ABSTRACT
To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.
Subject(s)
Animals , Female , Male , Mice , Carbon Tetrachloride , Disease Models, Animal , Ethanol , Liver Cirrhosis, Experimental , Metabolism , Pathology , Liver Neoplasms, Experimental , Metabolism , Pathology , Mice, Knockout , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad Proteins , Genetics , Metabolism , Smad4 Protein , Genetics , Metabolism , Tissue Inhibitor of Metalloproteinase-1 , Genetics , Metabolism , Transforming Growth Factor beta1 , Genetics , MetabolismABSTRACT
Objective: To develop a high efficient expression, purification system of recombinant arginine deiminase(ADI).Methods: cDNA fragment encoding for mycoplasma ADI was obtained by artificial synthesis and was cloned into prokaryotic expression vector(pBV220). The recombinant ADI was generated by the transformation of the recombinant vector into the host strain DH5?. Anion exchange and gel filtration chromatography was carried out for purification of the recombinant ADI. The biological activity of final product was detected by the assay of agrinine degradation in vitro. Results: A prokaryotic expression plasmid pBV220-ADI was generated successfully, and was identified by DNA sequencing; the recombinant protein was highly expressed in DH5?, the proportion of the recombinant protein is exceeded 35% of the whole protein. The inclusion bodies were solubilized with 6mol/L guanidine hydrochloride under reducing conditions in order to avoid incorrect disulfide-bond formation of the recombinant ADI molecules. Dilution and dialysis at lower degrees temperature were the optimum renaturation methods. After gel filtration, the purity and specific activity of rADI reached 95% and 80 IU/mg respectively. Conclusions: A set of protocols for high efficient rADI expression and purification has been established, which is simple, efficient and applicable.