蛋白包含两个以上的特异肽段[11]。根据谱图数进行差异蛋白筛选[12]。
1.6 统计学分析每个样品取两次技术重复的平均值用于统计学分析。采用自身对照的方式，将12、36、72 h鉴定到的蛋白分别与0 h进行比较。筛选差异蛋白的条件为：组间变化倍数≥1.5或≤0.67，双尾非配对t检验P < 0.05，高丰度组的平均蛋白谱图数≥3，且高丰度组每个样品的蛋白谱图都高于低丰度组。
1.7 Gene ontology分析使用DAVID 6.8 (https://david.ncifcrf.gov/)对3个模型鉴定到的差异蛋白进行生物学过程、细胞定位和分子功能3个方面的功能富集分析[13]。
2 结果与分析2.1 实验动物的行为学特征本研究中，在造模后对大鼠进行行为学观察。从行为学观察来看，对照组大鼠活动正常，饮食饮水正常，实验组与对照组相比，无明显的行为学差异。
2.2 腹腔注射微生物模型尿蛋白组学变化2.2.1 腹腔注射大肠杆菌模型尿蛋白组学变化在腹腔注射大肠杆菌的模型中，选取4只实验组大鼠的0、12、36、72 h的尿液蛋白，进行非标记LC-MS/MS质谱鉴定。该模型共鉴定到832个蛋白(≥2个特异性的多肽，蛋白水平FDR < 1%)，将12、36、72 h的尿蛋白分别与0 h进行比较，筛选标准：组间变化倍数FC≥1.5或≤0.67，P < 0.05，高丰度组的平均蛋白谱图数≥3，且高丰度组每个样品的蛋白谱图都高于低丰度组。
相对于0 h，在大肠杆菌注射后12 h鉴定到14个差异蛋白，其中12个上调，2个下调；在36 h鉴定到5个差异蛋白，其中3个上调，2个下调；在72 h鉴定到61个差异蛋白，其中50个上调，11个下调。不同时间点鉴定到的差异蛋白的详细信息列在表 1。用维恩图展示了不同时间点鉴定到的差异蛋白的重叠情况(图 1)，其中有1个蛋白在3个时间点连续被鉴定到。
表 1 腹腔注射大肠杆菌模型鉴定到的尿液差异蛋白Table 1 The differential proteins identified in E. coli intraperitoneal injection model
| Accession | Protein name | Trend | Fold change | P value | ||
| 12 h | 36 h | 72 h | ||||
| Q9QZA2 | Programmed cell death 6-interacting protein | ↑ | 1.57 | 1.69 | 2.11 | 1.50E-02 |
| Q641Z6 | EH domain-containing protein 1 | ↑ | 4.25 | 6.67 | 1.71E-04 | |
| P61589 | Transforming protein RhoA | ↑ | 2.75 | 2.25 | 1.45E-03 | |
| Q63751 | Vomeromodulin | ↑ | 2.61 | 3.14 | 7.05E-03 | |
| P85973 | Purine nucleoside phosphorylase | ↑ | 2.33 | 2.67 | 1.31E-03 | |
| P46413 | Glutathione synthetase | ↑ | 2.13 | 2.78 | 1.69E-03 | |
| Q9R063 | Peroxiredoxin-5, mitochondrial | ↑ | 1.83 | 2.28 | 9.08E-03 | |
| Q64319 | Neutral and basic amino acid transport protein rBAT | ↑ | 1.54 | 2.06 | 1.67E-02 | |
| Q6AYT0 | Quinone oxidoreductase | ↑ | 1.66 | 2.06 | 7.18E-03 | |
| Q99PS8 | Histidine-rich glycoprotein | ↓ | 0.37 | 0.36 | 4.77E-03 | |
| P10760 | Adenosylhomocysteinase | ↑ | 6.50 | 1.86E-03 | ||
| P11348 | Dihydropteridine reductase | ↑ | 2.07 | 9.28E-03 | ||
| Q497B0 | Omega-amidase NIT2 | ↑ | 2.00 | 3.21E-02 | ||
| P55159 | Serum paraoxonase/arylesterase 1 | ↑ | 1.75 | 1.67E-02 | ||
| P13596 | Neural cell adhesion molecule 1 | ↓ | 0.67 | 6.77E-03 | ||
| P19218 | Pancreatic secretory granule membrane major glycoprotein GP2 | ↓ | 0.66 | 1.21E-03 | ||
| P30120 | Metalloproteinase inhibitor 1 | ↑ | 8.33 | 7.71E-03 | ||
| D4ACX8 | Protocadherin-16 | ↓ | 0.28 | 1.68E-02 | ||
| O70377 | Synaptosomal-associated protein 23 | ↑ | ∞ | 3.94E-03 | ||
| P70502 | Solute carrier organic anion transporter family member 1A3 | ↑ | 31.00 | 4.15E-03 | ||
