Study On The Transactivation Function Of Recombinant Human Aumenter Of Liver Regeneration

The incidence of virus hepatitis and related cirrhosis is high in our country. These diseases threaten the lives of many patients and bring about heavy burdens to them. Up to date,there is no effective way to cure these diseases. Hepatocyte damage is found in patients with virus hepatitis and related cirrhosis. It is important to promote hepatocyte regeneration in the process of viral hepatitis treatment. Especially in severe patients. Aumenter of liver regeneration plays a key role in liver regeneration. Aumenter of liver regeneration is a protein factor which was cloned recently. Aumenter of liver regeneration can specifically stimulate the regeneration of cell which comes from liver . It was used to save the lives of acute hepatic function failure and chronic liver disease in animal models. But the molecular biological mechanisms is still unknown. We design this experiment to explicit the molecular biological mechanisms of recombinant human aumenter of liver regeneration. We hope this experiment can provide a theoretical basis to the therapeutics of virus hepatitis and related cirrhosis.Objective This study can be devided into two parts: (1) To clone and identify human genes transactivated by recombinant human aumenter of liver regeneration (ALR) by constructing a cDNA subtractive library with suppression subtractive hybridization (SSH) technique. (2) To investigate the relationship of recombinant human aumenter of liver regeneration (rhALR) and cyclin-dependent kinase 1 (cdk-1) gene and explain the molecular biological mechanisms of recombinant human aumenter of liver regeneration in gene expression regulations.Methods (1) Suppression subtractive hybridization and bioinformatics techniques were used for screening and cloning of the target genes transactivated by recombinant human aumenter of liver regeneration. The recombinant human ALR was prepared by standard procedure of fermentation and chromatography techniques. SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with BLASTn search after PCR. (2) Polymerase chain reaction (PCR) technique was employed to amplify the sequence of cdkl promoter using HepG2 cell genomic DNA as template, named cdklP, and the PCR product was cloned into pGEM-T vector. The cdc2P gene was cut from pGEM-T-cdklP by Kpnl and Xhol I, and then was cloned into pCAT3 basic, named pCAT3- cdklP. pCAT3- cdklP was transfected into the HepG2 cell line by FuGENE 6 transfection reagents. Then stimulated HepG2 cells with recombinant human aumenter of liver regeneration . The HepG2 cells transfected with pCAT3-basic was used as negative control. The activity of chloramphenicol acetyltransferase (CAT) in HepG2 cells transfected was detected by a enzyme-linked immunoassay (ELISA) kit after 48 hours, which reflected the transactivating function of recombinant human aumenter of liver regeneration to cdkl gene promoter.Results (1) The subtractive library of genes transactivated by recombinant human aumenter of liver regeneration was constructed successfully. The amplified library contains 30 positive clones. Colony PCR shows that these clones contain 200-1000 bp inserts. Sequence analysis was performed in 30 clones, and the full length sequences were obtained with bioinformatics method. Altogether 19 coding sequences were gotten, which consisted of 15 known and 4 unknown ones. (2) The expressive vector pCAT3-cdklP has been constructed and had been confirmed byrestriction enzyme digestion and sequencing. The expression of CAT in HepG2 cells transfected with pCAT3-cdklP and stimulated with recombinant human aumenter of liver regeneration was 39.4 times as higher as that of pCAT3-basic,and 3.2 times as higher as that of pCAT3-cdklP.Conclusion (1) The obtained sequences may be target genes transactivated by recombinant human aumenter of liver regeneration, among which some genes coding proteins involved in cell cycle regulation, tumor development, and metabolism. Advanced experiments need to be done to prove this finding. (2) It is suggested that recombinant human aumenter of liver regeneration can up-regulate cdkl gene promoter. These results provide a new evidence to explain the molecular biological mechanisms of recombinant human aumenter of liver regeneration in hepatocyte.