Proteomic analysis of the regenerating liver following 2/3 partial hepatectomy in rats

BACKGROUND: Liver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism. RESULTS: Protein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR. CONCLUSIONS: For the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.

The role of Kupffer cells in rat liver regeneration revealed by cell-specific microarray analysis.

Liver regeneration after partial hepatectomy is a process with various types of cells involved. The role of Kupffer cells (KCs) in liver regeneration is still controversial. In this study we isolated KCs from regenerating liver and conducted cell-specific microarray analysis. The results demonstrated that the controversial role of KCs in liver regeneration could be explained with the expression patterns of TGF-α, IL-6, TNF, and possibly IL-18 during liver regeneration. IL-18 may play an important role in negative regulation of liver regeneration. The functional profiles of gene expression in KCs also indicated that KC signaling might play a negative role in cell proliferation: signaling genes were down regulated before cell division. Immune response genes in KCs were also down regulated during liver regeneration, demonstrating similar expression profiles to that of hepatocytes. The expression patterns of key genes in these functional categories were consistent with the temporal functional profiles.

Alterations in DNA repair gene expression and their possible regulation in rat-liver regeneration

Rapidly proliferating tissue may require enhanced DNA repair capacity in order to avoid fixation of promutagenic DNA lesions to mutations. Partial hepatectomy (PH) triggers cell proliferation during liver regeneration (LR). However, little is known on how DNA repair genes change and how they are regulated at the transcriptional level during LR. In the present study, the Rat Genome 230 2.0 array was used to detect the expression profiles of DNA repair genes during LR, and differential expression of selected genes was confirmed by real-time RT-PCR. 69 DNA repair genes were found to be associated with LR, more than half of which distributed in a cluster characterized by a gradual increase at 24-72h and then returning to normal. The expression of base excision repair- and transcription-coupled repair-related genes was enhanced in the early and intermediate phases of LR, whereas the expression of genes related to HR, NHEJ and DNA cross-link repair, as well as DNA polymerases and related accessory factors, and editing or processing nucleases, were mainly enhanced in the intermediate phase. The expression changes of genes in DNA damage response were complicated throughout the whole LR. Our data also suggest that the expression of most DNA repair genes may be regulated by the cell cycle during LR.

Study on correlation of signal molecule genes and their receptor-associated genes with rat liver regeneration.

To investigate the effect of signal molecules and their receptor-associated genes on rat liver regeneration (LR) at the transcriptional level, the associated genes were originally obtained by retrieving the databases and related scientific publications; their expression profiles in rat LR were then checked using the Rat Genome 230 2.0 microarray. The LR-associated genes were identified by comparing gene expression difference between partial hepatectomy groups and operation-control groups. A total of 454 genes were proved to be LR related. The genes associated with the seven kinds of signal molecules (steroid hormones, fatty acid derivatives, protein and polypeptide hormones, amino acids and their derivatives, choline, cytokines, and gas signal molecules) were detected to be enriched in a cluster characterized by upregulated expression in LR. The number of genes related to the seven kinds of signal molecules was, in sequence, 63, 27, 100, 102, 16, 166, and 18. The 1027 frequencies of upregulation and 823 frequencies of downregulation in total as well as 42 types of different expression patterns suggest the complex and diverse gene expression changes in LR. It is presumed that signal molecules played an important role in metabolism, inflammation, cell proliferation, growth and differentiation, etc., during rat LR.

Correlation analysis of liver tumor-associated genes with liver regeneration.

AIM: To study at transcriptional level the similarities and differences of the physiological and biochemical activities between liver tumor (LT) and regenerating liver cells. METHODS: LT-associated genes and their expression changes in LT were obtained from databases and scientific articles, and their expression profiles in rat liver regeneration (LR) were detected using Rat Genome 230 2.0 array. Subsequently their expression changes in LT and LR were compared and analyzed. RESULTS: One hundred and twenty one LT-associated genes were found to be LR-associated. Thirty four genes were up-regulated, and 14 genes were down-regulated in both LT and regenerating liver; 20 genes up-regulated in LT were down-regulated in regenerating liver; 21 up-regulated genes and 16 down-regulated genes in LT were up-regulated at some time points and down-regulated at others during LR. CONCLUSION: Results suggested that apoptosis activity suppressed in LT was still active in regenerating liver, and there are lots of similarities and differences between the LT and regenerating liver at the aspects of cell growth, proliferation, differentiation, migration and angiogenesis.

