PDX-1 Protein Counteracts Apoptosis Induced By Cytokine And Palmitic Acid In Rat Islets Of Langerhans

Diabetes mellitus is a disease characteristicd by hyperglycemia, owing to insufficient insulin or/and insulin resistance, present therapy can not avoid diabetic complication. No matter type 1 or 2 diabetes, the apoptosis of beta-cells of Langerhans plays a prominent role in the pathogenesis of diabetes. Pancreatic duodenum homeobox-1 (pdx-1) is a main transcription regulator of insulin gene, and is necessary for pancreatic development and beta-cell specific gene expression. It is certain that when apoptosis occurs pdx-1 is translocated from nuclei to cytoplasm of the cells. But whether ectopic PDX-1 protein has the antiapoptosis effects is not clear. Since pdx-1 protein has protein transduction domains (PTDs) that allow proteins to be translocated across the membrane and into cell without endocytosis, there has been increasing interest in their potential for the delivery of bioactive peptides and proteins into eukaryotic cells as a valuable strategy for the transduction of therapeutic proteins into patients. Therefore we study whether the pdx-1 protein has antiapoptosis effects induced by cytokine and palmatic acid on primary rat islet of Langerhans through the technology of nest transcription polymerase chain reaction, gene expression, protein purification and Western blotting. Since PDX-1 plays such a crucial role in pancreatic development, fi-cell differentiation, and maintenance of normal β-cell function by its regulation of multiple important β-cell genes, if the protein has the antiapoptosis effect, it will provide extensive theory basis for PDX-1 protein practice in the diabetic prevention and treatment. The study includes three parts.1, Pdx-1 gene coloring and expression plasmid construction. Male Wistar rat pancreas were digested by collagenase XI properly, islet of Langerhans were pufificated by uncontinous density gradients through different concentions of Ficoll 400. Handpicked the purified islets under anatomy microscope,obtained islet cell total RNA using Trizol, after purificattion and identification,coloned a piece of gene countaining pdx-1 by RT and nest PCR . Using this gene as temblate, full-length rat Pdx-1 cDNA was amplified by PCR using appropriate primers the gene was identified by aragose electrophoresis and sequence measurement. Pdx-1 gene...