인간단뇌세포에서분화된신경줄기세포를이용한백서의척수재생

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1 대한정형외과학회지 : 제 42 권제 3 호 2007 J Korean Orthop Assoc 2007; 42: 인간단뇌세포에서분화된신경줄기세포를이용한백서의척수재생 김상림 정광훈 이광복 제주대학교의과대학정형외과학교실, 제주대학교병원정형외과 Spinal Cord Regeneration in Rat using Neural Stem Cell Differentiated from Human Telencephalon Sang-Rim Kim, M.D., Kwang Hoon Chung, M.D., and Kwang-Bok Lee, M.D. Department of Orthopedic Surgery, Cheju National University College of Medicine, Cheju National University Hospital, Jeju, Korea Purpose: To evaluate the effect of neural stem cells differentiated from a human telencephalon on the neural regeneration in the severed spinal cord. Materials and Methods: The 1 st surgery involving the insertion of plastic membrane in the transected cord was performed to prevent spontaneous healing of adult female rats (n=20, g) with a complete spinal cord transection. The media was inserted only after removing the previously inserted plastic membrane in the control group (n=6). In the experimental group (n=14), media and neural stem cell (1 ) were transplanted after removing the membrane, and immunohistochemical staining was performed. The experimental group was perfused transcardially 5 weeks after the 2 nd surgery, and the level of neural cell regeneration determined by immunohistochemical staining. In behavioral analysis, the Basso-Beatie-Bresnahan (BBB) scores of the control and experimental group were compared weekly from immediately after the injury until 5 weeks post-injury after the 2 nd surgery. Results: Immunohistochemical stain revealed no neural regeneration in the control group. On the other hand, the survival of transplanted human neural stem cells and remarkable neural regeneration (differentiate to neuron and astrocyte) were observed in the experimental group. In the BBB locomotor scale, the experimental group showed significant recovery in terms of control; and the score increased from postoperative 2 weeks to 3 weeks, and reached a plateau from 3 weeks to 5 weeks. Conclusion: The effect of neural stem cells differentiated from human telencephalon on cord regeneration does not produce functional recovery in the BBB locomotor scale, but there is slight recovery of the muscle function. The survival of transplanted human neural stem cells and the possibility of differentiation to neurons or astrocytes were observed. Key W ords: N eural stem cell, C ord injury, N eural regeneration 서론 3,10,12-16,19),,. 통신저자 : 이광복제주시삼도 2 동 154 제주대학교병원정형외과 TEL: FAX: osdr2815@cheju.ac.kr * 본논문의요지는 2005 년도대한정형외과학회추계학술대회에서발표되었음. * 이논문은 2005 년도제주대학교학술연구지원사업에의하여연구되었음. Address reprint requests to Kwang-Bok Lee, M.D. Department of Orthopedic Surgery, Cheju National University Hospital, 154, Samdo 2-dong, Jeju , Korea Tel: , Fax: osdr2815@cheju.ac.kr 324

2 인간신경줄기세포를이용한백서의척수재생 325.,,.,.,,. 대상및방법 1. 신경줄기세포획득 (fetal telencephalon), insulin, tranferrin, selenium, hydrocortisone, T3, βfgf UBC (University of British Columbia) 1 serum-free medisa (F3 cell)., Lac Z (encoding) 5). 2. 동물수술및술후처치 (NIH), (,, ), 6).,. 8 Sprague-Dawley (n=20, g), (ketamine) 25 mg/ml (rompun) 1.3 gm/ml, (forcep), cefazolin 50 mg/kg. T9-T10,, 11 (scalpel) (knife). 1.5 mm, Nylon, (metal clip) 1. 1, (cage) , 1 Fig. 1. Diagram summarizing the system of all procedures in this experiment.

