Maxillofacial Plastic and Reconstructive Surgery

  • 제27권5호
  • /
  • Pages.446-456
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  • 2005
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  • 2288-8101(pISSN)
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  • 2288-8586(eISSN)
대한악안면성형재건외과학회 (Korean Association of Maxillofacial Plastic and Reconstructive Surgeons)
권호 표지

말초신경재생을 위한 hNGF-$\beta$ recombinant Adenovirus의 제작 및 수종세포주에서 신경성장인자의 발현

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

  • 고은봉 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 정헌종 (건국대학교 의과대학 소아과학교실) ;
  • 안강민 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 김윤태 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 박희정 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 성미애 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 김남열 (연세대학교 의과대학 약리학교실) ;
  • 유상배 (연세대학교 의과대학 약리학교실) ;
  • 명훈 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 황순정 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 김명진 (서울대학교 치과대학 구강악안면외과학교실) ;
  • 김성민 (강릉대학교 치과대학 구강악안면외과학교실) ;
  • 장정원 (연세대학교 의과대학 약리학교실) ;
  • 이종호 (서울대학교 치과대학 구강악안면외과학교실)
  • 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)
  • 발행 : 2005.09.30
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초록

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.

키워드

참고문헌

  1. Lundborg G : A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance. J Hand Surg 25 : 391, 2000 https://doi.org/10.1053/jhsu.2000.4165
  2. Seckel BR : Enhancement of peripheral nerve regeneration. Muscle Nerve 13 : 785, 1990 https://doi.org/10.1002/mus.880130904
  3. May F, Weidner N, Hartung R : Tissue engineering of erectile nerves. Urologe A 43 : 1242, 2004 https://doi.org/10.1007/s00120-004-0692-5
  4. Boyd JG, Gordon T : Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury. Mol Neurobiol 27 : 277, 2003 https://doi.org/10.1385/MN:27:3:277
  5. Griffin CG, Letourneau PC : Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor. J Cell Biol 86 : 156, 1980 https://doi.org/10.1083/jcb.86.1.156
  6. Kaechi K, Ikegami R, Nakamura N, Nakajima M, Furukawa Y, Furukawa S : 4-Methylcatechol, an inducer of nerve growth factor synthesis, enhances peripheral nerve regeneration across nerve gaps. J Pharmacol Exp Ther 272 : 1300, 1995
  7. Lee AC, Yu VM, Lowe JB 3rd, Brenner MJ, Hunter DA, Mackinnon SE, Sakiyama-Elbert SE : Controlled release of nerve growth factor enhances sciatic nerve regeneration. Exp Neurol 184 : 295, 2003 https://doi.org/10.1016/S0014-4886(03)00258-9
  8. Rush RA, Mayo R, Zettler C : The regulation of nerve growth factor synthesis and delivery to peripheral neurons. Pharmacology & Therapeutics 65 : 93, 1995 https://doi.org/10.1016/0163-7258(94)00059-C
  9. Anton ES, Weskamp G, Reichardt LF, Matthew WD : Nerve growth factor and its low-affinity receptor promote Schwann cell migration. Proc Natl Acad Sci USA Mar 29; 91 : 2795, 1994
  10. Thomas PK : The cellular response to nerve injury. III. The effect of repeated crush injuries. J Anat 106 : 463, 1970
  11. Liu HM, Lei HY, Kao KP : Correlation between NGF levels in wound chamber fluid and cytological localization of NGF and NGF receptor in axotomized rat sciatic nerve. Exp Neurol 132 : 24, 1999 https://doi.org/10.1016/0014-4886(95)90055-1
  12. Verma IM, Weitzman MD : Gene Therapy: Twenty-First Century Medicine. Annu Rev Biochem 19, 2004
