【摘要】： 離體大鼠肝臟灌流是一項(xiàng)廣泛應(yīng)用于肝臟生理功能研究的技術(shù),在合適的灌流條件下,灌流肝臟可在一定的時(shí)間窗內(nèi)保持結(jié)構(gòu)完整。蛋白質(zhì)組學(xué)技術(shù)具有大規(guī)模、高通量的特點(diǎn)。本論文第一部分將這兩種技術(shù)結(jié)合,嘗試在器官水平上全面完整地研究肝臟的分泌功能。在該類型研究中,主要有兩大問(wèn)題：一是如何保持肝臟器官的結(jié)構(gòu)完整,二是控制灌流液中的血污染。我們一方面調(diào)整并控制了器官灌流條件來(lái)減少灌流損傷和降低血污染,另一方面以灌流液中的谷丙轉(zhuǎn)氨酶量來(lái)評(píng)判肝臟損傷情況,以灌流液中的免疫球蛋白IgG量作為灌流液血污染程度的指標(biāo)。我們選取谷丙轉(zhuǎn)氨酶水平較低和肉眼觀沒(méi)有明顯血污染的灌流液進(jìn)行了后續(xù)研究。同時(shí)Western Blot和后續(xù)的質(zhì)譜鑒定均顯示灌流液中的IgG水平較低,提示灌流液中血污染較少。 應(yīng)用二維毛細(xì)管液相色譜(強(qiáng)陽(yáng)+反相)與質(zhì)譜聯(lián)用方法,在鑒定假陽(yáng)性率約1%的基礎(chǔ)上,共獲得了886個(gè)灌流液蛋白鑒定。在這些蛋白中有423個(gè)蛋白被Swiss-Prot注釋,其中分泌蛋白的比例相對(duì)于肝臟全組織蛋白組數(shù)據(jù)有高達(dá)5倍的富集；在富集指數(shù)方法富集的部分,分泌蛋白的比例則有約10倍的富集。應(yīng)用信號(hào)肽預(yù)測(cè)方法和富集指數(shù)方法,我們共獲得357個(gè)可能分泌蛋白,其中有49個(gè)非經(jīng)典途徑分泌蛋白被富集指數(shù)方法以較高可信度富集。在這些分泌蛋白中,我們發(fā)現(xiàn)有71個(gè)蛋白參與信號(hào)傳導(dǎo)通路,其中61個(gè)蛋白在胞外發(fā)揮作用。通過(guò)對(duì)這些蛋白的功能分析,我們實(shí)現(xiàn)了在器官水平和蛋白質(zhì)組學(xué)廣度上研究肝臟分泌功能。 灌流液含有在灌流條件下器官水平的組織間隙液蛋白,其富含疾病標(biāo)志物。正常大鼠肝臟灌流液蛋白中胞內(nèi)蛋白部分為肝臟灌流損傷所致。在這些蛋白中,我們發(fā)現(xiàn)許多蛋白有文獻(xiàn)報(bào)道與損傷相關(guān)或已為肝臟損傷標(biāo)志物。我們認(rèn)為在離體器官灌流液中尋找潛在的標(biāo)志物而在血液或尿液中進(jìn)行驗(yàn)證,可能是尋找疾病標(biāo)志物的較好策略。在論文的第二部分,我們建立了Walker-256腫瘤細(xì)胞肝臟轉(zhuǎn)移模型,通過(guò)比較模型組與對(duì)照組之間的離體大鼠肝臟灌流液的差異,獲得了該腫瘤細(xì)胞轉(zhuǎn)移后肝臟灌流液中的差異蛋白。結(jié)果顯示：110個(gè)蛋白在模型組中增高,其中分泌蛋白為46個(gè)；70個(gè)蛋白在模型組中降低,分泌蛋白為60個(gè)。下降的蛋白主要為分泌蛋白且為常見(jiàn)的肝臟分泌蛋白,提示腫瘤轉(zhuǎn)移后肝臟的分泌功能下降,這可能與Walker-256轉(zhuǎn)移所致惡液質(zhì)有關(guān)。我們挑取了4個(gè)在灌流液中明顯增高的蛋白,應(yīng)用Western Blot方法在血清中進(jìn)行驗(yàn)證,擬探討離體大鼠肝臟灌流液在肝臟疾病標(biāo)志物研究中的價(jià)值。
[Abstract]:Isolated rat liver perfusion is a technique widely used in the study of liver physiological function. Under suitable perfusion conditions, the perfused liver can keep its structure intact within a certain time window. Proteomics technology has the characteristics of large-scale and high-throughput. In the first part of this thesis, we try to study the secretive function of liver at the organ level by combining these two techniques. In this type of study, there are two main problems: one is how to keep the structure of liver organs intact, and the other is to control the blood pollution in perfusate. On the one hand, we adjust and control organ perfusion conditions to reduce perfusion injury and reduce blood pollution, on the other hand, we judge the liver damage by the amount of glutamic transaminase in perfusion fluid. The level of immunoglobulin IgG in perfusion fluid was used as the index of the degree of blood contamination of perfusion fluid. A follow-up study was carried out with a low level of alanine aminotransferase (alt) and no obvious blood contamination in the naked eye. At the same time, Western Blot and subsequent mass spectrometric analysis showed that the level of IgG in perfusion fluid was lower, suggesting that there was less blood pollution in perfusion fluid. By using two-dimensional capillary