Erythropoietin produced by genetic-modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons
Journal
Brain and Behavior
Journal Volume
5
Journal Issue
8
Pages
e00356
Date Issued
2015
Author(s)
Abstract
Background: Erythropoietin (EPO) has potent neuroprotective effects. The short-term delivery of high-dose EPO seemed to improve patients' neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future invivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level. Methods: A mouse EPO cDNA was subcloned into the pCMS-EGFP vector and transfected into NIH/3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO-overexpressing cell line, EPO-3T3-EGFP, was established. To further confirm the neuroprotective abilities of secreted EPO from EPO-3T3-EGFP cells, a cell model of neurodegeneration, PC12-INT-EGFP, was applied. Results: The expression level of EPO was highly elevated in EPO-3T3-EGFP cells, and an abundant amount of EPO secreted from EPO-3T3-EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, the survival rate of PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-INT-EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF-M, phosphorylated-NF-M, and the α-INT-EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment. Conclusion: The established EPO-overexpressing NIH/3T3 cell line, EPO-3T3-EGFP, may provide a material for future studies of cell-based therapies for neurodegenerative diseases via the secretion of EPO on a short-term, high-dose, regional basis. ? 2015 The Authors.
SDGs
Other Subjects
alpha internexin; complementary DNA; erythropoietin; propidium iodide; complementary DNA; erythropoietin; neuroprotective agent; recombinant protein; 3T3 cell line; animal cell; Article; cell culture; cell differentiation; cell viability; controlled study; degenerative disease; enzyme linked immunosorbent assay; fibroblast; genetic transfection; immunocytochemistry; mouse; nerve degeneration; neurologic disease; nonhuman; plasmid; priority journal; real time polymerase chain reaction; reverse transcription polymerase chain reaction; time-lapse video microscopy; Western blotting; animal; biosynthesis; genetics; human; metabolism; molecular cloning; nerve degeneration; procedures; protein engineering; Animals; Cloning, Molecular; DNA, Complementary; Erythropoietin; Humans; Mice; Nerve Degeneration; Neuroprotective Agents; NIH 3T3 Cells; Protein Engineering; Recombinant Proteins
Publisher
John Wiley and Sons Ltd
Type
journal article