3 326 김상림 정광훈 이광복. (n=14) (thrombin), (fibrinogen), (collagen) (media) (Tachocom R ) cm ( ), (cyclosporin) 1, 3. (n=6) ( ), (Fig. 1)., % sucrose (immersing) (longitudinal sectioning, 25-μm ). (, F3 cell) B-gal, (neuron) Neurofilament, (astrocyte) GFAP,. 3. 면역조직화학적검사 2 5 phosphate buffered saline (PBS) 4% (paraformaldhyde), 1 cm 4. 임상적분석 2,, open-field Basso- Beattie-Bresnahan (BBB) locomotor rating scale 3) Table 1. BBB (Basso-Beatie-Bresnahan) Locomotor Rating Scale 0 No observable hindlim (HL) movement 1 Slight movement of one or two joints, usually the hip and/or knee 2 Extensive movement of one joint or extensive movement of one joint and slight movement of one other joint 3 Extensive movement of two joints 4 Slight movement of all three joints of the HL 5 Slight movement of two joints and extensive movement of the third 6 Extensive movement of two joints and slight movement of the third 7 Extensive movement of all three joints of the HL 8 Sweeping with no weight support or plantar placement of the paw with no weight support 9 Plantar placement of the paw with weight support in stance only (i.e, when stationary) or occasional, frequent, or consistent weight-supported dorsal stepping and no plantar stepping 10 Occasional weight-supported plantar steps; no FL-HL coordination 11 Frequent to consistent weight-supported plantar steps and no FL-HL coordination 12 Frequent to consistent weight-supported plantar steps and occasional FL-HL coordination 13 Frequent to consistent weight-supported plantar steps and frequent FL-HL coordination 14 Consistent weight-supported plantar steps, consistent FL-HL coordination, and predorminant paw position during locomotion is rotated (internally or externally) when it makes initial contact with the surface as well as just before it is lifted off at the end of stance; or frequent plantar stepping, consistent FL-HL coordination, and occasional dorsal stepping 15 Consistent plantar stepping, consistent FL-HL coordination, and no toe clearance or occasional toe clearance during forward limb advancement; predorminant paw position is pararelle to the body at initial contact 16 Consistent plantar stepping, consistent FL-HL coordination, and toe clearance occurs frequently during forward limb advancement; predorminant paw position is pararelle at initial contact and rotated at lift off 17 Consistent plantar stepping, consistent FL-HL coordination, and toe clearance occurs frequently during forward limb advancement; predorminant paw position is pararelle at initial contact and lift off 18 Consistent plantar stepping, consistent FL-HL coordination, toe clearance occurs consistently during forward limb advancement; predorminant paw position is pararelle at initial contact and rotated at lift off 19 Consistent plantar stepping, consistent FL-HL coordination, toe clearance occurs frequently during forward limb advancement; predorminant paw position is pararelle at initial contact and lift off, and tail is down part or all of the time 20 Consistent plantar stepping and consistent coordinated gait, consistent toe clearance, predorminant paw position is pararelle at initial contact and lift off, and trunk instability; tall consistently up 21 Consistent plantar stepping and consistent coordinated gait, consistent toe clearance, predorminant paw position is pararelle throughout stance, and consistent trunk staility; tall consistently up

4 인간신경줄기세포를이용한백서의척수재생 , 4.., (coordination) (Table 1). student t-test. 결과 1. 수술부위의육안적소견 1, A B Fig. 2. Photograph showing the injured spinal cord (asterix), and the insertion and removal of the plastic membrane (curved arrows) during the 1st surgery (A) and 2nd surgery (B). Showing the wide defect and surrounding scar tissue of the spinal cord result from the inserted plastic membrane. Fig. 3. Images showing β-gal (+) cell as a red color (B), Neurofilament (+) cell as a green color (A), and double staining (C) with two antibodies. A B C Fig. 4. Image showing β-gal (+) cell as a red color (B), GFAP (+) cell as a green color (A) and double staining (C) with two antibodies.