| P18757 | Cystathionine gamma-lyase | ↑ | 19.33 | 5.60E-03 | ||
| Q4KLZ6 | Triokinase/FMN cyclase | ↑ | 10.25 | 1.52E-02 | ||
| P53790 | Sodium/glucose cotransporter 1 | ↑ | 10.00 | 2.76E-03 | ||
| Q3ZAV1 | Solute carrier family 22 member 12 | ↑ | 10.00 | 1.23E-02 | ||
| P06760 | Beta-glucuronidase | ↑ | 9.00 | 5.68E-04 | ||
| P46720 | Solute carrier organic anion transporter family member 1A1 | ↑ | 7.17 | 2.87E-03 | ||
| Q63355 | Unconventional myosin-Ic | ↑ | 6.88 | 1.01E-03 | ||
| Q5M7T9 | Threonine synthase-like 2 | ↑ | 6.75 | 2.04E-03 | ||
| Q62687 | Sodium-dependent neutral amino acid transporter B(0)AT3 | ↑ | 6.60 | 2.99E-03 | ||
| Q7TQ94 | Deaminated glutathione amidase | ↑ | 6.00 | 1.11E-03 | ||
| Q9WUW9 | Sulfotransferase 1C2A | ↑ | 5.85 | 1.53E-03 | ||
| Q711G3 | Isoamyl acetate-hydrolyzing esterase 1 homolog | ↑ | 5.20 | 3.76E-03 | ||
| Q9WTW7 | Solute carrier family 23 member 1 | ↑ | 4.94 | 1.36E-03 | ||
| P09606 | Glutamine synthetase | ↑ | 4.63 | 1.04E-02 | ||
| Q80W57 | ATP-binding cassette sub-family G member 2 | ↑ | 4.63 | 6.06E-03 | ||
| Q63424 | Solute carrier family 15 member 2 | ↑ | 4.11 | 5.87E-04 | ||
| Q9ESG3 | Collectrin | ↑ | 3.57 | 1.52E-02 | ||
| Q9Z0W7 | Chloride intracellular channel protein 4 | ↑ | 3.56 | 2.38E-03 | ||
| O08557 | N(G), N(G)-dimethylarginine dimethylaminohydrolase 1 | ↑ | 3.29 | 2.31E-03 | ||
| P25093 | Fumarylacetoacetase | ↑ | 3.13 | 8.35E-03 | ||
| P19468 | Glutamate--cysteine ligase catalytic subunit | ↑ | 3.09 | 1.43E-03 | ||
| Q02974 | Ketohexokinase | ↑ | 2.69 | 1.87E-04 | ||
| P50399 | Rab GDP dissociation inhibitor beta | ↑ | 2.68 | 5.46E-03 | ||
| Q9JJ40 | Na(+)/H(+) exchange regulatory cofactor NHE-RF3 | ↑ | 2.54 | 1.83E-03 | ||
| Q63530 | Phosphotriesterase-related protein | ↑ | 2.47 | 6.29E-03 | ||
| P48508 | Glutamate--cysteine ligase regulatory subunit | ↑ | 2.38 | 4.46E-04 | ||
| P50398 | Rab GDP dissociation inhibitor alpha | ↑ | 2.30 | 1.61E-02 | ||
| P38918 | Aflatoxin B1 aldehyde reductase member 3 | ↑ | 2.27 | 1.59E-02 | ||
| O35763 | Moesin | ↑ | 2.24 | 4.81E-04 | ||
| Q64602 | Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial | ↑ | 2.21 | 3.28E-03 | ||
| P54311 | Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 | ↑ | 1.89 | 2.34E-02 | ||
| Q9JJ19 | Na(+)/H(+) exchange regulatory cofactor NHE-RF1 | ↑ | 1.82 | 3.10E-03 | ||
| P00884 | Fructose-bisphosphate aldolase B | ↑ | 1.81 | 4.03E-03 | ||
| Q5XI73 | Rho GDP-dissociation inhibitor 1 | ↑ | 1.77 | 4.01E-02 | ||
| P31977 | Ezrin | ↑ | 1.75 | 1.94E-03 | ||
