Transcriptome Atlas Of Stellate Cells In Rat Liver Regeneration

The liver is the vital body's organs, and its powerful regenerative capacity is also a hotpot to scholars. The mechanism of liver regeneration (LR) was studied with 2/3 rat hepatectomy model which established by Higgins and Anderson and liver tissue generally, but the liver is composed of a variety of cells. If various cells of regenerating liver were isolated, it will make the researches more clear, simplified and easy. Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells, locates in the Disse space between hepatic cord and sinusoidal walls. The protrusions on their surface attached to the outer surface of liver sinusoidal endothelial cells and hepatocytes. HSCs contained rough endoplasmic reticulum, Golgi complex and many different sizes of lipid droplets, and had many functions such as vitamin A storage and transport, fat absorption; damage response and repair in liver and hepatocytes. It is a professional antigen presenting cells, which can devour apoptotic bodies and regulating immune and inflammatory in liver. It is also major secreting cells of liver extracellular matrix and chemokines and other cytokines. A mass of evidences showed that HSCs play the key role in liver regeneration. Although more and more researches on HSCs has reported recently, there are no much studies on the changes of physical activity in HSCs during liver regeneration in genome-wide. In addition, new technology of genome-wide studied on hepatic stellate cells are emerging, but the understanding to the role of HSCs in liver regeneration was mainly from traditional research methods. Therefore, if we could detect the dynamic expression profile of HSCs in liver regeneration, it is possible to reveal the role of stellate cells in rat liver regeneration.Rat 2/3 hepatectomy (PH) model was made following Higgins et a1., hepatic cells were scattered by two-step perfusion and hepatic stellate cells were isolated and purified by density gradient centrifugation with 60% percol1 and immunomagnetic beads. Immunocytochemistry method was used to qualitify and localize desmin (DES) and vimentin (VIM) in liver tissue, isolated hepatic cells, and purified hepatic stellate cells. The expressions of DES and VIM were quantified using RT-PCR. The results showed that DES and VIM positive hepatic stellate cells account up more than 95% of the total hepatic stellate cells; mRNA levels of DES and VIM were stable in the purified hepatic stellate cells in rat regenerating liver (RL), and also was the content of the corresponding proteins.The gene expression profiling of hepatic stellate cells in rat regenerating liver at 10 recovery time points including 0, 2, 6, 12, 24, 30, 36, 72, 120 and 168 h after PH were checked employing Rat Genome 230 2.0 microarray. The reliability of chip results was evaluated by RT-PCR and Western blot methods. There expression patterns and functions in rat liver regeneration were analyzed using bioinformatics and system biology, and it was found that 2609 genes were related to liver regeneration, including 1186 up-regulated genes, 1212 down-regulated genes, and 211 up-/down-regulated genes. The gene expression profiles at different time points was analyzed by PCA indicated that gene expression profiles of 9 point times are distincted to the three groups, which is 6, 24 and 30 h; 2, 12 and 36 h; 72, 120 and 168 h; and mainly up-regulated in the group of 2, 12 and 36h.To investigate the role of above 1183 novel genes in LR, we firstly obtained the putative full-length cDNA of novel genes using in silico cloning method, then searched the amino acid sequences encoded by cDNA sequences by open reading frame analysis based on ORF finder program. Following above procedure, we finally obtained 936 predicted protein products of novel genes. Then we predicted the subcellular localization of 936 predicted amino acid sequence by API-SVM method using the dataset of rat protein subcellular localization developed by our lab. The results showed that a majority of novel genes were predictedly localized in cytoplasm, nucleus, plasma membrane and mitochondria.The synergies changes of 1426 known genes which involved 60 classes physiological activities in hepatic stellate cells of regenerating liver showed that transmembrane receptor protein tyrosine kinase signaling pathway, cellular component organization, cell growth, cell differentiation, apoptosis, carbohydrate catabolismm and so on were enhanced in the priming phase of liver regeneration; SMO signaling pathway, nucleic acid catabolism, carbohydrate biosynthesis, protein degradation, collagen biosynthesis, neurotransmitter transport, organic acid transport, ion transport and so on were enhanced in the progress phase; protein kinase signaling pathways, cell proliferation, cofactor metabolism and so on were enchanced in the termination phase; lipid transport, vesicle-mediated transport were enhanced in the priming and progress phase. However, cell adhesion and cytokine biosynthesis were weakened in the progress phase of liver regeneration; immune response-activating signal transduction, nitric oxide mediated signal pathway, amino acids and their derivatives biosynthesis, lipid transport, neurotransmitter transport and oxygen transport were decreased in the termination phase; nitric oxide mediated signal transduction and small GTP mediated signaling pathway were weakened in the priming and progress phase of liver regeneration.This study demonstrates that the detection results of Rat Genome 230 2.0 microarray are reliablie; hepatic stellate cells in regenerating liver mainly begin to proliferate at 36 h after PH, and regulates by transmembrane receptor protein tyrosine kinase signaling pathways and cytokine-mediated the signaling pathway; the increase of insulin-like growth factor binding protein will promote the growth of HSCs significantly; HSCs was stimulated by growth hormone will immediately display differentiation characteristics, but Notch signaling pathway can inhibit the differentiation of HSCs along with the process of liver regeneration.