5 328 김상림 정광훈 이광복 (scar tissue) (granulation tissue) (Fig. 2A, B). 2. 면역조직화학검사의분석, (n=14) Lac Z B-gal (TRITC, ) (Fig. 3B), Neurofilament (FITC, ) (Fig. 3A), (Fig. 3C).. (astrocyte) GFAP (FITC,, Fig. 4A) B-gal (Fig. 4B), (Fig. 4C, Table 2). Table 2. Summary of the Data for Immunohistochemical Staining and the BBB Locomotor Scores Number Weight (g) Operation β-gal* GFAP Neuro filament BBB (Rt) BBB (Lt) Media only , 0, 1, 1, 1, 1 0, 2, 2, 2, 2, Media only , 1, 3, 3, 3, 3 0, 1, 1, 1, 1, Media only , 1, 2, 2, 2, 2 0, 0, 0, 0, 0, Media only , 6, 9, 9, 10, 10 5, 7, 9, 9, 10, Media only , 0, 0, 0, 0, 0 1, 2, 2, 2, 2, Media only , 4, 7, 8, 8, 9 0, 0, 0, 0, 0, Stem cell + + No 0, 0, 0, 0, 0, 2 1, 3, 9, 9, 9, Stem cell + + No 0, 0, 0, 0, 0, 0 1, 2, 3, 3, 3, Stem cell + + No 1, 3, 3, 3, 3, 3 1, 1, 9, 11, 11, Stem cell + + No 3, 12, 19, 19, 19, 19 0, 0, 11, 14, 14, Stem cell + + No 0, 0, 0, 6, 6, 7 7, 7, 15, 15, 15, Stem cell + + No 3, 8, 8, 13, 13, 14 7, 10, 14, 16, 16, Stem cell + + No 14, 16, 18, 19, 19, 19 3, 3, 9, 9, 10, Stem cell + No + 0, 1, 1, 3, 3, 4 0, 0, 0, 0, 0, Stem cell + No + 0, 0, 3, 3, 3, 3 0, 3, 3, 3, 3, Stem cell + No + 0, 0, 0, 1, 1, 1 1, 3, 6, 6, 6, Stem cell + No + 0, 0, 0, 3, 3, 3 2, 2, 14, 15, 15, Stem cell + No + 2, 2, 2, 10, 13, 14 4, 9, 9, 16, 18, Stem cell + No + 0, 0, 1, 2, 2, 2 0, 0, 1, 2, 2, Stem cell + No - 0, 0, 0, 0, 1, 1 0, 1, 1, 3, 3, 3 *β-gal: β-galactosidase, GFAP: glial fibillic acidic protein. A BBB locomotor scores Control Experiment Rt. lower extremity B BBB locomotor scores Control Experiment Lt. lower extremity Weeks Weeks Fig. 5. Image showing the behavioral analysis with the BBB locomotor scores of the right (A) and left (B) lower extremities.

6 인간신경줄기세포를이용한백서의척수재생 임상적행동분석결과 BBB 1.17, , , , , , 1.00, , , , , BBB 1.64, , , , , , 1.93, , , , , BBB (student t-test, p=0.014, p=0.010), 2 3, (Fig. 5A, B). 고찰,,,,, 1,2,4,7,9,17,18,21,22,). (multipotent),,, 1,2,6,10,15)., (glial),,, (create bridges),, 15), (long tract). 8,10,11,15,20),,, (oligodendrocyte)..,. Lac Z,, Lac Z B-gal 5). B-gal,. Neurofilament GFAP ,.,,. 10,12,14,16) 2 3 (peak),.,..

7 330 김상림 정광훈 이광복.,,.,.,,..,,,,.,,, BBB,.,. (cyclosporin),.,.. 결론,,. 참고문헌 1. Alessandri G, Pagano S, Bez A, et al: Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet, 364: , Ankeny DP, McTigue DM, Jakeman LB: Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats. Exp Neurol, 190: 17-31, Basso DM, Beattie MS, Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma, 12: 1-21, Blesch A, Lu P, Tuszynski MH: Neurotrophic factors, gene therapy, and neural stem cells for spinal cord repair. Brain Res Bull, 57: , Chu K, Kim M, Jung KH, et al: Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats. Brain Res, 1023: , de Castro R Jr, Tajrishi R, Claros J, Stallcup WB: Differential responses of spinal axons to transection: influence of the NG2 proteoglycan. Exp Neurol, 192: , Diener PS, Bregman BS: Fetal spinal cord transplants support growth of supraspinal and segmental projections after cervical spinal cord hemisection in the neonatal rat. J Neurosci, 18: , Diers-Fenger M, Kirchhoff F, Kettenmann H, Levine JM, Trotter J: AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia. Glia, 34: , Caggiano AO, Zimber MP, Ganguly A, Blight AR, Gruskin EA: Chondroitinase ABCI improves locomotion and bladder function following contusion injury of the rat spinal

