Summary:The expression of basic fibroblast growth factor (bFGF) in rat liver fibrosis and hepatic stellate cells (HSCs) and the relationship between the expression of bFGF and rat liver fibrogenesis were studied. Sixt...Summary:The expression of basic fibroblast growth factor (bFGF) in rat liver fibrosis and hepatic stellate cells (HSCs) and the relationship between the expression of bFGF and rat liver fibrogenesis were studied. Sixty male SD rats (230-260 g) were divided into 4 groups randomly (the 0 week group, 1 week group, 4 week group and 8 week group). Liver fibrosis was induced by subcutaneous injection of carbon tetrachloride. The sections of rats' liver in each group were tested by Van-Gieson (V-G) staining and immunohistochemistry. The expression of bFGF mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). HSCs were isolated by the combined methods of collagenase IV perfusion and density gradient centrifugation. The expression of bFGF protein in cultured HSCs was detected by Western blot. Images of immunohistochemistry detection, agarose gel electrophoresis of RT-PCR and SDS-polyacrylamide gel electrophoresis of Western blot were analyzed semiquantitatively by image-analyzing system. The results were analyzed by statistics. The results showed that the fibers were gradually increased in the sections of rat liver with the prolongation of the model induction. At the end of the 8th weeks, liver fibrosis was formed. The expression of bFGF detected by immunohistochemistry showed a similar tendency of gradual increase. At the end of the 8th weeks, the bFGF expression could be observed in many regions in sections and the strongest expression was in interstitial cells including HSCs and some hepatocytes in regions around the portal area and central veins. Also there was moderate expression widely in extracellular matrix (ECM). In RT-PCR detection and Western blot detection of HSCs cultured in vitro, the similar tendency of gradual increase was evident either. It is suggested that bFGF is related with liver fibrosis of rats closely and may be a fibrogenesis factor of liver. bFGF possibly regulates liver fibrogenesis through regulating metabolism of extracellular matrix (ECM) by autocrine and paracrine stimulation.展开更多
Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem c...Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells （MSCs）. Methods：The HGF cDNA was amplificated from the expression plasmid pCMV-HGF, and was subcloned into the adenovirus shuttle plasmid pDC316-IRESEGFP vector containing a green fluorescence protein （GFP） reporter gene. Virus Ad-HGF was produced by homologous recombination in HEK293 package cells. Bone marrow derived MSCs were harvested and cultured, and then were transduced with Ad-HGF. The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression. And HGF/MSCs were generated. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody. Results： The recombinant, named pDC316-HGF-IRES-eGFP, was digested with restriction enzyme, and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation. GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15 × 10^10pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 96.89% of HGF/MSCs were GFP positive. Peak concentration levels of hHGF（103ng/mL） in the cultured supernatants were detected on day 2 post-transduction, and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion：Our data demonstrated that the adenovirus expression＇vector system pDC316-HGF-IRES-EGFP has been constructed successfully, and their effective expressions also have been obtained in MSCs. This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.展开更多
Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the cont...Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.展开更多
BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor...BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.展开更多
Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoi...Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.展开更多
AIM To explore the exact interaction between Notch and transforming growth factor(TGF)-β signaling in liver fibrosis. METHODS We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood m...AIM To explore the exact interaction between Notch and transforming growth factor(TGF)-β signaling in liver fibrosis. METHODS We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells(PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-β inhibitor for 24 h. The m RNA levels of Notch and TGF-β signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-β proteins was analyzed by western blotting.RESULTS Compared to control rats, Notch and TGF-β signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-β inhibitor suppressed Notch and TGF-β signal transducer in PBMCs of model rats. DAPT reduced m RNA and protein expression of TGF-β signaling, such as TGF-β1 and Smad3. TGF-β inhibitor also downregulated Notch1, Hes1 and Hes5, and m RNA and protein expression of the Notch signaling pathway.CONCLUSION Notch and TGF-β signaling play a role in liver fibrosis. TGF-β signaling upregulates Notch signaling, which promotes TGF-β signaling.展开更多
AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransform...AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.展开更多
AIM:To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fmslike tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC),and the blocking e...AIM:To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fmslike tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC),and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells.METHODS:Immunohistochemical staining for VEGF,FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae.A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000.Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein.The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltertrazolium bromide (MTT) assay.Cellular apoptosis was detected using flow cytometry (FCM).RESULTS:The expression of VEGF,FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P=0.008,P=0.000,P=0.000).The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P=0.009 and P=0.025,respectively).Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels.VEGF-siRNA cell growth inhibition was assessed by the MTT assay,and the tumor cell proliferation rate was significantly different at 24,48,and 72 h after transfection.FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak.CONCLUSION:VEGF,FLT-1 and FLK-1 are associated with colorectal carcinogenesis.siRNA silencing of the VEGF gene suppresses proliferation,and induces apoptosis in HCT116 cells.The results suggest that VEGF may be a new gene therapy target for colorectal cancer.展开更多
Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth...Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% ( P <0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased ( P <0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced ( P <0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC ( P <0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.展开更多
文摘Summary:The expression of basic fibroblast growth factor (bFGF) in rat liver fibrosis and hepatic stellate cells (HSCs) and the relationship between the expression of bFGF and rat liver fibrogenesis were studied. Sixty male SD rats (230-260 g) were divided into 4 groups randomly (the 0 week group, 1 week group, 4 week group and 8 week group). Liver fibrosis was induced by subcutaneous injection of carbon tetrachloride. The sections of rats' liver in each group were tested by Van-Gieson (V-G) staining and immunohistochemistry. The expression of bFGF mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). HSCs were isolated by the combined methods of collagenase IV perfusion and density gradient centrifugation. The expression of bFGF protein in cultured HSCs was detected by Western blot. Images of immunohistochemistry detection, agarose gel electrophoresis of RT-PCR and SDS-polyacrylamide gel electrophoresis of Western blot were analyzed semiquantitatively by image-analyzing system. The results were analyzed by statistics. The results showed that the fibers were gradually increased in the sections of rat liver with the prolongation of the model induction. At the end of the 8th weeks, liver fibrosis was formed. The expression of bFGF detected by immunohistochemistry showed a similar tendency of gradual increase. At the end of the 8th weeks, the bFGF expression could be observed in many regions in sections and the strongest expression was in interstitial cells including HSCs and some hepatocytes in regions around the portal area and central veins. Also there was moderate expression widely in extracellular matrix (ECM). In RT-PCR detection and Western blot detection of HSCs cultured in vitro, the similar tendency of gradual increase was evident either. It is suggested that bFGF is related with liver fibrosis of rats closely and may be a fibrogenesis factor of liver. bFGF possibly regulates liver fibrogenesis through regulating metabolism of extracellular matrix (ECM) by autocrine and paracrine stimulation.
