【作者】 李逊斗；

【导师】 高友鹤；

【作者基本信息】 北京协和医学院 ， 病理与病理生理学， 2014， 博士

【摘要】 传统的理论认为食物蛋白进入人体后,首先要在体内消化酶的作用下水解成氨基酸、二肽及三肽才能被肠道吸收和利用。近年来,越来越多的研究发现某些蛋白质能够以片段甚至完整形式穿过肠道粘膜屏障进入体内,虽然量比较小不足以起到营养学价值,但这些蛋白能却够保持其生物学活性并发挥相应的作用。本研究尝试采用蛋白质组学方法全面系统地鉴定食物中能够完整进入体内的蛋白质。淋巴管比血管的通透性更高,且淋巴液的流速低及成分相对简单,有利于外源性蛋白的鉴定。我们构建了一种新的大鼠肠道淋巴瘘管模型,并收集了4只大鼠食用牛奶前后的肠道淋巴液样品。然后分别采用离心超滤法(30kD cutoff)和不同溶解度法(DS)富集了淋巴液中的低分子量蛋白和多肽,经一维凝胶电泳分离和基于分子量大小的胶内酶切后,通过LC-MS/MS的方法对产生的多肽进行质谱鉴定。在数据库检索时,通过牛和大鼠种属特异性多肽来确定淋巴液中来源于牛奶的蛋白。在饮食牛奶后的大鼠淋巴液中,共鉴定到9个牛源性蛋白(FDR<1%),其中6个蛋白在两只不同的大鼠中均被鉴定到,另外有4个蛋白在采用两种不同富集方法得到的样本中都得到鉴定。这些牛源性蛋白都在不同分子量区间得到鉴定,提示它们可能以不同大小的片段或完整的形式进入淋巴液中。大部分牛源性蛋白是牛奶中的高丰度蛋白,比如p-乳球蛋白和酪蛋白。这9个蛋白中有7个是牛奶的主要过敏原。本研究中我们试图寻找这些能够进入淋巴液牛源性蛋白的共同特征,将为口服蛋白药物的开发和探寻预防食物过敏的新方法提供理论依据。生物标志物是和某种特定的生理或病理生理过程相联系的可监测的变化。血液是机体内环境的重要组成部分,有多种机制来维持血液成分的相对稳定,以保证机体正常的生命活动。尿液是经由泌尿系统及尿路排出体外的排泄物,完全没有稳定的必要性和机制,因此尿液能够更灵敏地反映机体的变化。与此同时,这也决定了尿液非常容易受到多种因素的影响,比如年龄、性别、药物、运动及饮食等。在尿蛋白生物标志物的研究中,为了得到疾病特异的差异蛋白,必须要排除这些非疾病因素对尿蛋白质组的影响,因此研究常见因素对尿蛋白质组的影响是非常必要的。药物是尿蛋白质组的影响因素之一。在进行尿生物标志物的研究中,不能干扰患者的正常治疗,因此研究药物的影响显得尤为重要。利尿剂是目前临床最常用的药物之一,特别是用于心脏及肾脏疾病的治疗。本研究以大鼠为研究对象,探讨了三种常用利尿剂(呋塞米、氢氯噻嗪和螺内酯)对尿蛋白质组的影响。通过代谢笼收集大鼠服用常规治疗剂量利尿剂前后的尿液样本,然后采用液相色谱串联质谱的方法(LC-MS/MS)分析尿蛋白质组的变化。经Progenesis LC-MS软件进行非标记定量及严格筛选(FDR<1%,P<0.05,变化倍数≥2,同时谱图数≥5)后,在呋塞米、氢氯噻嗪和螺内酯三组大鼠用药前后尿液中分别鉴定到7、5和2个具有显著差异的蛋白,且这三组中的差异蛋白各不相同。这14个蛋白中有10个已经被认为是疾病的生物标志物,而且大部分差异蛋白的人同源蛋白都是正常人尿核心蛋白质组的成员,这些蛋白标志物在将来的临床应用中以及新的生物标志物的研究中要慎重考虑利尿剂的影响。本研究结果提示在将来的尿蛋白质组生物标志物研究中,在进行数据分析时要考虑到利尿剂的影响,同时也为利尿剂对肾脏蛋白处理功能影响的机制研究提供了线索。此外,本策略同样适用于其他常见药物对尿蛋白质组影响的研究。随着年龄的增长,肾脏的功能会逐渐降低,肾小球滤过滤(GFR)从40岁到80岁会有20-25%的下降。老年人由于机体功能的衰退容易发生多种疾病,在进行这些疾病尿生物标志物的研究时,要排除自然衰老状态下尿蛋白质组学的变化。本研究中我们采用与利尿剂相似的策略,定量比较了9只青年大鼠(2个月龄)和9只老年大鼠(20个月龄)的尿蛋白质组的差异。两组大鼠中共鉴定到374个蛋白,其中具有显著差异的蛋白有37个,在老年组中升高的有17个,下降的有20个。其中有21个蛋白已经被报道为疾病的生物标志物。另外这37个蛋白中有28个具有人同源蛋白,其中14个是正常人尿核心蛋白质组的成员。利用人类蛋白质表达数据库(Human protein Atlas)对这些蛋白进行组织定位后发现,这28个蛋白可高表达于40个不同的组织器官中。其中高表达差异蛋白数量最多的组织是肾脏,体现了尿液能够很好地反映肾脏的功能。除此之外,消化系统(特别是小肠中)、脑部以及肺部高表达差异蛋白的数量也比较多,提示尿液也可能反映这些系统的功能变化,将来可用于寻找这些系统疾病的生物标志物。肾细胞癌(renal cell carcinoma, RCC)是最常见的成人肾脏肿瘤,其中85%的组织学类型为肾透明细胞癌(clear cell RCC)。早期肾癌经治疗后5年存活率可高达89%,但由于早期肾癌缺乏特异性症状、体征及实验室检查指标,30%以上的肾癌患者确诊时己经到了晚期阶段,5年存活率不足5%,因此肾癌的早期筛查和诊断尤为重要。目前肾癌诊断主要依靠B超、CT、磁共振等影像学检查,还没有可以应用于临床的特异性生物标志物。尿液与肾脏有着密不可分的关系,能够直接地反映肾脏的状态,非常适合作为寻找肾脏疾病生物标志物的样品来源。本研究中,我们通过非标记定量蛋白质组学方法分析了4例肾透明细胞癌患者手术前后尿液变化,发现具有显著差异且在所有患者中变化趋势一致的蛋白有4个。目前我们正在对其中的两个蛋白通过ELISA方法进行大规模验证。肾癌尿液生物标志物的研究将为肾癌早期筛查和诊断提供一种有效的方法。更多还原

