RESUMO
Neurogenin 3 is necessary for endocrine cell development in the embryonic pancreas and has been shown to induce transdifferentiation duct cells from adult pancreas toward a neuro-endocrine phenotype. Here we discovered that the demethylating agent 5'-Azadeoxycytidine (AZA) induced Ngn3 expression and endocrine differentiation from the PANC-1 human ductal cell line. The expression of markers specific to mature islet cells, i.e., glucagon and somatostatin, was also observed. In addition, we demonstrated that growth factors (betacellulin and soluble factors released during pancreas embryogenesis) increased the level of maturation. Our studies revealed that the PANC-1 model system may provide a basis for elucidating the ductal/endocrine differentiation.
Assuntos
Azacitidina/análogos & derivados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Transdiferenciação Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Ilhotas Pancreáticas/citologia , Proteínas do Tecido Nervoso/biossíntese , Ductos Pancreáticos/efeitos dos fármacos , Azacitidina/farmacologia , Diferenciação Celular , Linhagem Celular , Decitabina , Humanos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Fatores de Transcrição/biossínteseRESUMO
BACKGROUND: In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. RESULTS: This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability). As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. CONCLUSIONS: Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.