| P07861 | Neprilysin | ↑ | 1.68 | 4.30E-04 | ||
| Q99MA2 | Xaa-Pro aminopeptidase 2 | ↑ | 1.65 | 3.53E-03 | ||
| P07314 | Glutathione hydrolase 1 proenzyme | ↑ | 1.63 | 2.79E-03 | ||
| P60711 | Actin, cytoplasmic 1 | ↑ | 1.52 | 4.08E-03 | ||
| P04073 | Gastricsin | ↓ | 0.67 | 1.85E-03 | ||
| P01015 | Angiotensinogen | ↓ | 0.64 | 3.61E-03 | ||
| P97605 | Ephrin-A5 | ↓ | 0.62 | 9.99E-04 | ||
| P10959 | Carboxylesterase 1C | ↓ | 0.60 | 6.69E-04 | ||
| Q1WIM1 | Cell adhesion molecule 4 | ↓ | 0.54 | 7.34E-03 | ||
| Q9QZQ5 | CCN family member 3 | ↓ | 0.43 | 1.67E-02 | ||
| P24594 | Insulin-like growth factor-binding protein 5 | ↓ | 0.41 | 5.16E-03 | ||
| P27590 | Uromodulin | ↓ | 0.40 | 1.44E-03 | ||
| Q99MH3 | Hepcidin | ↓ | 0.29 | 1.16E-04 | ||
| Q63108 | Carboxylesterase 1E | ↓ | 0.18 | 3.04E-03 | ||
表选项
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| 图 1 腹腔注射大肠杆菌模型中12、36、72 h鉴定的差异蛋白的维恩图 Fig. 1 The Venn diagram of differential proteins identified at 12, 36, and 72 h in E. coli intraperitoneal injection model. |
| 图选项 |
2.2.2 腹腔注射金黄色葡萄球菌模型尿蛋白组学变化在腹腔注射金黄色葡萄球菌的模型中，选取3只实验组大鼠的0、12、36、72 h的尿液蛋白，进行非标记LC-MS/MS质谱鉴定。该模型共鉴定到798个蛋白(≥2个特异性的多肽，蛋白水平FDR < 1%)。相对于0 h，在金黄色葡萄球菌注射后的12 h鉴定到9个差异蛋白，其中6个上调，3个下调；在36 h鉴定到11个差异蛋白，其中6个上调，5个下调；在72 h鉴定到17个差异蛋白，其中10个上调，7个下调。不同时间点鉴定到的差异蛋白的详细信息列在表 2。用维恩图展示了不同时间点鉴定到的差异蛋白的重叠情况(图 2)，其中有2个蛋白在3个时间点连续被鉴定到。
表 2 腹腔注射金黄色葡萄球菌模型鉴定到的尿液差异蛋白Table 2 The differential proteins identified in S. aureus intraperitoneal injection model
| Accession | Protein name | Trend | Fold change | P value | ||
| 12 h | 36 h | 72 h | ||||
| Q66H12 | Alpha-N-acetylgalactosaminidase | ↑ | 2.44 | 1.78 | 2.61 | 6.52E-03 |
| P04639 | Apolipoprotein A-Ⅰ | ↓ | 0.48 | 0.48 | 0.45 | 1.15E-02 |
| O88766 | Neutrophil collagenase | ↑ | 3.23 | 2.62 | 1.43E-02 | |
| Q9WTW7 | Solute carrier family 23 member 1 | ↓ | 0.65 | 0.59 | 2.70E-02 | |
| P02625 | Parvalbumin alpha | ↑ | 3.09 | 1.74E-03 | ||
| Q9ES87 | Prostasin | ↑ | 2.00 | 1.73E-02 | ||
| Q63416 | Inter-alpha-trypsin inhibitor heavy chain H3 | ↑ | 1.67 | 2.54E-02 | ||
| P18292 | Prothrombin | ↑ | 1.52 | 2.20E-02 | ||
| Q80WF4 | Transmembrane protein 132A | ↓ | 0.37 | 3.27E-02 | ||
| P45592 | Cofilin-1 | ↑ | 2.57 | 2.54E-02 | ||
| P85973 | Purine nucleoside phosphorylase | ↑ | 2.55 | 1.61E-03 | ||
| Q7TQ94 | Deaminated glutathione amidase | ↑ | 2.29 | 3.83E-02 | ||
| P42854 | Regenerating islet-derived protein 3-gamma | ↑ | 1.95 | 2.74E-02 | ||
| P97574 | Stanniocalcin-1 | ↑ | 1.67 | 2.49E-02 | ||
