食物中能够进入淋巴液的蛋白尿蛋白质组影响因素及肾癌标志物的研究

发布时间：2018-05-27 10:46

本文选题：淋巴液 + 食物蛋白　；参考：《北京协和医学院》2014年博士论文

【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(FDR1%),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(FDR1%,P0.05,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。
[Abstract]:The traditional theory holds that after the food protein enters the human body, it is first to be dissolved into the amino acid by the digestive enzyme in the body, and the two peptide and the three peptide can be absorbed and utilized by the intestine. In recent years, more and more studies have found that some proteins can penetrate into the intestinal mucosal barrier in fragments and even complete form, although the amount is relatively small. In order to play a nutritive value, these proteins can maintain their biological activity and play a corresponding role.
In this study, the proteomics method was used to comprehensively and systematically identify the proteins that could enter the body completely and systematically. The lymphatic vessels were more permeable than the blood vessels, and the flow velocity of the lymph was low and the composition was relatively simple, which was beneficial to the identification of exogenous proteins. A new rat model of intestinal lymphatic fistula was constructed, and 4 of them were collected and collected. The intestinal lymph samples before and after milk were used in rats. Then the low molecular weight proteins and peptides in the lymph were enriched by centrifuge ultrafiltration (30kD cutoff) and different solubility methods (DS). After the separation by one dimensional gel electrophoresis and the gel internal enzyme based on the molecular weight, the mass spectra of the produced peptides were carried out by LC-MS/MS method. Identification. In the database retrieval, the protein derived from the milk in the lymph was determined by the specific polypeptide of the cow and the rat. In the rat lymph after the diet milk, 9 bovine protein (FDR1%) were identified, of which 6 proteins were identified in two different rats, and the other 4 proteins were enriched with two different enrichment. All of these bovine derived proteins are identified in different molecular weight intervals, suggesting that they may enter the lymph in different sizes or in complete form. Most bovine derived proteins are high abundance proteins in milk, such as p- lactoglobulin and casein. 7 of these 9 proteins are cattle. The main allergen of milk. In this study we are trying to find the common features that can enter the lymph cow - derived proteins, which will provide a theoretical basis for the development of oral protein drugs and the exploration of new ways to prevent food allergies.
A biomarker is a monitoring change associated with a particular physiological or pathophysiological process. Blood is an important part of the body's environment. There are various mechanisms to maintain the relative stability of the blood components to ensure the normal life of the body. Urine is excreted from the urinary system and urinary tract and is completely absent. There is a stable necessity and mechanism so that urine can be more sensitive to the changes in the body. At the same time, it also determines that urine is very vulnerable to a variety of factors, such as age, sex, medicine, exercise and diet. In the study of urinary protein biomarkers, it is necessary to eliminate the specific differential proteins of the disease. The influence of these non disease factors on urinary proteome is therefore very necessary to study the effects of common factors on urinary proteome.
Drug is one of the factors affecting the proteinuria group. In the study of urinary biomarkers, it can not interfere with the normal treatment of the patients. Therefore, it is very important to study the influence of drugs. Diuretic is one of the most commonly used drugs at present, especially in the treatment of heart and kidney disease. The effects of three commonly used diuretics (furosemide, hydrochlorothiazide and spironolactone) on urine protein groups were collected. Urine samples were collected from rats before and after taking conventional treatment doses of diuretics through metabolic cages, and the changes in proteinuria group were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The non labeling by Progenesis LC-MS software was carried out. After the quantitative and strict screening (FDR1%, P0.05, change multiple more than 2, and more than 5), in the urine of furasim, hydrochlorothiazide and spironolactone, three groups of proteins with significant differences were identified, respectively, and the difference proteins in the three groups were not the same. 10 of these 14 proteins were considered to be disease organisms. The markers, and most of the differentially protein human homologous proteins are members of the normal human urine core protein group. These protein markers should be carefully considered in future clinical applications and in the study of new biomarkers. The results suggest that in the study of the biomarkers that will be used in the urine protein group, The effects of diuretics on the effects of diuretics and the mechanism for the effect of diuretics on renal protein treatment were also taken into account. In addition, this strategy also applies to the study of the effects of other common drugs on the proteinuria group.
As the age increases, the function of the kidney decreases gradually, and the glomerular filtration filtration (GFR) decreases from 40 to 80 years. The elderly are prone to a variety of diseases due to the decline of the body's function. In the study of the urinary biomarkers in these diseases, the changes in proteinuria in the natural aging state should be excluded. We used a diuretic similar strategy to quantify the differences in the urine protein groups of 9 young rats (2 months of age) and 9 old rats (20 months old). Two groups of rats identified 374 proteins, of which 37 were significantly different, 17 in the old group and 20 in the decline. 21 of them had been found. 28 of the 37 proteins have human homologous proteins, and 14 of them are members of the normal human urine core protein group. The protein expression database (Human protein Atlas) is used to locate these proteins, and these 28 proteins can be highly expressed in 40 different tissues. In addition, the amount of high expression of differential proteins in the digestive system (especially in the small intestine), the brain and the lungs is much more, suggesting that urine can also reflect the functional changes of these systems and can be used in the future. Look for the biomarkers of these systemic diseases.
Renal cell carcinoma (RCC) is the most common adult renal tumor, of which 85% of the histologic types are clear cell carcinoma of the kidney (clear cell RCC). The 5 year survival rate of early renal carcinoma can be as high as 89%. However, due to the lack of specific symptoms, signs and laboratory tests, more than 30% of renal cancer patients have been diagnosed with early renal cancer. In the late stage, the survival rate of 5 years is less than 5%, so the early screening and diagnosis of renal cancer is particularly important. The diagnosis of renal cancer is mainly based on B-ultrasound, CT, magnetic resonance imaging and other specific biomarkers. There is a close relationship between urine and kidney, which can directly reflect the state of the kidney. In this study, we analyzed the urine changes in 4 patients with renal clear cell carcinoma before and after surgery in this study. We found that there were significant differences in and 4 of the changes in all patients. The two proteins are tested in a large scale through the ELISA method. The study of urine biomarkers of renal cancer will provide an effective method for early screening and diagnosis of renal cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

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