AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombin...AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals.RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50μg/kg pcDNA3-ALR DNA.The effect of intravenous injection of recombinant pcDNA3ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid,the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P＜0.01).CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level ofAST and ALT in CCl4-intoxicated rats.展开更多
AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cant...AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37℃, 10% FCS in DMEM was replacedin each well. After the treatment repeated twice to threetimes in each concentration of cantide, hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis, respectively. Telomerase aclivity was determinedby TRAP-ELISA assay. CPP32- and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment.RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P＜0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P＜0.01), respectively. By contrast, 8%and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.展开更多
基金Supported by the Natural Science Foundation of Hebei Province, No. 302489
文摘AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl4) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 μg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl4 administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 μg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl4 after 4 h of CCl4 administration, respectively. Rats living over 96 h were considered as survivals.RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 μg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50μg/kg pcDNA3-ALR DNA.The effect of intravenous injection of recombinant pcDNA3ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid,the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P＜0.01).CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level ofAST and ALT in CCl4-intoxicated rats.
基金the National Natural Science Foundation of China, No.39870879the Special Funds for Major State Basic Research of China,No.G 1998051103
文摘AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37℃, 10% FCS in DMEM was replacedin each well. After the treatment repeated twice to threetimes in each concentration of cantide, hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis, respectively. Telomerase aclivity was determinedby TRAP-ELISA assay. CPP32- and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment.RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P＜0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P＜0.01), respectively. By contrast, 8%and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.
