本文選題：淋巴液 + 食物蛋白��； 參考：《北京協(xié)和醫(yī)學院》2014年博士論文

【摘要】：傳統(tǒng)的理論認為食物蛋白進入人體后,首先要在體內消化酶的作用下水解成氨基酸、二肽及三肽才能被腸道吸收和利用。近年來,越來越多的研究發(fā)現(xiàn)某些蛋白質能夠以片段甚至完整形式穿過腸道粘膜屏障進入體內,雖然量比較小不足以起到營養(yǎng)學價值,但這些蛋白能卻夠保持其生物學活性并發(fā)揮相應的作用。 本研究嘗試采用蛋白質組學方法全面系統(tǒng)地鑒定食物中能夠完整進入體內的蛋白質。淋巴管比血管的通透性更高,且淋巴液的流速低及成分相對簡單,有利于外源性蛋白的鑒定。我們構建了一種新的大鼠腸道淋巴瘺管模型,并收集了4只大鼠食用牛奶前后的腸道淋巴液樣品。然后分別采用離心超濾法(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,經一維凝膠電泳分離和基于分子量大小的膠內酶切后,通過LC-MS/MS的方法對產生的多肽進行質譜鑒定。在數(shù)據(jù)庫檢索時,通過牛和大鼠種屬特異性多肽來確定淋巴液中來源于牛奶的蛋白。在飲食牛奶后的大鼠淋巴液中,共鑒定到9個牛源性蛋白(FDR1%),其中6個蛋白在兩只不同的大鼠中均被鑒定到,另外有4個蛋白在采用兩種不同富集方法得到的樣本中都得到鑒定。這些牛源性蛋白都在不同分子量區(qū)間得到鑒定,提示它們可能以不同大小的片段或完整的形式進入淋巴液中。大部分牛源性蛋白是牛奶中的高豐度蛋白,比如p-乳球蛋白和酪蛋白。這9個蛋白中有7個是牛奶的主要過敏原。本研究中我們試圖尋找這些能夠進入淋巴液牛源性蛋白的共同特征,將為口服蛋白藥物的開發(fā)和探尋預防食物過敏的新方法提供理論依據(jù)。 生物標志物是和某種特定的生理或病理生理過程相聯(lián)系的可監(jiān)測的變化。血液是機體內環(huán)境的重要組成部分,有多種機制來維持血液成分的相對穩(wěn)定,以保證機體正常的生命活動。尿液是經由泌尿系統(tǒng)及尿路排出體外的排泄物,完全沒有穩(wěn)定的必要性和機制,因此尿液能夠更靈敏地反映機體的變化。與此同時,這也決定了尿液非常容易受到多種因素的影響,比如年齡、性別、藥物、運動及飲食等。在尿蛋白生物標志物的研究中,為了得到疾病特異的差異蛋白,必須要排除這些非疾病因素對尿蛋白質組的影響,因此研究常見因素對尿蛋白質組的影響是非常必要的。 藥物是尿蛋白質組的影響因素之一。在進行尿生物標志物的研究中,不能干擾患者的正常治療,因此研究藥物的影響顯得尤為重要。利尿劑是目前臨床最常用的藥物之一,特別是用于心臟及腎臟疾病的治療。本研究以大鼠為研究對象,探討了三種常用利尿劑(呋塞米、氫氯噻嗪和螺內酯)對尿蛋白質組的影響。通過代謝籠收集大鼠服用常規(guī)治療劑量利尿劑前后的尿液樣本,然后采用液相色譜串聯(lián)質譜的方法(LC-MS/MS)分析尿蛋白質組的變化。經Progenesis LC-MS軟件進行非標記定量及嚴格篩選(FDR1%,P0.05,變化倍數(shù)≥2,同時譜圖數(shù)≥5)后,在呋塞米、氫氯噻嗪和螺內酯三組大鼠用藥前后尿液中分別鑒定到7、5和2個具有顯著差異的蛋白,且這三組中的差異蛋白各不相同。這14個蛋白中有10個已經被認為是疾病的生物標志物,而且大部分差異蛋白的人同源蛋白都是正常人尿核心蛋白質組的成員,這些蛋白標志物在將來的臨床應用中以及新的生物標志物的研究中要慎重考慮利尿劑的影響。本研究結果提示在將來的尿蛋白質組生物標志物研究中,在進行數(shù)據(jù)分析時要考慮到利尿劑的影響,同時也為利尿劑對腎臟蛋白處理功能影響的機制研究提供了線索。此外,本策略同樣適用于其他常見藥物對尿蛋白質組影響的研究。 隨著年齡的增長,腎臟的功能會逐漸降低,腎小球濾過濾(GFR)從40歲到80歲會有20-25%的下降。老年人由于機體功能的衰退容易發(fā)生多種疾病,在進行這些疾病尿生物標志物的研究時,要排除自然衰老狀態(tài)下尿蛋白質組學的變化。本研究中我們采用與利尿劑相似的策略,定量比較了9只青年大鼠(2個月齡)和9只老年大鼠(20個月齡)的尿蛋白質組的差異。兩組大鼠中共鑒定到374個蛋白,其中具有顯著差異的蛋白有37個,在老年組中升高的有17個,下降的有20個。其中有21個蛋白已經被報道為疾病的生物標志物。另外這37個蛋白中有28個具有人同源蛋白,其中14個是正常人尿核心蛋白質組的成員。利用人類蛋白質表達數(shù)據(jù)庫(Human protein Atlas)對這些蛋白進行組織定位后發(fā)現(xiàn),這28個蛋白可高表達于40個不同的組織器官中。其中高表達差異蛋白數(shù)量最多的組織是腎臟,體現(xiàn)了尿液能夠很好地反映腎臟的功能。除此之外,消化系統(tǒng)(特別是小腸中)、腦部以及肺部高表達差異蛋白的數(shù)量也比較多,提示尿液也可能反映這些系統(tǒng)的功能變化,將來可用于尋找這些系統(tǒng)疾病的生物標志物。 腎細胞癌(renal cell carcinoma, RCC)是最常見的成人腎臟腫瘤,其中85%的組織學類型為腎透明細胞癌(clear cell RCC)。早期腎癌經治療后5年存活率可高達89%,但由于早期腎癌缺乏特異性癥狀、體征及實驗室檢查指標,30%以上的腎癌患者確診時己經到了晚期階段,5年存活率不足5%,因此腎癌的早期篩查和診斷尤為重要。目前腎癌診斷主要依靠B超、CT、磁共振等影像學檢查,還沒有可以應用于臨床的特異性生物標志物。尿液與腎臟有著密不可分的關系,能夠直接地反映腎臟的狀態(tài),非常適合作為尋找腎臟疾病生物標志物的樣品來源。本研究中,我們通過非標記定量蛋白質組學方法分析了4例腎透明細胞癌患者手術前后尿液變化,發(fā)現(xiàn)具有顯著差異且在所有患者中變化趨勢一致的蛋白有4個。目前我們正在對其中的兩個蛋白通過ELISA方法進行大規(guī)模驗證。腎癌尿液生物標志物的研究將為腎癌早期篩查和診斷提供一種有效的方法。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.11

【參考文獻】

相關期刊論文 前2條

1 Xundou Li;Mindi Zhao;Menglin Li;Lulu Jia;Youhe Gao;;Effects of Three Commonly-used Diuretics on the Urinary Proteome[J];Genomics,Proteomics & Bioinformatics;2014年03期

2 GAO YouHe;;Urine—an untapped goldmine for biomarker discovery?[J];Science China(Life Sciences);2013年12期

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本文編號：1941731