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1 대한척추외과학회지제 14 권제 1 호 Journal of Korean Spine Surg. Vol. 14, No. 1, pp 25~33, 2007 퇴행비후된척추후관절이황색인대의섬유화및골화에미치는영향 이광일 # 김향 장주웅 # 전흥재 + 김현민 ++ 박시영 $ 김슬기 이환모 김학선 문성환 연세대학교의과대학정형외과학교실, ( 주 ) 코리아본뱅크생체재료의공학연구소 #, 고려대학교의과대학정형외과학교실 $, 연세대학교공과대학기계공학과 +, 연세대학교공과대학재료공학과 ++ The Effect of Synovial Fluid from Degenerated Facet on Hypertrophy and Ossification of the Ligamentum Flavum Abstract Kwang-Il Lee, M.S. #, Hyang Kim, M.S., Ju-Woong Jang, Ph.D. #, Heoung-Jae Chun, Ph.D. +, Hyun-Min Kim, Ph.D. ++, Si-Young Park, M.D. $, Sul-Ki Kim, M.D., Hwan-Mo Lee, M.D., Hak-Sun Kim, M.D., Seong-Hwan Moon, M.D. Department of Orthopedic Surgery, Yonsei University College of Medicine, Institute for Biomaterial Research, Korea Bone Bank Co. Lt #, Department of Orthopedic Surgery, Korea University College of Medicine $, Department of Mechanical Engineering +, Yonsei University, School of Advanced Materials Engineering ++, Yonsei University Study Design: In vitro experimental study Objectives: To examine the effect of a synovial supernatant on the cell viability, osteogenic phenotype, mrna expression of the types collagen and various transcriptional factors on osteogenesis in ligamentum flavum (LF) cells stimulated with synovial fluid from a degenerated facet joint. Literature Review: In degenerative lumbar spinal stenosis, hypertrophied LF or osteoarthritic hypertrophy of a facet joint often causes neurogenic claudication. The facet joint is a synovial joint with hyaline cartilage on each side. Therefore, osteoarthritis of a facet joint eventually occurs with aging and other degenerative conditions of the spine. In lumbar spinal degeneration, inflammatory mediators or cytokines are released from the facet joint tissue, which consequently affects the adjacent LF because the LF covers posterolateral aspect of the spinal canal near facet joints. However, there are no reports on the relationship between a degenerated facet joint fluid and the LF in the lumbar spine. Materials and Methods: LF surgical specimens were obtained from patients with a lumbar spine stenosis, and the cells were isolated by enzymatic digestion. Each of the synovium tissues were weighed and recorded. Each tissue was cut into small pieces with a pair of scissors and then washed 3 times with PBS. The washed tissue pieces were then cultured for 96 hr at 37 C, 5% CO 2 in DMEM/F % FBS with a density of 200 mg/ml medium. The supernatant was collected after 96 hr. In order to Address reprint requests to Seong-Hwan Moon, M.D. Department of Orthopaedic Surgery, Yonsei University, College of Medicine #134 Shinchon-Dong, Soedaemun-ku, Seoul, , Korea Tel: , Fax: , shmoon@yumc.yonsei.ac.kr 본연구는과학기술부기초과학연구사업 R , 연세대학교의과대학 Brain Korea 21 Project 에의해지원되었습니다