| P31211 | Corticosteroid-binding globulin | ↓ | 0.55 | 1.47E-04 | ||
| Q06496 | Sodium-dependent phosphate transport protein 2A | ↓ | 0.52 | 1.61E-02 | ||
| Q99PS8 | Histidine-rich glycoprotein | ↓ | 0.37 | 4.47E-02 | ||
| P47853 | Biglycan | ↓ | 0.24 | 3.29E-02 | ||
| P14942 | Glutathione S-transferase alpha-4 | ↑ | 4.40 | 1.91E-02 | ||
| P25093 | Fumarylacetoacetase | ↑ | 2.77 | 4.52E-02 | ||
| O35952 | Hydroxyacylglutathione hydrolase, mitochondrial | ↑ | 2.18 | 3.14E-02 | ||
| P26051 | CD44 antigen | ↑ | 2.05 | 3.65E-03 | ||
| P01835 | Ig kappa chain C region, B allele | ↑ | 1.72 | 3.00E-02 | ||
| Q80WD1 | Reticulon-4 receptor-like 2 | ↑ | 1.68 | 2.20E-02 | ||
| P82450 | Sialate O-acetylesterase | ↑ | 1.67 | 3.02E-03 | ||
| P14630 | Apolipoprotein M | ↑ | 1.59 | 4.54E-03 | ||
| P54311 | Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 | ↓ | 0.61 | 4.37E-02 | ||
| P54313 | Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 | ↓ | 0.56 | 3.11E-02 | ||
| Q6MG71 | Choline transporter-like protein 4 | ↓ | 0.53 | 2.73E-02 | ||
| Q6RUV5 | Ras-related C3 botulinum toxin substrate 1 | ↓ | 0.47 | 1.94E-02 | ||
| P08010 | Glutathione S-transferase Mu 2 | ↓ | 0.29 | 3.16E-02 | ||
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| 图 2 腹腔注射金黄色葡萄球菌模型中12、36、72 h鉴定的差异蛋白的维恩图 Fig. 2 The Venn diagram of differential proteins identified at 12, 36, and 72 h in S. aureus intraperitoneal injection model. |
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2.2.3 腹腔注射白色念球菌模型尿蛋白组学变化在腹腔注射白色念球菌的模型中，选取3只实验组大鼠的0、12、36、72 h的尿液蛋白，进行非标记LC-MS/MS质谱鉴定。该模型共鉴定到696个蛋白(≥2个特异性的多肽，蛋白水平FDR < 1%)。相对于0 h，在白色念球菌注射后的12 h鉴定到11个差异蛋白，其中3个上调，8个下调；在36 h鉴定到14个差异蛋白，其中2个上调，12个下调；在72 h鉴定到24个差异蛋白，其中4个上调，20个下调。不同时间点鉴定到的差异蛋白的详细信息列在表 3。用维恩图展示了不同时间点鉴定到的差异蛋白的重叠情况(图 3)，其中有1个蛋白在3个时间点连续被鉴定到。
表 3 腹腔注射白色念球菌模型鉴定到的尿液差异蛋白Table 3 The differential proteins identified in C. albicans intraperitoneal injection model
| Accession | Protein name | Trend | Fold change | P value | ||
| 12 h | 36 h | 72 h | ||||
| Q80WF4 | Transmembrane protein 132A | ↓ | 0.50 | 0.35 | 0.35 | 1.73E-02 |
| P30152 | Neutrophil gelatinase-associated lipocalin | ↑ | 2.23 | 3.96 | 2.21E-02 | |
| P50398 | Rab GDP dissociation inhibitor alpha | ↓ | 0.63 | 0.28 | 1.32E-02 | |
| P50137 | Transketolase | ↓ | 0.46 | 0.11 | 1.06E-02 | |
| P10758 | Lithostathine | ↑ | 7.00 | 6.67 | 3.99E-02 | |
| P25113 | Phosphoglycerate mutase 1 | ↓ | 0.63 | 0.18 | 2.85E-02 | |
| Q5U2Q3 | Ester hydrolase C11orf54 homolog | ↓ | 0.63 | 0.46 | 2.13E-02 | |
