인회석 박막 피복 도관과 Brain-derived neurotrophic factor(BDNF) 유전자 이입 슈반세포를 이용한 백서 좌골신경 재생에 관한 연구

• 영문명: SCIATIC NERVE REGENERATION USING CALCIUM PHOSPHATE COATED CONDUIT AND BRAIN-DERIVED NEUROTROPHIC FACTOR GENE-TRANSFECTED SCHWANN CELL IN RAT
• 발행기관: 대한구강악안면외과학회
• 저자명: 최원재(Won-Jae Choi) 안강민(Kang-Min Ahn) 황순정(Soon-Jeong Hwang) 정필훈(Pill-Hoon Choung) 김명진(Myung-Jin Kim) 김남열(Nam-Yeol Kim) 유상배(Sang-Bae Yoo) 장정원(Jeong-Won Jahng) 김현만(Hyun-Man Kim) 김중수(Joong-Soo Kim) 김윤희(Yun-Hee Kim) 김성민(Soung-Min Kim) 오승재(Seung-Jae Oh) 김성준(Sung-Jun Kim) 양병은(Byoung-Eun Yang) 이종호(Jong-Ho Lee)
• 간행물 정보: 『대한구강악안면외과학회지』대한구강악안면외과학회지 제31권 제3호, 199~218쪽, 전체 20쪽
• 주제분류: 의약학 > 내과학
• 파일형태: PDF
• 발행일자: 2005.06.30

영문 초록

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with 􀝜-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x106) or BDNF-Ad infected Schwann cells(1x106) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54±4.0×106 and 9.66±9.6×106. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 μg/μl of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 μg/μl of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

키워드

Peripheral nerve regeneration Calcium phosphate coating Nerve conduit Schwann cell BDNF(Brain-derived Neurotrophic factor) Adenovirus