Title page for etd-0722117-235342


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URN etd-0722117-235342
Author Ying-Ying Shen
Author's Email Address No Public.
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Department Institute of Biomedical Sciences
Year 2016
Semester 2
Degree Master
Type of Document
Language English
Title Generation and Characterization of Recombinant Adenovirus Encoding Irisin
Date of Defense 2017-07-04
Page Count 55
Keyword
  • angiogenesis
  • gene therapy
  • obesity
  • myokines
  • Irisin
  • gluconeogenesis
  • Abstract Exercise represents one of the most effective approaches for control of obesity and metabolic syndromes. Irisin is a 112-residue myokine secreted by skeletal muscle through proteolytical cleavage from its precursor fibronectin type III domain containing 5 (FNDC5). Irisin stimulates brown fat-like development from white fat and thermogenesis through increasing mitochondria genesis and energy expenditure, thereby reducing body weight and insulin resistance. Because of its anti-obesity effects and evolutionary conservation, Irisin has been proposed as a promising therapeutic agent for metabolic diseases since its discovery in 2012. To combat the obesity syndrome, gene therapy approached may be required for long-term management of patients with metabolic diseases. Thus, the present study aims to generate the recombinant adenovirus vectors for Irisin production in various types of cells/organs, thereby evaluating their therapeutic potential and mechanism. Recombinant irisin was expressed and purified from E. coli with an apparent molecular weight of 14 kDa. The anti-irisin antibody was raised by periodical injection of recombinant irisin into rabbit and purified from serum using protein G Sepharose affinity chromatography. For gene delivery, adenovirus vector encoding FNDC5 (Ad-FNDC5) and irisin (Ad-irisin) were generated and purified by cesium chloride ultracentrifugation. To investigate the profile of FNDC5/irisin expression in different types of cells, endothelial EA.hy926, muscle C2C12, hepatocytes Clone-9, and embryonic kidney HEK293 cells were employed for adenovirus gene delivery. By immunoblot analysis and enzyme-linked immunoassay (ELISA), it was found that Ad-irisin effectively transduced and conferred irisin secretion in all four types of cells whereas Ad-FNDC5 evoked moderate irisin secretion only in C2C12 and Clone-9 cells. Infection with Ad-irisin enhanced the viability and migration of endothelial cells, supporting the pro-angiogenic function of irisin. Besides, Ad-irisin-infected hepatocytes exhibited elevated activities of AMPK/Akt and inhibition of PEPCK signaling, suggesting the role of irisin in gluconeogenesis in the livers. With the development of these irisin-based tools, future studies are warranted to elucidate the cellular function of irisin in different organs, thereby exploring the therapeutic potential of irisin therapy for various human diseases.
    Advisory Committee
  • Sheu, Jim Jinn-Chyuan - chair
  • Chen, Chun-Lin - co-chair
  • Tai, Ming-Hong - advisor
  • Files
  • etd-0722117-235342.pdf
  • Indicate in-campus at 0 year and off-campus access at 99 year.
    Date of Submission 2017-08-23

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