| Q8R5M3 | Leucine-rich repeat-containing protein 15 | ↓ | 0.61 | 0.48 | 3.14E-02 | |
| Q6P6V0 | Glucose-6-phosphate isomerase | ↓ | 0.48 | 0.45 | 7.18E-03 | |
| P04642 | L-lactate dehydrogenase A chain | ↓ | 0.39 | 0.36 | 4.46E-02 | |
| P17559 | Uteroglobin | ↑ | 1.92 | 1.32E-02 | ||
| P08721 | Osteopontin | ↑ | 1.86 | 4.57E-02 | ||
| O08628 | Procollagen C-endopeptidase enhancer 1 | ↓ | 0.64 | 2.32E-02 | ||
| Q6X936 | Kin of IRRE-like protein 1 | ↓ | 0.56 | 1.61E-02 | ||
| Q9Z1Y3 | Cadherin-2 | ↓ | 0.51 | 8.58E-03 | ||
| Q793F9 | Vacuolar protein sorting-associated protein 4A | ↓ | 0.44 | 7.49E-03 | ||
| P97710 | Tyrosine-protein phosphatase non-receptor type substrate 1 | ↓ | 0.37 | 2.24E-02 | ||
| Q9R1T3 | Cathepsin Z | ↓ | 0.64 | 2.32E-02 | ||
| O88767 | Protein/nucleic acid deglycase DJ-1 | ↓ | 0.59 | 2.54E-02 | ||
| Q5XI73 | Rho GDP-dissociation inhibitor 1 | ↓ | 0.59 | 3.80E-02 | ||
| P16617 | Phosphoglycerate kinase 1 | ↓ | 0.48 | 4.91E-02 | ||
| Q9Z2Y9 | Klotho | ↓ | 0.25 | 4.23E-02 | ||
| Q63270 | Cytoplasmic aconitate hydratase | ↓ | 0.21 | 6.07E-03 | ||
| Q920A6 | Retinoid-inducible serine carboxypeptidase | ↑ | 1.72 | 3.11E-02 | ||
| Q4QQW8 | Putative phospholipase B-like 2 | ↑ | 1.68 | 4.58E-02 | ||
| Q6P7A9 | Lysosomal alpha-glucosidase | ↑ | 1.60 | 1.19E-02 | ||
| P62630 | Elongation factor 1-alpha 1 | ↓ | 0.67 | 1.61E-02 | ||
| P62260 | 14-3-3 protein epsilon | ↓ | 0.65 | 2.23E-02 | ||
| P07340 | Sodium/potassium-transporting ATPase subunit beta-1 | ↓ | 0.64 | 2.32E-02 | ||
| P27139 | Carbonic anhydrase 2 | ↓ | 0.62 | 3.15E-02 | ||
| Q9WTQ2 | Podocalyxin | ↓ | 0.57 | 3.16E-02 | ||
| P41562 | Isocitrate dehydrogenase [NADP] cytoplasmic | ↓ | 0.55 | 3.84E-02 | ||
| P01026 | Complement C3 | ↓ | 0.55 | 7.96E-03 | ||
| P19112 | Fructose-1, 6-bisphosphatase 1 | ↓ | 0.53 | 3.07E-02 | ||
| Q9R066 | Coxsackievirus and adenovirus receptor homolog | ↓ | 0.46 | 3.25E-02 | ||
| Q9QWJ9 | Neuropilin-1 | ↓ | 0.44 | 2.42E-02 | ||
| P42123 | L-lactate dehydrogenase B chain | ↓ | 0.39 | 3.50E-02 | ||
| P51635 | Aldo-keto reductase family 1 member A1 | ↓ | 0.37 | 3.89E-02 | ||
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| 图 3 腹腔注射白色念球菌模型中12、36、72 h鉴定的差异蛋白的维恩图 Fig. 3 The Venn diagram of differential proteins identified at 12, 36 and 72 h in C. albicans intraperitoneal injection model. |