2 대한척추외과학회지 Vol. 14, No. 1, 2007 measure quantitatively the proliferation of cells, the AlamarBlue assay was used. The total cellular RNA was extracted from the cells and amplification reactions specific to the following types of cdna were performed: the osteogenic master transcription factors, Dlx5, Runx2, osterix, and types collagen and osteocalcin. Alkaline phosphatase staining for the biochemical assay and western blotting for osteocalcin protein expression were performed. Results: Human LF cells cultured with the supernatant from the facet synovium showed a slightly stronger AlamarBlue staining than the intensity of the control culture. RT-PCR revealed the upregulation of the osteogenic master transcription factors, Dlx5, Runx2, and osterix in the synovium supernatant group from one hour to 72 hours, and an increase in osteocalcin, types collagen I, III, V, XI levels from one hour to one week. LF cells cultured with the supernatant from the facet synovium showed positive staining for alkaline phosphatase. The level of the osteocalcin protein in the LF cells cultured with the supernatant from the facet synovium was higher than the control group. Conclusions: The supernatant of the facet joint from patients with degenerative spinal stenosis affects LF cells by increasing the level of cellular proliferation, upregulating the mrna expression of osteocalcin, types of collagen, osteogenic transcription factors, positive alkaline phosphatase staining, and osteocalcin protein expression. Therefore, degenerated synovial fluid from the facet joint is an important mechanism of LF hypertrophy and ossification. Key Words: Ligamentum flavum, Facet joint, Synovium, Synovial fluid, Spinal stenosis 서 론 퇴행성요추협착증은중앙의척추관, 신경근이주행하는후관절의전내측에있는신경관또는추간공이좁아지면서신경에압박이오고, 물리적화학적변화를일으켜요통이나하지에여러가지복합된신경증상을일으키는질환이다 1). 요추협착증의주요병인으로는돌출된추간판, 후관절의염증성비후, 그리고황색인대의섬유화에따른비후를들수있다. 이중요추후관절은초자연골로이루어진활액막관절로서개개의관절가동범위는적지만복합운동을하기때문에전체적으로상당한운동범위을보인다. 요추후관절의관절면은주로시상면에서 45 도정도로이루어져있으며, 그형태는원형내지타원형으로편평하거나곡선형태이고, 주로신전과굴곡운동에관여하고있다. 척추후관절의활막은통증에예민한조직이며, 척추에가해지는하중의 1/4 정도가후관절로전달된다. 따라서요추후관절의관절염은나이가들면서점차적으로진행되고이로인한척추의퇴행현상도나타난다. 퇴행성요추병변이심해지면염증성 cytokine 들이요추후관절로부터배출됨으로써인접한황색인대의퇴행성변화에영향을끼칠수도있다 2,3,4,5,6). 퇴행된추간판이황색인대의비후및골화에영향을끼칠수있다는연관성을제시하는보고는있지만 7,8,9), 퇴행비후된후관절과황색인대의비후및골화간의관계에대한연구는없었다. 따라서본연구에서는시험관내에서분리배양한황색인대세포를척추협착증환자로부터퇴행성척추후관절의활액막을추출하였고, 그것을조직배양하여추 출한활액막액을세포에자극시켰을때, 시간이지남에따라활액막액이황색인대세포의독성및증식도, 교원질생성및골형성에어떤영향을끼치는지에대하여알아보았다. 연구대상및방법 1. 세포배양 요추척추관협착증으로감압술을시행받은 10 명의환자들에게서수술시퇴행비후된척추황색인대를채취하였다. 황색인대세포배양은이전에보고된방법을이용하였다 7). 자세히기술하면채취된황색인대조직을멸균된 DPBS (Dulbecco s Phosphate Buffered Saline, Gibco- BRL, Grand island, NY) 로한차례닦아서환자의혈액성분을제거한후, 5% 의우태아혈청 (Fetal bovine serum, Gibco-BRL, Grand Island, NY) 이들어있는 Dulbecco s Modified Eagel s medium (DMEM 배지 ) 에서미세가위를이용하여조직을잘게잘랐다. 잘게자른황색인대조직단편을 250 U/ml 의제 1A 형 collagenase (Sigma, St. Louis, MO, USA) 가함유된 DMEM 배지에서 2 시간동안효소처리하였다. 효소처리한황색인대조직을 5% 의우태아혈청이들어있는배지로두차례씻어낸후, 10% 우태아혈청과 100 unit/ml penicillin, 100 ug/ml streptomycin (all antibiotics from Gibco-BRL, Grand Island, NY) 이들어있는 DMEM 배지로교반하여 37 C, 5% CO 2 배양기에서배양하였다. 배지는 3 일에한번씩교환하였다. 계대수