[The origination and action of the hepatic stems cells].

Oval cells are small cells with scant cytoplasm and ovoid-shaped nuclei. These cells, probably deriving from the bone marrow or the cell population associated with the bone marrow, are activated hepatic stem cell. The morphological characteristic of the hepatic stem cells is similar to the oval cells and its biochemistry marker is c-kit/CD45/TRE19. In this paper, the knowledge about the hepatic stem cells and their functions responsible for liver development, liver regeneration, carcinogenesis and relation to other cell are reviewed.

[Cloning and analysis of rat heat shock factor binding protein 1 cDNA].

Heat shock factor binding protein 1(HSBP1) is a nuclear-localized, novel, conserved, low molecular weight (< 100 residues) transcriptional factor, which may repress the activity of the heat shock factor 1 (HSF1) by binding HSF1 active trimerization domain. HSBP1 gene have been cloned in human and mouse, but not reported in rat. In this paper, a pair of consensus degenerate primers were designed based on N-terminal and C-terminal conservative amino acid sequence. Using RT-PCR method, hsbp1 gene fragment was amplified and cloned from total RNA extracted from rat C6 glioma cells. Then the EST was probed to isolate the rat full-length hsbp1 cDNA by in situ hybridization from a rat C6 glioma cells cDNA library. The full-length hsbp1 was deposited in GenBank (accession No. AY522937). It was blasted in RGD (rat genome database) and was localized in 19q12 and composed of four extrons and three introns. The distance between the first extron and the fourth extron was 5829bp. Then its Uinigene was searched, results showed HSBP1 existed widely in all kinds of organs and tissues, the data suggested that it may play a important roles in physiological activity. In addition, the sequence similarity and phylogenetic relationship were compared with DNAman tool. The result showed the relationship is consistent between the similarity of amino acid sequence and phylogenetic evolution from morphological of those species which were nearly in evolution.

Shedding of TNFR1 in regenerative liver can be induced with TNF alpha and PMA.

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated. METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-alpha, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFalpha was studied by detecting apoptotic index. RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNFalpha, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNFalpha for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNFalpha and plasma membrane from hepatocytes affected with TNFalpha for 2 h or from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21 % to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too, but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats. CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFalpha. Membrane-anchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.

[Heredity and evolution of ADAM].

ADAM (a disintegrin and metalloproteinase), or named MDC (metalloproteinase/disintegrin/cysteine-rich) is a family of glycoproteins in cell surface, which was found in recent years and consists of a signal peptide, a propetide, a metalloproteinase, a disintegrin, a cysteine-rich domain, and an epidermal growth factor (EGF)-like domain, a transmembrane region, and a cytoplasmic tail. The research results about their origin, heredity, evolution and evolutionary relationship are summarized in this paper.

Modification and Degradation of the C(6) Rat Glioma Cellulr HSP68 in Vitro and in Vivo.

The biological modification and degradation of C(6) in vitro and in vivo were analyzed with renaturation electrophoresis, Western blot and autoradiography. The results show: (1) the C(6) cellular neutral proteases hardly take part in HSP68 degradation; (2) HSP68 degradation involves cytosol ATP-binding proteases and lysosomal acidic proteases. The ATP-binding proteases play an important role in starting HSP68 degradation and in degrading HSP68 into large polypeptide fragments. The lysosomal acidic proteases are mainly involved in the complete degradation of the large polypeptide fragments; (3) while HSP68-gene is induced through heat shock to express HSP68, a gene is also induced to express 65 kD ATP-binding protease, which not only specifically degrade HSP68 but also modify it to have serine protease activity; (4) the degradation products of HSP68 in vivo are similar to those in vitro.