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2.3 差异蛋白的功能注释2.3.1 腹腔注射大肠杆菌模型差异蛋白的功能注释使用DAVID数据库对该模型鉴定到的总计69个差异蛋白从生物学过程、细胞成分和分子功能3个方面进行功能富集分析(图 4)。在生物学过程中(图 4A，表 4)，这些差异蛋白倾向于参与谷氨酸代谢、谷胱甘肽生物合成、细胞蛋白分解代谢的正调控、肌动蛋白细胞骨架组织的调节和谷胱甘肽代谢等过程。在细胞成分中(图 4B)，大多数的差异蛋白来源于细胞外的外泌体。在分子功能中(图 4C)，这些差异蛋白倾向于受体结合、不依赖钠的有机阴离子跨膜转运蛋白活性和谷氨酸-半胱氨酸连接酶活性等功能。
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| 图 4 腹腔注射大肠杆菌模型中差异蛋白的功能注释 Fig. 4 Functional analysis of differential proteins in E. coli intraperitoneal injection model. (A) Biological process. (B) Cellular component. (C) Molecular function. |
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表 4 腹腔注射大肠杆菌模型中差异蛋白参与的生物学过程Table 4 Biological process of differential proteins in E. coli intraperitoneal injection model
| Biological process | P value | Count | Uniprot |
| Glutamate metabolic process | 1.80E-07 | 5 | P09606, Q64602, P48508, P07314, P19468 |
| Glutathione biosynthetic process | 6.00E-06 | 4 | P48508, P07314, P19468, P46413 |
| Aging | 2.30E-05 | 9 | P01015, P13596, P30120, P48508, P07314, Q99MH3, P19468, P24594, P46413 |
| Gland morphogenesis | 4.90E-04 | 3 | O35763, P31977, Q9JJ19 |
| Positive regulation of cellular protein catabolic process | 7.50E-04 | 3 | O35763, P31977, Q9JJ19 |
| Establishment of epithelial cell apical/basal polarity | 7.50E-04 | 3 | O35763, Q99MH3, P31977 |
| Regulation of actin cytoskeleton organization | 9.90E-04 | 4 | Q9Z0W7, P18757, P48508, P19468 |
| Glutathione metabolic process | 9.90E-04 | 4 | P61589, P31977, P97605, Q99PS8 |
| Sodium-independent organic anion transport | 3.10E-03 | 3 | P70502, Q3ZAV1, P46720 |
| Regulation of cell size | 3.70E-03 | 3 | O35763, P31977, Q9JJ19 |
| Leukocyte cell-cell adhesion | 4.00E-03 | 3 | O35763, P27590, P31977 |
| Cellular response to follicle-stimulating hormone stimulus | 4.60E-03 | 3 | P48508, P19468, P97605 |
| Negative regulation of cell migration | 6.70E-03 | 4 | Q9Z0W7, P24594, Q5XI73, Q9JJ19 |
| Cellular response to thyroxine stimulus | 7.50E-03 | 2 | P48508, P19468 |
| Negative regulation of cell growth | 1.10E-02 | 4 | P01015, P18757, Q9QZQ5, Q99PS8 |
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2.3.2 腹腔注射金黄色葡萄球菌模型差异蛋白的功能注释同上，使用DAVID数据库对该模型鉴定到的总计31个差异蛋白从生物学过程、细胞成分和分子功能3个方面进行功能富集分析(图 5)。在生物学过程中(图 5A，表 5)，这些差异蛋白倾向于参与谷胱甘肽代谢、糖皮质激素代谢、高密度脂蛋白颗粒组装、反向胆固醇运输和高密度脂蛋白颗粒重塑等过程。在细胞成分中(图 5B)，大多数的差异蛋白来源于细胞外的外泌体和细胞外空间。在分子功能中(图 5C)，这些差异蛋白倾向于丝氨酸型内肽酶抑制剂的活性、脂质转运蛋白活性和谷胱甘肽结合等功能。
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| 图 5 腹腔注射金黄色葡萄球菌模型中差异蛋白的功能注释 Fig. 5 Functional analysis of differential proteins in S. aureus intraperitoneal injection model. (A) Biological process. (B) Cellular component. (C) Molecular function. |