【Abstract】 Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow’s milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by1D-SDS-PAGE, digested in-gel based on molecular weight, and identified using nano-LCMS/MS. In the postprandial rat intestinal lymph, nine bovine-specific proteins (false discovery rate≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were high-abundant proteins in the milk, such as β-lactoglobulin and caseins. Seven of the nine identified bovine-specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross-barrier transport or translocation.Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including age, gender, medications, exercises, etc. Careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F; hydrochlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the criteria of P≤ 0.05, a fold change≥2, a spectral count≥5and false positive rate (FDR)<1%,14proteins (seven for F, five for H, and two for S) were identified by Progenesis LC-MS. The human orthologs of most of these14proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics.The kidney shows a quantifiable decrease in function with age. The glomerular filtration rate declines with20-25%from40to80years of age. Furthermore, the capacity of the kidney to concentrate urine reduces progressively with age. Urine samples from9young rats (2months) and9old rats (20months) were analyzed by label-free quantitative proteomics.37proteins were significantly changed between the two groups,17proteins were up-regulated in old group, and20proteins were down-regulated. Most of these proteins were found in the human core urinary proteome. Compared these proteins with human protein atlas database, they are highly or strongly expressed in40different tissues. Among these tissues, kidney has the most highly or strongly expressed proteins, which may due to its natural relationship with urine. The number of highly or strongly expressed proteins in brain, lung and intestines is relatively high, implying that urinary proteins may reflect the status of these organs.Renal cell carcinoma (RCC) is the most common neoplasm in adult kidney, making up3%of all adult malignancies. Histopathologically, about85%of RCC is clear-cell carcinoma. Early diagnosis of kidney-localized RCC is associated with a quite favorable prognosis with a five-year survival rate of about89%. Unfortunately, patients often present with few signs, symptoms, or laboratory abnormalities, and are frequently (-30%) diagnosed at the metastatic stage, when the prospects for cure are dismal (9%five-year survival rate).The clinical diagnosis of RCC is often confirmed by imaging studies, including ultrasonic, MRI and CT. There are currently no biomarkers available for the diagnosis of RCC. Because of the natural relationship of urine and kidney, urine is particularly suitable as a source of kidney disease biomarkers. We have identified4candidate markers by label-free quantitative proteomics from urine samples of4ccRCC patients. And then, the dis-regulated proteins are validated by ELISA in a large scale.We expect to find some usefull biomarkers of ccRCC for early diagnosis.更多还原