  13. Yi Y, Hahm SH, Lee KH : Retroviral gene therapy: safety issues and possible solutions Curr Gene Ther 5 : 25, 2005 https://doi.org/10.2174/1566523052997514
  14. Haddada H, Cordier L : Perricaudet M.Gene therapy using adenovirus vectors. Curr Top Microbiol Immunol 199 : 297, 1995
  15. Smith GM, Romero MI : Adenoviral-mediated gene transfer to enhance neuronal survival, growth, and regeneration. J Neurosci Res 55 : 147, 1999 https://doi.org/10.1002/(SICI)1097-4547(19990115)55:2<147::AID-JNR2>3.0.CO;2-8
  16. Brody SL, Crystal RG : Adenovirus-mediated in vivo gene transfer. Ann N Y Acad Sci 716 : 90, 1994 https://doi.org/10.1111/j.1749-6632.1994.tb21705.x
  17. Le Gal La Salle G, Robert JJ, Berrard S, Ridoux V, Stratford-Perricaudet LD, Perricaudet M, Mallet J : An adenovirus vector for gene transfer into neurons and glia in the brain. Science 259 : 988, 1993 https://doi.org/10.1126/science.8382374
  18. Klein RL, Hamby ME, Gong Y, Hirko AC, Wang S, Hughes JA, King MA, Meyer EM : Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain. Exp Neurol 176 : 66, 2002 https://doi.org/10.1006/exnr.2002.7942
  19. Abe K, Setoguchi Y, Hayashi T, Itoyama Y : In vivo adenovirus- mediated gene transfer and the expression in ischemic and reperfused rat brain. Brain Res. 763 : 191, 1997 https://doi.org/10.1016/S0006-8993(97)00389-2
  20. Borsani G, Silani V : Human fetal brain beta-nerve growth factor cDNA: molecular cloning of 5'and 3' untranslated regions. Neurosci Lett 130 : 279, 1991 https://doi.org/10.1016/0304-3940(91)90415-P
  21. Kim SM, Lee JH : Improved protocol for Schwann cell isolation and proliferation from rat dorsal root ganglia in vitro. J Kor Maxillofac Plast Reconstr Surg 25 : 265, 2003
  22. Smith GM, Romero MI : Adenoviral-mediated gene transfer to enhance neuronal survival, growth, and regeneration. J Neur Res 55 : 147, 1997
  23. Graham FL, Prevec L : Methods for construction of adenovirus vectors. Mol Biotechnol 3 : 207, 1995 https://doi.org/10.1007/BF02789331
  24. Smith GM, Berry R, Yang Y, Tanelian D : Electrophysiological analysis of dorsal root ganglion neurons preand pos-coexpression of green fluorescent protein and functional 5-HT3 receptor. J Neurophysiol 77 : 3115, 1997 https://doi.org/10.1152/jn.1997.77.6.3115
  25. Smith JG, Raper SE, Wheeldon EB, Hackney D, Judy K, Wilson JM, Eck SL : Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self-limiting inflammatory response. Human Gene Ther 8 : 943, 1997 https://doi.org/10.1089/hum.1997.8.8-943
  26. Jimenez JC, Tyson DR, Dhar S, Nguyen T, Hamai Y, Bradshaw RA, Evans GR : Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration. Plast Reconstr Surg 113 : 605, 2004 https://doi.org/10.1097/01.PRS.0000101066.76548.E8
  27. Koda M, Hashimoto M, Murakami M, Yoshinaga K, Ikeda O, Yamazaki M, Koshizuka S, Kamada T, Moriya H, Shirasawa H, Sakao S, Ino H : Adenovirus vector-mediated in vivo gene transfer of brain-derived neurotrophic factor (BDNF) promotes rubrospinal axonal regeneration and functional recovery after complete transection of the adult rat spinal cord. J Neurotrauma 21 : 329, 2004 https://doi.org/10.1089/089771504322972112
  28. Joung I, Kim HS, Hong JS, Kwon H, Kwon YK : Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury. Mol Cells 31 : 540, 2000