Maxillofacial Plastic and Reconstructive Surgery

Korean Association of Maxillofacial Plastic and Reconstructive Surgeons (대한악안면성형재건외과학회)
권호 표지

EFFECT OF NERVE GROWTH FACTOR GENE INJECTION ON THE NERVE REGENERATION IN RAT LINGUAL NERVE CRUSH-INJURY MODEL

백서 설신경 압박손상모델에서 신경성장인자 유전자 주입이 신경재생에 미치는 영향

  • Gao, En-Feng (Department of Oral & Maxillofacial Surgery, Dental Research Institute, Graduate School of Dentistry, Seoul National University) ;
  • Chung, Hun-Jong (Department of Pediatrics, Chung-Ju Medical Hospital, Kon-Kuk University) ;
  • Ahn, Kang-Min (Department of Oral & Maxillofacial Surgery, School of Medicine, Ulsan University, Asan Medical Center) ;
  • Kim, Soung-Min (Department of Oral & Maxillofacial Surgery, School of Dentistry, Kangnung National University) ;
  • Kim, Yun-Hee (Department of Biology, Kyung-Hee University) ;
  • Jahng, Jeong-Won (Department of Oral & Maxillofacial Surgery, Dental Research Institute, Graduate School of Dentistry, Seoul National University) ;
  • Lee, Jong-Ho (Department of Oral & Maxillofacial Surgery, Dental Research Institute, Graduate School of Dentistry, Seoul National University)
  • 고은봉 (서울대학교 치과대학 구강악안면외과학교실 및 치학연구소) ;
  • 정헌종 (건국대학교 의과대학 소아과학교실) ;
  • 안강민 (성균관대학교 의과대학 구강악안면외과) ;
  • 김성민 (강릉대학교 치과대학 구강악안면외과) ;
  • 김윤희 (경희대학교 이과대학 생물학과) ;
  • 장정원 (서울대학교 치과대학 구강악안면외과학교실 및 치학연구소) ;
  • 이종호 (서울대학교 치과대학 구강악안면외과학교실 및 치학연구소)
  • Published : 2006.09.30
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Abstract

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

Keywords

References

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