AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vec...AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.展开更多
AIM： To study the effect of interleukin-10 （IL-10） on the expression of transforming growth factor β1 （TGF-β1） in hepatic fibrosis rats and the anti-fibrotic role of exogenous IL-10. METHODS： Hepatic fibrosis ...AIM： To study the effect of interleukin-10 （IL-10） on the expression of transforming growth factor β1 （TGF-β1） in hepatic fibrosis rats and the anti-fibrotic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by carbon tetrachloride administered （CCh） intraperitoneally. The experiment was performed in two stages. In the first stage, 60 SD rats were divided randomly into normal control group I（GNI, n = 8）, hepatic fibrosis group（GC, n = 28）and IL-10 intervened group（GI, n = 24）. At the beginning of the 7^th and 11^th wk, hepatic stellate cells （HSCs） were isolated, reverse transcription-polymerase chain reation （RT-PCR） and immunocytochemistry were performed to detect the expression of TGF-β1 in HSCs. Histological examination was used to determine the degree of hepatic fibrosis. In the second stage, 47 SD rats were divided randomly into normal control group 2 （GN2, n = 6）and CCh group（GZ, n = 41）. At the end of the 9th week, rats in GZ group were allocated randomly into model group（GM, n = 9）, IL-10 treatment group（GT, n = 9） and recovered group （GR, n = 9）. At the end of the 12^th week, all rats were sacrificed. RT-PCR and immuno- histochemistry were performed to detect the expression of TGF-β1 in liver tissue. ELISA was used to assay serum TGF-β1 levels. RESULTS： Hepatic fibrosis developed in rats with the increase of the injection frequency of CCI4. In the first stage, hepatic fibrosis developed and HSCs were isolated successfully. At the 7^th and 11^th week, TGF-β1 mRNA in GC group increased significantly compared with that in GN1（P = 0.001/0.042） and GI groups（P = 0.001/0.007）, whereas there was no significant difference between the two groups. The levels of TGF-β1 at the beginning of the 7^th wk was higher than that of the 11^th wk （P = 0.049）.Immunocytochemistry results of TGF-β1 were consistent with the above findings. In the second stage, TGF-β1 increased significantly in GM group compared to GN2. Alter treatment with IL-10, TGF-β1 declined obviously. The expression of TGF-β1 decreased in GR group but was still higher than that in GT group. CONCLUSION： The levels of TGF-β1 are increased in hepatic fibrosis rats and decreased alter treatment with exogenous IL-10. IL-10 may play an anti-fibrotic role by suppressing TGF-β1 expression.展开更多
AIM:The transforming growth factor-beta (TGF-β)/Smad signaling pathway system plays a prominent role in the control of cell growth and extracellular matrix formation in the progression of liver fibrogenesis. Smad pro...AIM:The transforming growth factor-beta (TGF-β)/Smad signaling pathway system plays a prominent role in the control of cell growth and extracellular matrix formation in the progression of liver fibrogenesis. Smad proteins can either positively or negatively regulate TGF-β responses. In this study, the therapeutic effects of Chinese traditional compound decoction, JinSanE, and the changes of TGF-β/Smad signaling pathway system in carbon tetrachloride (CCl4)-induced rat experimental liver fibrosis were examined. METHODS: Seventy-two healthy Wistar rats were assigned to groups including normal control group, CCl4 model group, JinSanE treatment group I and JinSanE treatment group II. Each group contained 18 rats. All groups, except the normal control group, received CCl4 subcutaneous injection for 8 wk. Rats in JinSanE groups I and II were orally treated with JinSanE daily at the 1st and 5th wk, respectively, after exposure to CCl4. The expression of TGF-β1 and TGF-β1 type II receptor (TRII) mRNA in the liver was determined by reverse transcription polymerase chain reaction, and the expression of TGF-β1, Smad3 and Smad7 by immunohistochemistry. The liver histopathology was also examined by HE staining and observed under electron microscope. The activities of several serum fibrosis-associated enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the levels of serum hyaluronic acid (HA) were assayed. RESULTS: Hepatic fibrosis caused by CCl4 was significantly inhibited in the JinSanE-treated groups. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the JinSanE-treated groups than in the model control group. The expression of TGF-β1, TRII and Smad3 was significantly higher in the model group than that in the JinSanE-treated groups, and the active/total TGF-β1 ratio in the JinSanE groups was suppressed. Expression of TRII mRNA and Smad3 proteins snowed a distribution pattern similar to that of TGF-β1 with a direct correlation in terms of the degree of hepatic fibrosis. The amount of positive staining Smad7 cells was significantly less in the model group than in the JinSanE-treated groups and the normal group. The contents of ALT, AST and HA were significantly lower in the JinSanE-treated groups than those in the model group. CONCLUSION: Traditional Chinese medicine, JinSanE, prevents the progression of hepatic damage and fibrosis through the inhibition of TGF-β1, TRII and Smad3 signal proteins, and increases expression of Smad7 signal protein in vivo.展开更多
