Primary Culture And Identification Of Porcine Preadipocyte And Tissue Expression Distribution Of Porcine SMAF1, ATGL Gene

Primary culture and identification of porcine preadipocyte is pre-requisite for pig adipogenesis and adipocyte function study in vitro. Undersdanding the tissue expression distribution of genes related to adipogenesis and adipocyte function is also the most importent task to study gene function. The present study aimed to establish the model for primary culture of porcine preadipocyte, to investigate the tissue expression distribution of prcine SMAF1(small adipocyte factor 1) and ATGL(adipose triglyceride lipase) genes in vivo, Landrace×Large White, Meishan and Large White were used in this experiment. SMAF1 gene was isolated and identified with comparative genomics and bioinformatic approaches, etc.The main results are as following:1 The primary culture and identification of porcine preadipocyte1.1 Cell suspensions were prepared by collagenase digestion from subcutaneous adipose tissue of young healthy subjects and induced to differentiate into mature adipocytes. Morphological changes accompanying differentiation were observed under light microscopy. After 24 hours of cell suspension were seeded into flask, the adherent cells appeared spindle shape. During the following days, the mophology of preadipocytes changed gradually. Cell acquired a round shape and numerous lipid droplets of variable-size filled-up cytoplasm. During the nenxt stage of differentiation, the lipid droplets fused together, forming large globules. On day 6 of culture, most of the cells were completely filled with lipid droplets. After 7 days of culture, some cells were floated from the bottom of flask and dead gradually.1.2 Biological characterization of cells was detected with cell growth curve. Cell grew slowly during 1～2 days of the preadipocytes plated. Cells then entered exponential growth phase. On day 6 of culture, cells growth entered plateau phase. After 7 days of culture, the numbers of live cells decreased gradually.1.3 The differentiated cells were stained with Oild red-O. The numbers and the volume of Oild red O-contained cells increased with the change of differentiated time. After 7 days of culture, the number of stained cells were begin to reduce.2 Cloning of porcine SMAF1 geneThe porcine SMAF1 gene was cloned by degenerate PCR, The PCR products for porcine SMAF1, generated from pocine adipose tissue, covered intact open reading frame which was identified with GeneScan software and via manual inspection. The length of this cDNA sequence was 256bp (GenBank DQ191892). The sequence for pig was siminar to those of the human(86%)and mouse(78 %). The amino acid sequence of porcine SMAF1 was deduced from the cDNA sequence. It is a protein with 81 amino acid and a molecular weight of 9.5kDa. The SMAF1 amino acid sequence of pig was similar to those of the human(81%), mouse(67%), rat (70%) and bovine (84%).3 The tissue expression distribution of pig SMAF1...