AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells(HSCs)and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of ...AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells(HSCs)and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation.Total RNA and mRNA of quiescent HSCs,and cultureactivated HSCs were extracted,quantified and reversely transcripted into cDNA.The global gene expression profile was analyzed by microarray with Affymetrix rat genechip.Differentially expressed genes were annotated with Gene Ontology(GO)and analyzed with Kyoto encyclopedia of genes and genomes(KEGG)pathway using the Database for Annotation,Visualization and Integrated Discovery.Microarray data were validated by quantitative real-time polymerase chain reaction(qRTPCR).The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi.The expression of Wnt5a in fibrotic liver of a carbon tetrachloride(CCl4)-induced fibrosis rat model was also analyzed with Western blotting.RESULTS:Of the 28 700 genes represented on this chip,2566 genes displayed at least a 2-fold increase or decrease in expression at a P<0.01 level with a false discovery rate.Of these,1396 genes were upregulated,while 1170 genes were downregulated in culture-activated HSCs.These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms.The most enriched GO terms included response to wounding,wound healing,regulation of cell growth,vasculature development and actin cytoskeleton organization.KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs.Wnt5a was significantly increased in cultureactivated HSCs as compared with quiescent HSCs.qRTPCR validated the microarray data.Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation,downregulated expressions of typeⅠcollagen and transforming growth factor-β1.Wnt5a was upregulated in the fibrotic liver of a CCl4-induced fibrosis rat model.CONCLUSION:Wnt5a is involved in the activation of HSCs,and it may serve as a novel therapeutic target in the treatment of liver fibrosis.展开更多
Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results,...Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results, obtained in different laboratories using different platforms, is very relevant nowadays. For revelation of problems ofmicroarrays, the comparative analysis of hepatic and renal gene expression profiles (GEP) was carried out by using swine protein annotated oligonucleotide microarrays (SPAM). Revealed differences in GEP between kidney and liver of pigs were correlated with functional and histological distinctions of these organs. It was shown that sources of errors in the comparative analysis of organ-specific GEP could be connected to the cross hybridization of one probe to transcripts (cDNA of mRNA) of different genes and to individual variability in gene expression between animals, related with the changeability of influences of exo- and endogenous regulation factors.展开更多
基金Supported by Research Grant for Health Science and Technology of Pudong Health Bureau of Shanghai,No.PKJ2009-Y16
文摘AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells(HSCs)and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation.Total RNA and mRNA of quiescent HSCs,and cultureactivated HSCs were extracted,quantified and reversely transcripted into cDNA.The global gene expression profile was analyzed by microarray with Affymetrix rat genechip.Differentially expressed genes were annotated with Gene Ontology(GO)and analyzed with Kyoto encyclopedia of genes and genomes(KEGG)pathway using the Database for Annotation,Visualization and Integrated Discovery.Microarray data were validated by quantitative real-time polymerase chain reaction(qRTPCR).The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi.The expression of Wnt5a in fibrotic liver of a carbon tetrachloride(CCl4)-induced fibrosis rat model was also analyzed with Western blotting.RESULTS:Of the 28 700 genes represented on this chip,2566 genes displayed at least a 2-fold increase or decrease in expression at a P<0.01 level with a false discovery rate.Of these,1396 genes were upregulated,while 1170 genes were downregulated in culture-activated HSCs.These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms.The most enriched GO terms included response to wounding,wound healing,regulation of cell growth,vasculature development and actin cytoskeleton organization.KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs.Wnt5a was significantly increased in cultureactivated HSCs as compared with quiescent HSCs.qRTPCR validated the microarray data.Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation,downregulated expressions of typeⅠcollagen and transforming growth factor-β1.Wnt5a was upregulated in the fibrotic liver of a CCl4-induced fibrosis rat model.CONCLUSION:Wnt5a is involved in the activation of HSCs,and it may serve as a novel therapeutic target in the treatment of liver fibrosis.
文摘Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results, obtained in different laboratories using different platforms, is very relevant nowadays. For revelation of problems ofmicroarrays, the comparative analysis of hepatic and renal gene expression profiles (GEP) was carried out by using swine protein annotated oligonucleotide microarrays (SPAM). Revealed differences in GEP between kidney and liver of pigs were correlated with functional and histological distinctions of these organs. It was shown that sources of errors in the comparative analysis of organ-specific GEP could be connected to the cross hybridization of one probe to transcripts (cDNA of mRNA) of different genes and to individual variability in gene expression between animals, related with the changeability of influences of exo- and endogenous regulation factors.