离体大鼠肝脏灌流液蛋白质组研究
发布时间：2019-04-02 13:39
【摘要】： 离体大鼠肝脏灌流是一项广泛应用于肝脏生理功能研究的技术,在合适的灌流条件下,灌流肝脏可在一定的时间窗内保持结构完整。蛋白质组学技术具有大规模、高通量的特点。本论文第一部分将这两种技术结合,尝试在器官水平上全面完整地研究肝脏的分泌功能。在该类型研究中,主要有两大问题：一是如何保持肝脏器官的结构完整,二是控制灌流液中的血污染。我们一方面调整并控制了器官灌流条件来减少灌流损伤和降低血污染,另一方面以灌流液中的谷丙转氨酶量来评判肝脏损伤情况,以灌流液中的免疫球蛋白IgG量作为灌流液血污染程度的指标。我们选取谷丙转氨酶水平较低和肉眼观没有明显血污染的灌流液进行了后续研究。同时Western Blot和后续的质谱鉴定均显示灌流液中的IgG水平较低,提示灌流液中血污染较少。 应用二维毛细管液相色谱(强阳+反相)与质谱联用方法,在鉴定假阳性率约1%的基础上,共获得了886个灌流液蛋白鉴定。在这些蛋白中有423个蛋白被Swiss-Prot注释,其中分泌蛋白的比例相对于肝脏全组织蛋白组数据有高达5倍的富集；在富集指数方法富集的部分,分泌蛋白的比例则有约10倍的富集。应用信号肽预测方法和富集指数方法,我们共获得357个可能分泌蛋白,其中有49个非经典途径分泌蛋白被富集指数方法以较高可信度富集。在这些分泌蛋白中,我们发现有71个蛋白参与信号传导通路,其中61个蛋白在胞外发挥作用。通过对这些蛋白的功能分析,我们实现了在器官水平和蛋白质组学广度上研究肝脏分泌功能。 灌流液含有在灌流条件下器官水平的组织间隙液蛋白,其富含疾病标志物。正常大鼠肝脏灌流液蛋白中胞内蛋白部分为肝脏灌流损伤所致。在这些蛋白中,我们发现许多蛋白有文献报道与损伤相关或已为肝脏损伤标志物。我们认为在离体器官灌流液中寻找潜在的标志物而在血液或尿液中进行验证,可能是寻找疾病标志物的较好策略。在论文的第二部分,我们建立了Walker-256肿瘤细胞肝脏转移模型,通过比较模型组与对照组之间的离体大鼠肝脏灌流液的差异,获得了该肿瘤细胞转移后肝脏灌流液中的差异蛋白。结果显示：110个蛋白在模型组中增高,其中分泌蛋白为46个；70个蛋白在模型组中降低,分泌蛋白为60个。下降的蛋白主要为分泌蛋白且为常见的肝脏分泌蛋白,提示肿瘤转移后肝脏的分泌功能下降,这可能与Walker-256转移所致恶液质有关。我们挑取了4个在灌流液中明显增高的蛋白,应用Western Blot方法在血清中进行验证,拟探讨离体大鼠肝脏灌流液在肝脏疾病标志物研究中的价值。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341
本文编号：2452606
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341
【参考文献】
相关期刊论文 前1条
1 王琦;时高峰;彰俊杰;杜煜;王亚宁;李月考;杨丽;刘辉;张金香;;大鼠Walker-256肝微小转移癌模型的建立及病理学、影像学的初步研究[J];中国医学影像技术;2006年04期
，本文编号：2452606
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