基金the grant from Chinese Academy of Sciences,No.KSCX2-2-204
文摘AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
基金Supported by Natural Science Foundation of Fujian Province, No. 2005D094 and No. C0410025
文摘AIM： To study the effect of interleukin-10 （IL-10） on the expression of transforming growth factor β1 （TGF-β1） in hepatic fibrosis rats and the anti-fibrotic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by carbon tetrachloride administered （CCh） intraperitoneally. The experiment was performed in two stages. In the first stage, 60 SD rats were divided randomly into normal control group I（GNI, n = 8）, hepatic fibrosis group（GC, n = 28）and IL-10 intervened group（GI, n = 24）. At the beginning of the 7^th and 11^th wk, hepatic stellate cells （HSCs） were isolated, reverse transcription-polymerase chain reation （RT-PCR） and immunocytochemistry were performed to detect the expression of TGF-β1 in HSCs. Histological examination was used to determine the degree of hepatic fibrosis. In the second stage, 47 SD rats were divided randomly into normal control group 2 （GN2, n = 6）and CCh group（GZ, n = 41）. At the end of the 9th week, rats in GZ group were allocated randomly into model group（GM, n = 9）, IL-10 treatment group（GT, n = 9） and recovered group （GR, n = 9）. At the end of the 12^th week, all rats were sacrificed. RT-PCR and immuno- histochemistry were performed to detect the expression of TGF-β1 in liver tissue. ELISA was used to assay serum TGF-β1 levels. RESULTS： Hepatic fibrosis developed in rats with the increase of the injection frequency of CCI4. In the first stage, hepatic fibrosis developed and HSCs were isolated successfully. At the 7^th and 11^th week, TGF-β1 mRNA in GC group increased significantly compared with that in GN1（P = 0.001/0.042） and GI groups（P = 0.001/0.007）, whereas there was no significant difference between the two groups. The levels of TGF-β1 at the beginning of the 7^th wk was higher than that of the 11^th wk （P = 0.049）.Immunocytochemistry results of TGF-β1 were consistent with the above findings. In the second stage, TGF-β1 increased significantly in GM group compared to GN2. Alter treatment with IL-10, TGF-β1 declined obviously. The expression of TGF-β1 decreased in GR group but was still higher than that in GT group. CONCLUSION： The levels of TGF-β1 are increased in hepatic fibrosis rats and decreased alter treatment with exogenous IL-10. IL-10 may play an anti-fibrotic role by suppressing TGF-β1 expression.
基金Supported by the Basic Research Program of Hubei Province Education Bureau, No. 2003X113
文摘AIM:The transforming growth factor-beta (TGF-β)/Smad signaling pathway system plays a prominent role in the control of cell growth and extracellular matrix formation in the progression of liver fibrogenesis. Smad proteins can either positively or negatively regulate TGF-β responses. In this study, the therapeutic effects of Chinese traditional compound decoction, JinSanE, and the changes of TGF-β/Smad signaling pathway system in carbon tetrachloride (CCl4)-induced rat experimental liver fibrosis were examined. METHODS: Seventy-two healthy Wistar rats were assigned to groups including normal control group, CCl4 model group, JinSanE treatment group I and JinSanE treatment group II. Each group contained 18 rats. All groups, except the normal control group, received CCl4 subcutaneous injection for 8 wk. Rats in JinSanE groups I and II were orally treated with JinSanE daily at the 1st and 5th wk, respectively, after exposure to CCl4. The expression of TGF-β1 and TGF-β1 type II receptor (TRII) mRNA in the liver was determined by reverse transcription polymerase chain reaction, and the expression of TGF-β1, Smad3 and Smad7 by immunohistochemistry. The liver histopathology was also examined by HE staining and observed under electron microscope. The activities of several serum fibrosis-associated enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the levels of serum hyaluronic acid (HA) were assayed. RESULTS: Hepatic fibrosis caused by CCl4 was significantly inhibited in the JinSanE-treated groups. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the JinSanE-treated groups than in the model control group. The expression of TGF-β1, TRII and Smad3 was significantly higher in the model group than that in the JinSanE-treated groups, and the active/total TGF-β1 ratio in the JinSanE groups was suppressed. Expression of TRII mRNA and Smad3 proteins snowed a distribution pattern similar to that of TGF-β1 with a direct correlation in terms of the degree of hepatic fibrosis. The amount of positive staining Smad7 cells was significantly less in the model group than in the JinSanE-treated groups and the normal group. The contents of ALT, AST and HA were significantly lower in the JinSanE-treated groups than those in the model group. CONCLUSION: Traditional Chinese medicine, JinSanE, prevents the progression of hepatic damage and fibrosis through the inhibition of TGF-β1, TRII and Smad3 signal proteins, and increases expression of Smad7 signal protein in vivo.
