Effects Of Gene Of Pancreatitis-Associated Protein Pap On Rat Liver Regeneration

Pancreatitis-associated protein(PAP) is an inflammatory protein what was found in pancreatic juicefrom pancreatitis, to promote mitosis, the anti-apoptotic, anti-inflammatory and promote cell adhesion toextracellular matrix and so on. In order to study its role in liver regeneration, According to the mRNAsequence of rat pap gene, pap gene was cloned and constructed into the vector. On this basis, interferefragments were designed and two interference vectors pGenesil-1.0-pap(289) and pGenesil-1.0-pap(485),three test vectors pGenesil-1.0-pap(289)-pap,pGenesil-1.0-pap(485)-pap and pGenesil-1.0-HK-pap wereestablished, and pap(485) was slected by methods of the tail vein hydraulic testing the role of each vector.The expression vector pEGFP-N1-pap and its control pEGFP-N1, interference vector pGenesil-1.0-pap-(485) and its control pGenesil-1.0-HK transfected into hepatoma cell BEL-7402with lipofectamine, andselection of stably transfected cell lines to the use of G418. Then, all kinds of cell lines above were detectedusing the following methods: cell morphology observed in vivo and by HE staining; cell growth curve bycell counting and MTT assay; cell proliferation detected by PCNA immunocytochemistry; cell apoptosisdetected by Hoechst33258staining; cell colony formation rate detected by Methyl cellulose semi-solidmedium; cell resistance to drugs detected by MTT assay. These laid the foundation for further study of papin liver regeneration and provided information to reveal the mechanism of liver regeneration molecules. Invivo experiments, the pEGFP-N1-pap, pGenesil-1.0-pap(289) and pGenesil-1.0-pap(485) employed thetechnique of hydrodynamics-based transgening were injected into the remnant livers of rat after2/3partialhepatectomy(PH), and the mortality of rats, the morphological structure of regenerating livers, livercoefficient and liver regeneration rate were analyzed using statistical methods.The results of vitro experiments showed the nucleus of BEL-7402cell transfected with expressionvectors pEGFP-N1-pap significantly increased and looked like spindle; EGFP fusion proteins with papwere mainly distributed around the nucleus nuclear membrane, while no significant changes were observedin other transfected cell lines with EGFP mainly distributed in the nucleus; Growth curve and MTT assayshowed that the proliferation of cells transfected with pEGFP-N1-pap was obvious; PCNAimmunocytochemistry and Hoechst33258staining showed that plasmid pEGFP-N1-pap could promote cell proliferation and inhibit apoptosis; The degree of malignancy of cell lines transfected with pEGFP-N1-papwas increased; MTT assay showed that pap gene can promote BEL-7402cell growth, but no significanteffect on its resistance to drugs. The results of vivo experiments showed that liver coefficient and liverregeneration rate of rats transfected with pEGFP-N1-pap were higher than PH control at120h, but that ofrats transfected with siRNA vectors were lower than the control. In short, pap gene may promote cellproliferation, increase the role of malignancy of hepatoma cell, but has no apparent resistance on hepatomacell as anticancer agents; pap may be involved in promoting the process of liver regeneration.