말초신경재생을 위한 hNGF-β recombinant Adenovirus의 제작 및 수종세포주에서 신경성장인자의 발현
Construction of hNGF-betarecombinant adenovirus and screening of its expression after transfection into various cell lines
고은봉, 정헌종, 안강민, 김윤태, 박희정, 성미애, 김남열, 유상배, 명훈, 황순정, 김명진, 김성민, 장정원, 이종호,
소속 상세정보
고은봉 ( Gao En-Feng ) - 서울대학교 치과대학 구강악안면외과학교실
정헌종 ( Chung Hun-Jong ) - 건국대학교 의과대학 소아과학교실
안강민 ( Ahn Kabg-Min ) - 서울대학교 치과대학 구강악안면외과학교실
김윤태 ( Kim Youn-Tae ) - 서울대학교 치과대학 구강악안면외과학교실
박희정 ( Park Hee-Jung ) - 서울대학교 치과대학 구강악안면외과학교실
성미애 ( Sung Mi-Ae ) - 서울대학교 치과대학 구강악안면외과학교실
김남열 ( Kim Nam-Yeol ) - 연세대학교 의과대학 약리학교실
유상배 ( Yoo Sang-Bae ) - 연세대학교 의과대학 약리학교실
명훈 ( Myoung Hoon ) - 서울대학교 치과대학 구강악안면외과학교실
황순정 ( Hwang Soon-Jung ) - 서울대학교 치과대학 구강악안면외과학교실
김명진 ( Kim Myung-Jin ) - 서울대학교 치과대학 구강악안면외과학교실
김성민 ( Kim Soung-Min ) - 강릉대학교 치과대학 구강악안면외과학교실
장정원 ( Jang Jeong-Won ) - 연세대학교 의과대학 약리학교실
이종호 ( Lee Jong-Ho ) - 서울대학교 치과대학 구강악안면외과학교실
KMID : 0360120050270050446
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-betarecombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-betagene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-betawhich was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-betarecombinant 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-betarecombinat 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-betarecombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-betarecombinant 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-betagene transfected cells.
키워드
NGF;Schwann cell;Peripheral nerve regeneration;Adenoviral vector
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