liquid chromatography (strong positive reversed phase) coupled with mass spectrometry, a total of 886 perfusate proteins were identified on the basis of the false positive rate of about 1%. Among these proteins, 423 proteins were annotated by Swiss-Prot, in which the proportion of secretory proteins was up to 5 times higher than that of the whole liver proteome data. In the enrichment index method, the proportion of secretory protein was about 10 times. By using the signal peptide prediction method and the enrichment index method, we obtained 357 possible secretory proteins, among which 49 non-classical pathway secretory proteins were enriched by the enrichment index method with high reliability. Among these secretory proteins, 71 proteins were found to be involved in signal transduction pathways, of which 61 proteins played an extracellular role. Through the functional analysis of these proteins, we have realized the study of liver secretory function at organ level and proteomics breadth. Perfusate contains tissue interstitial fluid protein at organ level under perfusion conditions, which is rich in disease markers. The part of intracellular protein in the perfusion fluid of normal rat liver is caused by liver perfusion injury. Among these proteins, we found that many proteins have been reported to be related to liver injury or have been used as markers of liver injury. We believe that searching for potential markers in vitro organ perfusion fluids and verifying them in blood or urine may be a better strategy for finding disease markers. In the second part of the thesis, we established the liver metastasis model of Walker-256 tumor cells, and compared the difference between the model group and the control group in isolated rat liver perfusion. The differentially expressed proteins in the liver perfusion fluid after metastasis of the tumor cells were obtained. The results showed that 110 proteins were increased in the model group, among which 46 were secretory proteins, 70 were decreased in the model group, and 60 proteins were secreted in the model group. The decreased proteins were mainly secretory proteins and common hepatic secretory proteins, suggesting that the secretion function of liver decreased after tumor metastasis, which may be related to cachexia caused by Walker-256 metastasis. In order to explore the value of isolated rat liver perfusion fluid in the study of liver disease markers, four proteins which were significantly increased in perfusate were selected and verified in serum by Western Blot method in order to explore the value of in vitro rat liver perfusion fluid in the study of liver disease markers.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王琦;時(shí)高峰;彰俊杰;杜煜;王亞寧;李月考;楊麗;劉輝;張金香;;大鼠Walker-256肝微小轉(zhuǎn)移癌模型的建立及病理學(xué)、影像學(xué)的初步研究[J];中國(guó)醫(yī)學(xué)影像技術(shù);2006年04期

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本文編號(hào)：2452606