基金supported by National Natural Science Foundation of China(No.30671992)a grant from the"135"Foundation of Jiangsu Province(No.135-10).
文摘Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells （MSCs）. Methods：The HGF cDNA was amplificated from the expression plasmid pCMV-HGF, and was subcloned into the adenovirus shuttle plasmid pDC316-IRESEGFP vector containing a green fluorescence protein （GFP） reporter gene. Virus Ad-HGF was produced by homologous recombination in HEK293 package cells. Bone marrow derived MSCs were harvested and cultured, and then were transduced with Ad-HGF. The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression. And HGF/MSCs were generated. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody. Results： The recombinant, named pDC316-HGF-IRES-eGFP, was digested with restriction enzyme, and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation. GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15 × 10^10pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 96.89% of HGF/MSCs were GFP positive. Peak concentration levels of hHGF（103ng/mL） in the cultured supernatants were detected on day 2 post-transduction, and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion：Our data demonstrated that the adenovirus expression＇vector system pDC316-HGF-IRES-EGFP has been constructed successfully, and their effective expressions also have been obtained in MSCs. This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.
基金Supported by Grants from the Ministerio de Ciencia e Innovación, MICINN, Spain (SAF2009-12477 to Sánchez A BFU2009-07219 and ISCIII-RTICC RD06/0020 to Fabregat I)+1 种基金AGAUR-Generalitat de Catalunya (2009SGR-312 to Fabregat I)UCM-BSCH (920359 to Sánchez A)
文摘Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to prolifera-tion, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the trans-forming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-βmediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expan-sion of liver stem cells. Hedgehog family ligands are nec-essary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell fac-tor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies.
基金Supported by the National Natural Science Foundation of China,No.81560104 and No.81860115
文摘BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.
文摘Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.
基金Supported by the Natural Science Foundation of Shandong Province,No.2014ZRB01466
文摘AIM To explore the exact interaction between Notch and transforming growth factor(TGF)-β signaling in liver fibrosis. METHODS We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells(PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-β inhibitor for 24 h. The m RNA levels of Notch and TGF-β signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-β proteins was analyzed by western blotting.RESULTS Compared to control rats, Notch and TGF-β signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-β inhibitor suppressed Notch and TGF-β signal transducer in PBMCs of model rats. DAPT reduced m RNA and protein expression of TGF-β signaling, such as TGF-β1 and Smad3. TGF-β inhibitor also downregulated Notch1, Hes1 and Hes5, and m RNA and protein expression of the Notch signaling pathway.CONCLUSION Notch and TGF-β signaling play a role in liver fibrosis. TGF-β signaling upregulates Notch signaling, which promotes TGF-β signaling.
基金the National Natural Science Foundation of China,No.39670287the Scientific Research Foundation for Doctorate Education,State Education Commission.No.96026530
文摘AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.
基金Supported by Natural Science Foundation of Anhui Province,No. 090413098
文摘AIM:To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fmslike tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC),and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells.METHODS:Immunohistochemical staining for VEGF,FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae.A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000.Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein.The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltertrazolium bromide (MTT) assay.Cellular apoptosis was detected using flow cytometry (FCM).RESULTS:The expression of VEGF,FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P=0.008,P=0.000,P=0.000).The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P=0.009 and P=0.025,respectively).Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels.VEGF-siRNA cell growth inhibition was assessed by the MTT assay,and the tumor cell proliferation rate was significantly different at 24,48,and 72 h after transfection.FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak.CONCLUSION:VEGF,FLT-1 and FLK-1 are associated with colorectal carcinogenesis.siRNA silencing of the VEGF gene suppresses proliferation,and induces apoptosis in HCT116 cells.The results suggest that VEGF may be a new gene therapy target for colorectal cancer.
文摘Background Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor β subunit (PDGFR-β) is the predominant signal transduction pathyway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-β mRNA in HSC and the effect on biological characteristics of HSC.Methods Expression vector of anti-PDGFR-β ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-β, α-smooth muscle actin, and typeⅠand type Ⅲ collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.Results The expression of PDGFR-β at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49%-57% ( P <0.05-0.01). The proliferation and α-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased ( P <0.05-0.01), and the type Ⅰ and type Ⅲ collagen synthesis were also reduced ( P <0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC ( P <0.01), and typical apoptotic cells could be found under transmission electron microscopy.Conclusions The anti-PDGFR-β ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-β expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-β expression.