8 인간신경줄기세포를이용한백서의척수재생 331 cord. J Neurotrauma, 22: , Galvin KA, Jones DG: Adult human neural stem cells for cell-replacement therapies in the central nervous system. Med J Aust, 177: , Hofstetter CP, Holmstrom NA, Lilja JA, et al: Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat Neurosci, 8: , Liu S, Qu Y, Stewart TJ, et al: Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci USA, 97: , Lu P, Jones LL, Tuszynski MH: BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol, 191: , Lu P, Jones LL, Snyder EY, Tuszynski MH: Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol, 181: , McDonald JW, Sadowsky C: Spinal-cord injury. Lancet, 359: , Myckatyn TM, Mackinnon SE, McDonald JW: Stem cell transplantation and other novel techniques for promotingrecovery from spinal cord injury. Transpl Immunol, 12: , Rabchevsky AG, Fugaccia I, Turner AF, Blades DA, Mattson MP, Scheff SW: Basic fibroblast growth factor (bfgf) enhances functional recovery following severe spinal cord injury to the rat. Exp Neurol, 164: , Sayer FT, Oudega M, Hagg T: Neurotrophins reduce degeneration of injured ascending sensory and corticospinal motor axons in adult rat spinal cord. Exp Neurol, 175: , Shihabuddin LS, Horner PJ, Ray J, Gage FH: Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci, 20: , Shihabuddin LS, Ray J, Gage FH: FGF-2 is sufficient to isolate progenitors found in the adult mammalian spinal cord. Exp Neurol, 148: , Yick LW, Cheung PT, So KF, Wu W: Axonal regeneration of Clarke's neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp Neurol, 182: , Zuo J, Neubauer D, Dyess K, Ferguson TA, Muir D: Degradation of chondroitin sulfate proteoglycan enhances the neurite- promoting potential of spinal cord tissue. Exp Neurol, 154: , = 국문초록 = 목적 : 인간단뇌에서분화된신경줄기세포의척수재생에대한효과에대해알아보고자하였다. 대상및방법 : 생후 8 주의암컷백서 (n=20, g) 에게척수손상모델중완전절단 (transection model) 을선택한후척수의자가재생 (spontaneous healing) 을방지하기위해플라스틱막 ( cm) 을삽입하여 1 차수술하였다. 대조군 (n=6) 은 2 차수술시삽입된플라스틱막을제거후 media 만삽입하였고, 실험군 (n=14) 은 media 와신경줄기세포 ( ) 를이식한후, 5 주째척수손상부위를얻어면역조직화학염색으로신경세포의재생여부를확인하였다. 임상적결과는 2 차수술후 1 주째부터 1 주간격으로 5 주째까지 BBB (Basso-Beattie-Bresnahan) locomotor scale 을이용하여비교분석하였다. 결과 : 면역조직화학염색상대조군은신경세포의재생은없었고, 실험군에서이식한인간신경줄기세포의생존및신경원세포와성상세포로분화를확인할수있었다. BBB locomotor scale 은대조군에비해실험군에서통계적으로유의한수준의회복을보였고, 술후 2 주에서 3 주째까지점수가상승되다가, 술후 3 주째부터는큰변화는없었다. 결론 : 인간줄기세포를이용한백서의척수재생정도는, 이식된신경줄기세포의생존및신경원세포나성상세포로분화가가능함을확인할수있었고, 임상적으로는어느정도근력회복을보이나기능적수준의회복은보이지않음을알수있었다. 색인단어 : 신경줄기세포, 척수손상, 신경재생