Perfusate From The Rat Liver Proteome Research

Isolated perfused rat liver was the physiologic model widely employed to study liver functions. In the first part of the dissertation, liver secretory function was studied by proteomic profiling of perfusates from the model, which preserved the intact hepatic structure. There are two major concerns in this type of study, the integrity of the liver and blood contamination in the perfusates. Perfusion conditions were carefully controlled and Alanine aminotransferase levels in the perfusates were monitored as indicators of liver integrity. The criteria for choosing the perfusates for further analyses were a low ALT level and no obvious blood contamination by visual observation. The amount of immunoglobulins identified in the perfusates by both western blot and MS/MS indicated low serum contamination. In total, 886 perfusate proteins were identified by SCX-RPLC-MS/MS with a false positive rate around 1%. Of these proteins,423 proteins had annotations in Swiss-Prot, of which secretory proteins were five-fold enriched to rat/mouse liver tissue proteome, while secretory proteins were around ten-fold enriched in the portion identified by the Enrichment Index method. In total,357 were likely secretory proteins identified by signal peptide prediction and/or the Enrichment Index method, and 49 proteins secreted through a non-classical pathway were identified by the Enrichment Index method at a high level of confidence. Of the 71 proteins involved in signal transduction,61 were related to extracellular signaling. These data provide a comprehensive view of liver secretory function on the organ level.The strategy can be used in pathophysiology research and biomarker discovery for diseases in the liver. The perfusate contains abundant tissue interstitial fluid proteins under perfusion conditions and then is a reservoir of disease biomarkers. Some injury-related proteins were identified in the perfusates and a few of them have been widely employed as biomarkers for liver injury in the clinic. It would be a good strategy for biomarker searching by discovering candidates in perfusates before the validation in blood or urine samples. A model of Walker-256 metastasis cancer in the rat liver was established in the second part of the dissertation. Some differentiated proteins in the perfusates of the model rats were identified by quantitative proteomic technology. In total, 110 proteins were elevated and 70 proteins were decreased in model perfusates, in which 46 secretory proteins were elevated and 60 secretory proteins were decreased. Most decreased proteins were well-known liver secretory proteins, which indicated the normal liver secretory function was inhibited by Walker-256 metastasis. And it could also be associated with the cachexia condition caused by the metastasis. Four significantly elevated proteins in model perfusates were chosen for further validation in serum by Western Blot method. It showed the value of perfusates in discovering liver diseases biomarkers.