| Objective: Augmenter of liver regeneration (ALR) cloned from liver of weaning rat is a novel hepatic stimulator. It was reported that ALR mRNA expression was correlated with DNA synthesis of hepatocytes after partial hepatectomy, and could significantly promote the survial rate of intoxicated hepatitis rats. As the specific mitogen, ALR could specifically enhance hepatocyte proliferation and might play an important role in recovery of liver injury. In this study, eukaryotic expression plasmid pcDNA3-ALR was constructed and its function on acute hepatic injury was investigated in rats. Methods: PCR was performed using plasmid pBV220-ALR as templat under the following conditions: 94℃ for 1 min, 56℃ for 1 min and 72℃ for 2 min for 30 cycles, and then 72℃ for 10 min. The ALR fragment digested by HindⅢand EcoR I was ligated with efficient eukaryotic expression vector pcDNA3 and transformed into the competent E.coli DH5?. The positive clone containing recombinant plasmid was selected in 2×YT culture, and theinsert of positive clone was identified by enzyme digestion and nucleotide sequencing. The recombinant plasmid DNA was used to treat the rats with acute hepatic injury. The model of acute hepatic failure rat was made by 50% CCl4 of 4ml/kg peritoneally. Forty rats were randomly divided into ALR therapy group and saline control group, which were injected peritoneally ALR gene (200μg/kg) and saline per 12h respectively 4h after CCl4 injection. Rats living over 96h were considered survival. To observe the protective effects of ALR gene on acute liver injury rat, thirty-six rats that were injected peritoneally with 50% CCl4 of 2ml/kg were divided into the flowing 6 group according to doses (50?g/kg and 200?g/kg ) and administration routes (veinous and peritoneal) of pcDNA3-ALR DNA 4h after CCl4 injection: group 1, model group; group 2, ALR gene of 50μg/kg was injected venally; group 3, ALR gene of 200?g/kg was injected veinally; group 4, ALR gene of 50?g/kg was injected peritoneally; group 5, ALR gene of 200?g/kg was injected peritoneally; group 6, ALR gene of 200?g/kg was injected peritoneally and veinously respectively. Another 4 rats that were not treated were as normal control. ALR gene was injected per 12h in all therapy groups for four times, and the rats were decapitated 12h after last injection. Concentration of AST and ALT in serum was determined by biochemical method, changes of liver histopathology observed by HE staining, and PCNA expression in livertissue detected using immuno-histochemistry staining in order to observe protective effects of ALR gene on CCl4-intoxicated rats. Chi-squared test and analysis of variance were carried out for statistics analysis of experiment results. Results: The sequence of target gene of recombinant plasmid pcDNA3-ALR constructed in this experiment was in accordance with the sequence of rat ALR cDNA reported. Recombinant pcDNA3-ALR plasmid was used to treat acute hepatic failure and acute liver injury of CCl4-induced rats in this study. The results showed that ALR gene could remarkedly enhance survival rate of acute hepatic failure rats. In the acute hepatic injury rats, ALR gene could reduce the degree of liver histopathology injury and the concentration of AST and ALT of serum, and promote hepatotic cell proliferation by increasing PCNA expression. The results also showed that the effect of ALR gene with 200?g/kg on acute liver injury in CCl4-intoxicated rats was potenter than that of ALR gene with 50?g/kg. The effect of ALR gene administration routes was as follows: injection of vein ＞mixture injection ＞abdominal injection. It was indicated that the vein injection might be considered as the first selection of gene therapy in this study. Conclusion: (1) The eukaryotic expression plasmid pcDNA3-ALR has been successfully constructed; (2) ALR gene may play an important role in relieving acute hepatic injury by enhancing survival rate of rats, promoting hepaticcell proliferation and reducing concentrati... |
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