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CONSTRUCTION OF HNGF-$\beta$ RECOMBINANT ADENOVIRUS & SCREENING OF ITS EXPRESSION AFTER TRANSFECTION INTO VARIOUS CELL LINES  

Gao, En-Feng (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Chung, Hun-Jong (Dept. of Pediatrics, Chung-Ju Medical Hospital, Kon-Kuk University)
Ahn, Kang-Min (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Kim, Yoon-Tae (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Park, Hee-Jung (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Sung, Mi-Ae (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Kim, Nam-Yeol (Dept. of Pharmacology, Yonsei University School of Medicine)
Yoo, Sang-Bae (Dept. of Pharmacology, Yonsei University School of Medicine)
Myoung, Hoon (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Hwang, Soon-Jung (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Kim, Myung-Jin (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Kim, Sung-Min (Dept. of Oral and Maxillofacial Sugery, Kang-Nung National University)
Jang, Jeong-Won (Dept. of Pharmacology, Yonsei University School of Medicine)
Lee, Jong-Ho (Dept. of Oral and Maxillofacial Sugery, Seoul National University)
Publication Information
Maxillofacial Plastic and Reconstructive Surgery / v.27, no.5, 2005 , pp. 446-456 More about this Journal
Abstract
Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.
Keywords
NGF; Schwann cell; Peripheral nerve regeneration; Adenoviral vector;
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