| Mammalian liver is one of the most important and complexest organs of the body, it undertakes the metabolism, synthesis of plasma proteins, storage of glycogen and vitamins, detoxification and hematopoietic function during embryonic development, and also takes part in immune defense response and other functions. Since Human Liver Proteome Project implemented. It has been taken a large number of proteomic researches on the human and mouse liver tissue and subcellular level, got many significant research results and accumulated the massive high confidence liver protein expression profiling data. With further research, many scientific questions, including the proteins compositions and modifications in different liver cells, and the cellular localization of proteins from the liver tissue/subcellular proteomics research, and studying the cell cycle,cell differentiation,apoptosis,cell regulatory networks and protein interactions within the dynamic cellular proteomics, and furthermore the pathogenesis of liver diseases, must be solved on the cellular proteomics study. Cellular proteomics research has become the new trend of the liver and important content. Construction of the liver different cell groups protein expression profiling is basis to solve the above scientific issues.Getting sufficient purity and quantity of the liver primary cells is basic need to carry out the liver cellular proteomics. In this study, modified Seglen method of in situ rat liver perfusion to isolate of liver cells, and established the improved two steps in situ circulatory perfusion method to separate the C57 mice liver cells. After perfusion, the C57mouse liver was cut and digested in vitro. Then the mouse hepatocytes were purified by differential centrifugal method. The purity of hepatocytes was detected by microscopic morphology,HE,PAS and immunocytochemical identifications, assayed the cell activity with trypan blue dying, and cultured the primary mouse hepatocytes in RPMI-1640 nutrients containing 20%FBS. Finally, hepatocytes with 95% purity about and 90% viability were harvested, and yield was 4.2～6.5×107cells from one mouse liver. Harvested hepatocytes can be cultivated continuously for above 7Ds, these hepatocytes completely fully meet the need for proteomics research.Obtained C57 mouse Nonparenchymal cells population, and C57 mouse hepatic stellate cells,Kupffer cells and sinusoidal endothelial cells was isolated by density gradient centrifugation method with the Nycodenz ?, PercollTM and OptiPrepTM density gradient media, flow cytometry and trypan blue dye tests was taken to detect the hepatic Nonparenchymal cells purity and activity, and estimated the cell recovery on blood counting chamber, and evaluated the differences of these methods. The results showed that, in the purification of hepatic stellate cells Nycodenz ? method has a distinct advantage, OptiPrepTM method has higher cell purity, cell viability and cell yield, in sorting non-parenchymal cells, and it is a reliable sorting methods. Furthermore, high-speed fluorescence-activated cell sorting was taken to separate the main non-parenchymal cells, compared with density gradient centrifugation and immunomagnetic beads methods, hepatic endothelial cells and Kupffer cells have the highest Cell purity, cell yield and cell activity by Moflo high-speed flow cytometry. And enough hepatic endothelial cells and Kupffer cells was gained for non-parenchymal cells proteomics research.For achievement the hepatic cell protein expression profiling, a nano-2D-LC technology system was established for tanglesome protein samples separation.In this study, Eksigent's Nano-2D-LC system, was established for the study of liver cells proteomics research in Shotgun strategy. Only 30μg protein sample was need for this system, and first mobile phase A (citric acid - ammonia) was devided into 10 gradients within pH 3.0 ~ 8.5 and 3.5 mM ~ 46.5 mM, the second mobile phase B (NH4Cl) was devided into 9 gradients within 2 mM ~ 2000 mM, 2% CAN +0.1% FA. Combined with LTQ-MS/MS and LTQ-FT-MS/MS (Thermo FinniganTM company) were identified with a full scan mass spectrometry (Full MS :400-2000, full scan 160min, MS / MS data collection 140min) and three sub-scan (Mass Range: m / z 400-650/600-800/800-2000, MS / MS data collection 140min) mode. 95% confidence and minimum two peptides match (95P2) were used for protein identificion within reverse mouse database. Combinated SDS-PAGE protein separation with nano-LC-LTQ-FT MS/MS analysis, taken the same mass spectrometry data acquisition mode, and identified proteins with same standard.Our C57 hepatocytes protein expression profiling contains 95% confidence and minimum two peptides matched 3703 proteins, 40 CYPs and 223 new found liver proteins. Comparative analysis of C57 mouse hepatocytes protein expression profiling, and found 724 plasma proteins and 21 liver interstitial fluid proteins was synthesized and secreted by hepatocytes, and 215 proteins was found to express in mouse fetal liver and adult liver. And compared with sub-cellular proteomics resultes, C57 mouse hepatic 2239 proteins have undoubtable subcellular localization. these include, 250 cytoplasm proteins , 37 Golgi body proteins, 734 microsomal proteins, 209 mitochondria proteins , 138 membrane proteins, 191 endosome proteins, 35 proteasome proteins, 645 endoplasmic reticulum, proteins in mouse liver cells. 1464 hepatic proteins have no clear subcellular location information.In conclusion, this study first successfully established a method of perfusion mice in suti liver to separation liver cells, and isolated C57 mouse hepatocytes by differential centrifugation method for hepatocytes proteomics. Gained sufficient hepatic sinusoidal endothelial cells and kupffer cells for these cellular proteomics study, by using Moflo high-speed fluorescence-activated cell sorting method, and set up a protein separation and identification system for mice hepatic cell proteomics research. Construction of the first C57 mouse hepatic protein expression profiling , obtained 95% confidence and minimum two peptides matched 3703 proteins, 40 CYPs and 223 new found liver proteins, combined with the mouse liver tissue and subcellular proteomics results, got the relevant information on mouse liver developmental proteins, plasma proteins, liver interstitial fluid proteins, and hepatic proteins sub-cellular localization . This study provides a helpful common mice liver cellular proteomics studying technology, and supplied a useful mice hepatocytes proteomics data for other liver scientific issues studies... |
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