Experimental Research On Iron Accumulation In Mouse H22 Hepatoma Cells Mediated By 24p3 Gene

Iron is typically acquired by mammalian cells through receptor-mediated endocytosis of Tf. However, there are kinds of iron delivery systems mediated by non-Tf-TfR pathway. Recently, a member of the Lipocalin family, 24p3/NGAL, was demonstrated to bind iron and was suggested to participate in a non-Tf dependent iron transport mechanism capable to deliver iron into the cytoplasm and further to regulate the expression of special iron responsive genes in the process of differentiation of renal epithelial cells and renal tissues injury repair. Mouse 24p3/SIP24 protein coded by 24p3 gene is an excretive protein. NGAL(Neutrophil Gelatinase-Associated Lipocalin) protein discovered in human neutrophilic granulocyte has a high homology with 24p3/SIP24 protein ( 60.2%).Some insights had been made into this function of 24p3/NGAL gene maily in renal epithelial cells and kidney tissues. However, there is no paper reporting that whether iron transporting by ways of 24p3/NGAL was ubiquitous in the organism. Liver was recognized as one of the most important organs in the regulation of iron metabolism. It is very significant to investigate this new iron transporting pathway in hepatocytes. To investigate this issue, we designed experiments to examine iron accumulation in mouse H22 hepatoma cells mediated by 24p3 gene in response to different iron concentration. Main methods and results were summarized below.Part 1 Construction and identification of pcDNA3.0/24p3 recommbinant plasmidAccording to mouse 24p3 gene sequence (NM:008491) registered in GenBank, a pair of oligonucleotides were designed as primers which contained digestion sequence of BamH I and EcoR I restriction endonuclease at two 5'ends respectively were designed. Complete CDS of 24p3 gene was amplified from mouse lung tissue by RT-PCR. pcDNA3.0/24p3 recombinant plasmid was constructed and was further verified by sequencing. The results demonstrated that inserted sequence was completely identical to registered CDS of 24p3 gene in GenBank (NM:008491).