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表 5 腹腔注射金黄色葡萄球菌模型中差异蛋白参与的生物学过程Table 5 Biological process of differential proteins in S. aureus intraperitoneal injection model
| Biological process | P value | Count | Uniprot |
| Glutathione metabolic process | 3.56E-03 | 3 | P14942, O35952, P08010 |
| Response to organic cyclic compound | 1.10E-02 | 4 | P08010, P97574, P26051, Q99PS8 |
| Glucocorticoid metabolic process | 1.19E-02 | 2 | P31211, P04639 |
| Ossification | 1.21E-02 | 3 | O88766, Q06496, P97574 |
| High-density lipoprotein particle assembly | 1.36E-02 | 2 | P14630, P04639 |
| Reverse cholesterol transport | 2.03E-02 | 2 | P14630, P04639 |
| High-density lipoprotein particle remodeling | 2.03E-02 | 2 | P14630, P04639 |
| Cytokine-mediated signaling pathway | 2.32E-02 | 3 | P26051, P47853, Q80WD1 |
| Fibrinolysis | 2.54E-02 | 2 | P18292, Q99PS8 |
| Negative regulation of endopeptidase activity | 2.64E-02 | 3 | Q63416, P31211, Q99PS8 |
| Positive regulation of focal adhesion assembly | 3.53E-02 | 2 | Q6RUV5, Q99PS8 |
| Regulation of cell morphogenesis | 4.36E-02 | 2 | P45592, Q6RUV5 |
| Lipoprotein metabolic process | 4.52E-02 | 2 | P14630, P04639 |
| Cholesterol efflux | 4.52E-02 | 2 | P14630, P04639 |
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2.3.3 腹腔注射白色念球菌模型差异蛋白的功能注释同上，使用DAVID对该模型鉴定到的总计38个差异蛋白从生物学过程、细胞成分和分子功能3个方面进行了功能富集分析(图 6)。在生物学过程中(图 6A，表 6)，这些差异蛋白倾向于参与碳水化合物代谢、糖质新生、糖酵解、醛分解和类固醇激素反应等过程。在细胞成分中(图 6B)，大多数的差异蛋白来源于细胞外的外泌体。在分子功能中(图 6C)，这些差异蛋白倾向于蛋白结合、单糖结合和乳酸脱氢酶活性等功能。
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| 图 6 腹腔注射白色念球菌模型中差异蛋白的功能注释(A：生物学过程；B：细胞成分；C：分子功能) Fig. 6 Functional analysis of differential proteins in C. albicans intraperitoneal injection model. (A) Biological process. (B) Cellular component. (C) Molecular function. |
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表 6 腹腔注射白色念球菌模型中差异蛋白参与的生物学过程Table 6 Biological process of differential proteins in C. albicans intraperitoneal injection mode
| Biological process | P value | Count | Uniprot |
| Carbohydrate metabolic process | 5.36E-06 | 6 | P04642, P42123, Q6P6V0, Q9Z2Y9, P19112, P16617 |
| Gluconeogenesis | 4.16E-05 | 4 | Q6P6V0, P19112, P16617, P25113 |
| Glycolytic process | 2.90E-03 | 3 | Q6P6V0, P16617, P25113 |
| Aldehyde catabolic process | 6.15E-03 | 2 | Q6P6V0, P51635 |
| Response to steroid hormone | 6.95E-03 | 3 | P41562, P27139, P08721 |
| Cell migration | 7.59E-03 | 4 | Q9QWJ9, P97710, Q9WTQ2, Q9Z1Y3 |
