Volume 1, Issue 1 (6-2002)                   JRUMS 2002, 1(1): 9-19 | Back to browse issues page

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Abstract:   (11221 Views)

  Neuropeptide Y Expressins, Encapsulated Endocrine Cell Line as a Model for Chronic Endocrine Manipulation


  M.Mahmoodi1, MA. Ghatei2


  1- Department of Biocheistry, Rafsanjan School of Medicine Rafsanjan, Iran

  2- Department of Medicine, ICSM, Hammersmith Campus, Du Cane Road, London W12 onn, Uk


  Background: Neuropeptide Y (NPY) is an endocrine peptide hormone that is produced by many parts of the bidy 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 sytem 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 ti use clonal endocrine cell line e.g, RIN 1056a.

  Material 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 4x 107 cells/ml in 1% alginate and loaded into the fibres (internal diameter 1mm). The fibre was heat sealed approximatly every 3mm and the alginate solidified by immersion in 0.05% Cac12 solution.

  Results: Individual 3mm sections were maintained in 1ml DMEM 10% FCS and retained their NPY screting activity for at least 28 days in vitro. Secreation of IR-NPY was measured by specific radioimmunossay. 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 cell in high levels.

  Keywords: Neuropeptide Y, RIN 1056a cell line, Gen Expression, cDNA


Type of Study: Research | Subject: Biochemistry
Received: 2005/12/18 | Published: 2002/06/15

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