كپسوله شده ، ترشح كننده هورمون نوروپيتيد به عنوان يك مدل از دستكاري ژنتيكي RIN6501a تهيه رده سلولي درون ريز

NEUROPEPTIDE Y EXPRESSING, ENCAPSULATED ENDOCRINE CELL LINE AS A MODEL FOR CHRONIC ENDOCRINE MANIPULATION

Background: Neuropeptide Y (NPY) is an endocrine peptide hormone that is produced by many parts of the body specially hypothalamous and has various physiological effects. Repeated intracerebroventricular (ICV) injections have shown NPY to be the most potent stimulator of feeding yet discovered. Chronic studies of many endocrine systems are frustrated by the lack of a reliable delivery system and the instability of many peptides. We have used transfected cell line to model chronic over expression of NPY. To achieve the correct processing of NPY it was necessary to use clonal endocrine cell line e.g, RIN 1056a. Materials and Methods: Full length of NPY cDNA was isolated by PCR, cloned into the expression vector pCEP4 and transfected into RIN 1056a cells. NPY gen expression was shown by total RNA extraction from transfected cells and Northern blotting, correct processing was also confirmed by reversed-phase FPLC and size exclusion chromatography. Since the syngeneic strains were not available for these cells, they had to be immunoisolated. Cells were micro-encapsulated in semipermiable PTFE hollow fibres (MWCO 500000) by resuspending freshly trypsinised cells at a concentration of 4* 10^7 cells/ml in 1% alginate and loaded into the fibres (internal diameter 1 mm). The fibre was heat. sealed approximatly every 3mm and the alginate solidified by brief immersion in 0.05% CaCI2 solution. Results: Individual 3mm sections were maintained in 1 ml DMEM/10% FCS and retained their NPY secreting activity for at least 28 days in vitro. Secretion of IR-NPY was measured by specific radioimmunoassay. Secretion of NPY was maintained for 28 days at approximately the same level with secretion maintained at 150 fmol/hour/fibre. Conclusion: Therefore we have successfully transfected this cell line with NPY cDNA for the first time and measured the production and secretion of NPY from transfected cells in high levels.