| The mammalian adult liver exerts a strong regenerative capacity in response to mechanical stimulus (i.e., surgical hepatectomy, ischemia/reperfusion) or chemical stimulus (i.e., CCL4 administration). Because the liver is composed of multiple cell populations, rebuilding of the liver after 2/3 partial hepatectomy (PH) requires the synergistical actions of various liver cell types. It is well documented that liver sinusoidal endothelial cells (LSECs) are the largest number of liver non-parenchymal cells and the second largest number among all resident liver cells next to hepatocytes, and play a pivotal role in liver regeneration (LR), especially in the initiation of regeneration. However, details about the relevance of LSECs with liver regeneration remains yet to be delineated. It is likely that the measurement of dynamic expression profiles of LSECs would reveal the underlying mechanism of the action of this cell on liver regeneration (LR). In this study, the animal model of 70% hepatectomy in rats was established according to the method described by Higgins and Anderson; the methods of two-step perfusion combined with collagenase digestion were used to disperse liver cells; Percoll centrifugation and immunomagentic bead sorting methods were applied to isolate and purify LSECs from regenerating liver at 10 different recovery time points; CD14 and endothelin-1 (ET-1) immunostaining were employed for identifying the isolated LSECs. The immunochemical results showed that the proportion of ET1- and CD14-positive cells among the isolated LSECs were both at least 95%. The comprehensive analysis of gene expression profiles of LSECs during rat LR was carried out using Affymetrix rat genome 230 2.0 microarray, which is composed of 11789 known genes and 13231 EST sequences (totally 25020 genes), accounting for about 85% of rat genome. To evaluate the reliability of microarray results, this study used quantitative real-time PCR technology to detect the dynamic transcriptional profiles of several selected genes in LSECs from rat regenerating liver, including UBC, GAPDH, JUN, TTR, MYC, PCNA, APOE, TRIM24, CD14 and ET-1. Analysis on microarray data demonstrated that 833 known genes and 796 unknown genes with unknown structure (totaling 1629 genes) were identified as LR-related.Of them, 833 known genes were classified into eight clusters based on their expression patterns by means of K-means clustering method. The features of eight expression patterns are as follows: (1) rapid increase at priming phase and then quick return; (2) significant increase at proliferation phase; (3) quick increase at priming phase and then gradual decrease, again increase at terminal phase; (4) gradual enhancement and persistence; (5) transient downregulation and rapid recovery to baseline; (6) quick downregulation and slow recovery; (7) gradual downregulation and quick recovery with the lowest level at 30 h; (8) slight increase at priming phase and then downregulation. Gene function analysis indicated that these 833 known genes participate in many biological activities, such as signal transduction, blood coagulation, blood vessel morphogenesis, cell metabolism, extraculluar matrix and organelle organization and biogenesis, cell growth, proliferation, differentiation, immunity and inflammation, detoxification, secretion of active substances, and others. Frequencies of the functionally categorized genes in each expression cluster were represented with their statistical significance evaluated by Fisher's exact test. Analyses of functional group enrichement combined with gene synergy showed that LSEC's major functions, i.e. blood coagulation, phagocytosis and transportation, significantly enriched in the cluster characterized by rapid activation and gradual reduction, exhibited the quick recovery of LSEC function after partial hepatectomy. The catergories"immunity&inflammation","defense response"and"the related signaling pathways"were enriched in clusters exhibiting the features of transient gene down-regulation and quick recovery, but they were not drastically changed in response to partial hepatectomy during almost the whole LR based on gene synergy. In addition, the"glycogen synthesis"and"glycolysis"groups were significantly distributed in those clusters marked by"significant increase at proliferation phase and gradual decrease"and"up-regulation at early phase and down-regulation at late phase", and as does"amino acid metabolism"functional group; while the"detoxification"functional group highly enriched in"up-regulation in early phase"became active at early phase, which is helpful in the elimination of waste substance released during LR. The recovery of expression levels of cell proliferation-involved genes after 30 h could favor the proliferation of LSECs during LR. Taken together, analyses of expression patterns of functionally classified genes and gene synergy gave insights into the potential mechanism of roles of LSECs in liver regeneration.To investigate the role of above 796 unknown genes in LR, we firstly obtained the putative full-length cDNA of unknown genes using in silico cloning method, then searched amino acid sequences encoded by cDNA sequences by open reading frame analysis based on ORF finder program. Following above procedure, we finally obtained 486 predicted protein products of unknown genes. Then we predicted the subcellular localization of 486 predicted amino acid sequence by API-SVM method using the dataset of rat protein subcellular localization made by our lab. The results showed that a majority of unknown genes were predictively localized in extracellular space, plasma membrane and nucleus, which inferred that the products encoded by these unknown genes possibly are dedicated to the composition of extracellular matrix, cell membrane formation and gene transcriptional regulation. Briefly, the above results may provide useful clues to confirm their biological functions by conducting the further experiments.
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