| Regulation of protein localization | 8.33E-03 | 3 | Q793F9, Q9Z1Y3, Q5XI73 |
| Cell-cell adhesion | 9.16E-03 | 4 | O88767, P41562, P04642, P62260 |
| Lactate metabolic process | 1.23E-02 | 2 | P04642, P42123 |
| NAD metabolic process | 1.43E-02 | 2 | P04642, P42123 |
| Cell adhesion | 1.62E-02 | 4 | P07340, Q9WTQ2, Q9Z1Y3, P08721 |
| Response to fibroblast growth factor | 1.83E-02 | 2 | Q9Z2Y9, P17559 |
| Single organismal cell-cell adhesion | 1.96E-02 | 3 | Q6X936, Q9R066, Q9Z1Y3 |
| Response to estrogen | 2.17E-02 | 3 | P04642, P27139, P01026 |
| Response to oxidative stress | 3.61E-02 | 3 | O88767, P41562, P30152 |
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2.4 三种微生物腹腔注射模型尿蛋白的比较对3种微生物腹腔注射模型鉴定到的尿液差异蛋白进行比较，重叠情况如图 7所示。从图中可以看出，3个模型鉴定到的差异蛋白没有重合，但是在腹腔注射大肠杆菌和金黄色葡萄球菌两个模型中共同鉴定到6个差异蛋白，包括S23A1、HRG、NIT1、FAAA、GBB1和PNPH；在腹腔注射大肠杆菌和白色念球菌两个模型中共同鉴定到2个差异蛋白，包括GDIA和GDIR1；在腹腔注射金黄色葡萄球菌和白色念球菌两个模型中共同鉴定到T132A这1个差异蛋白。对3种模型尿液差异蛋白富集到的生物学过程进行比较，发现大鼠腹腔注射不同的微生物时尿蛋白富集的生物学过程也不同。
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| 图 7 三种微生物腹腔注射模型鉴定到的差异蛋白的维恩图 Fig. 7 The Venn diagram of differential proteins identified by intraperitoneal injection models of three microorganisms. |
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3 讨论本研究使用大肠杆菌、金黄色葡萄球菌和白色念球菌建立了3种微生物腹腔注射大鼠模型，通过对尿液进行非标记LC-MS/MS质谱鉴定，探究不同微生物感染腹腔时尿蛋白的变化情况。结果显示，在大肠杆菌腹腔注射模型中共鉴定到69个差异蛋白，在金黄色葡萄球菌腹腔注射模型中共鉴定到31个差异蛋白，在白色念球菌腹腔注射模型中共鉴定到38个差异蛋白。
腹膜炎大多是由于腹腔感染了微生物而引起的[14]，有趣的是，在3个模型鉴定到的差异蛋白中有多个蛋白在之前的研究中被报道与腹膜炎有关。在腹腔注射大肠杆菌模型中，有4个蛋白与腹膜炎有关。血清对氧磷酶1 (PON1)是一种具有抗氧化作用的血清酶，有研究报道在细菌和病毒感染时PON1活性会降低[15]。金属蛋白酶抑制剂1 (TIMP1)作为腹膜透析患者腹膜损伤的生物标志物[16]。β-葡萄糖醛酸酶(BGL1)的活性被报道作为细菌性腹膜炎症的诊断指标[17]。有研究报道，血浆中铁调素(HEPC)在感染和炎症期间的浓度大幅升高，而本次研究中该蛋白下调[18]。在腹腔注射金黄色葡萄球菌模型中，有2个蛋白与腹膜炎有关。中性粒细胞胶原酶(MMP8)是一种组织基质降解酶，在炎症刺激下由白细胞释放，可能导致腹膜相关器官损伤，继发性腹膜炎患者的腹膜液样品中MMP-8水平显著升高[19]。CD44缺乏与大肠杆菌诱导的腹膜巨噬细胞释放促炎性因子和趋化因子的增强有关[20]。在腹腔注射白色念球菌模型中，有3个蛋白与腹膜炎有关。中性粒细胞明胶酶相关的脂蛋白(NGAL)是抗菌性先天免疫系统的重要组成部分，研究发现腹膜炎患者的腹膜液中NGAL水平升高，可能作为早期诊断的有用工具[21-22]。胰石蛋白(LITH)在区分腹膜炎严重程度和预测重症监护病房患者死亡方面具有很高的诊断准确性[23-24]。有研究发现，在发生腹膜炎的肝硬化患者中，腹水补体3 (CO3)水平显著降低[25]。这些尿蛋白将来有可能成为腹膜炎敏感的早期诊断标志物。
对3个模型尿液蛋白质组的结果进行比较后发现，3个模型尿蛋白差别显著，尿蛋白富集的生物学过程也不同，猜测这可能与3种微生物自身的特点和对腹腔感染的严重程度不同有关，这表明尿液可以反映腹腔被不同菌液感染时的状态。仅在大肠杆菌腹腔注射模型中富集到了白细胞黏附这一个与免疫相关的过程，其他模型的尿蛋白没有富集到与炎症或者免疫直接相关的生物学过程，猜测这可能与大鼠自身的免疫水平较强有关。值得注意的是，虽然本研究的3个动物模型是由两组研究人员进行的，使用了两种大鼠品系，但是我们采用的是被感染前后自身对照的方法来筛选差异蛋白，所以理论上差异蛋白主要与感染和刺激有关，与大鼠品系无关。本次研究是尝试利用尿蛋白区分不同微生物腹腔感染的初步探索，这些结果还需要在更大量的动物实验中进行验证。
4 结论我们发现不同的微生物注射到大鼠腹腔时尿蛋白质组差别显著，提示尿液可能具有良好的鉴别诊断潜能。
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