3 활액막액이황색인대에미치는영향 이광일외 가 2 일때 trypsin/edta 효소처리법으로인간황색인대세포를수집하여액체질소에냉동보관하였다. 2. 조직배양및상층액처리 요추척추관협착증환자 20 명에게서척추감압술을시행시퇴행된척추후관절활액막을채취하였다. 채취된활액막조직을일정량의 DPBS 가들어있는 Nunc tube 에넣어그수치의차이를이용해무게를확인한후, 200 mg/ml 의농도로 0.1% 우태아혈청이함유된 Dulbecco s Modified Eagle Medium and Hams F-12 medium (DMEM/F-12 배지 ) 을처리한후, 37 C, 5% CO 2 배양기에서 3 일간배양하였다 9). 배지는매일교환하였으며, 각각의상층액을 tube 에모아서 centrifuge 로조직의잔여물을걸러낸후상층액만모아서실험에사용하기전까지 -70 C 에냉동보관하였다. 실험에필요한상층액은녹여서 0.1% 우태아혈청과 10 mm Glycerophosphate 가함유된 α-mem 배지와 1:1 의비율로혼합한후, 각각의 well plate 에분주된황색인대세포에처리하여 37 C, 5% CO 2 배양기에서배양하였다. 3. AlamarBlue assay 를통한세포증식반응측정 96 well plate 에각각분주된황색인대세포에세포의대사활성을감지하기위한 10% AlamarBlue solution (Serotec Ltd, Oxford, UK) 을처리하여 37 C, 5% CO 2 배양기에서배양하면서시간 (6, 12, 24, 48, 72 시간 ) 이지남에따라 plate reader 를사용하여 570 nm 과 600 nm 파장에서의흡광도를측정하여시간에따른세포의증식및활액막상층액의독성여부를분석하였다 10). 4. Reverse Transcriptase-Polymerase Chain Reaction 을이용한 mrna 발현검사 RNeasy mini kit (QIAGEN, Maryland, USA) 를이용하여 Total RNA 를분리하였다. 황색인대세포의 Total RNA 1 μg 와 oligo (dt) 1 μl (Invitrogen, USA, 0.5 μg/μl) 와 3 차증류수를혼합하여 50 μl 으로혼합한후, AccuPower RT-premix (Bioneer, 대전, 한국 ) 에넣어 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 을하였다. 42 C 에서 60 분, 94 C 에서 5 분간반응하여 cdna 를합성하였고, 합성한 cdna 를에탄올로정제하여, 최종부피 20 μl 를만들었다. 이중각각의 cdna 1 μl 를취하여, sense primer 와 antisense primer 10 pmole, 3 차증류수를혼합하여 10 μl 의반응용액을만들어 Sapphire PCR-premix (Sapphire, USA) 에넣고, RT (Real Time)-PCR 을시행하였다. PCR 생성물은 2% agarose 젤에서전기영동을통해각유전자의발현을검출하였다 (Table 1, 2) 11,12,13,14). RT-PCR 에대한대조군으로 GAPDH 를사용하였으며 TINA 2.0 program 을통해각각의발현정도를비교분석하였다. Table 1. Sequences of primers used for reverse transcription-polymerase chain reaction to amplify various cdna Gene Sequence (5 3 ) Length Size (bp) Dlx5 TGA CAG GAG TGT TTG ACA GA 20 TGA TAC TGG TAG GGG TTG AG 20 Runx2 AGA TGG GAC TGT GGT TAC TG 20 GTA GCT ACT TGG GGA GGA TT 20 Osterix CCT TTA CAA GCA CTA ATG GG 20 CAC CAT GGA GTA GGA GTG TT 20 Osteocalcin CAC TCC TCG CCC TAT TGG CC 20 GCC AAC TCG TCA CAG TCC GG 20 Collagen type I CCT GTC TGC TTC CTG TTA AC 20 AGA GAT GAA TGC AAA GGA AA 20 Collagen type III CTG CCA TCC TGA ACT CAA GAG TGG 24 CCA TCC TCC AGA ACT GTG TAG G 22 Collagen type V GGA TGA GGA GGT GTT TGA 18 GCC CCT TCA CTG GTT TCA 18 Collagen type XI GCT GAA AGT GTA ACA GAG GG 20 GGT TCT CCT TTC TGT CCT TT 20 Beta actin GGC GGA CTA TGA CTT AGT TG 20 AAA CAA CAA TGT GCA ATC AA bp 321 bp 367 bp 237 bp 182 bp 447 bp 345 bp 452 bp 238 bp

4 대한척추외과학회지 Vol. 14, No. 1, Western Blot analysis 를이용한 osteocalcin 발현검사 Lysis buffer (0.5% Triton X-100, 10 mm HEPES, 150 mm NaCl, 0.02% sodium azide, protease inhibitor mixture) (Sigma) 를이용하여 cell lysate 를만들고이를 10% tricine- SDS gel 을이용하여분리하고전이하였다. Osteocalcin 를분석하기위해 1 TBST with 5% 탈지유에 block 후 blotted membrane 을 rabbit anti-osteocalcin antibody (1:10,000 dilution)(chemicon international, Temecula, CA) 에 1 시간노출시켰다. Membrane 은 1 TBST 으로세척후 secondary antibody (1:10,000 dilution of goat anti-rabbit IgG, horseradish perocidase conjugated, Santa Cruz, CA) 에노출시키고상온에서 45 분간보관하였다. Immuno-reactive bands 는 1 TBST 로 3 번세척후 ECL kit (Amersham Pharmacia, Piscataway, NJ) 를이용하여분석하였다. 황색인대세포에 0.1% 우태아혈청, 10 mm β-glycerophosphate 이함유된 α-mem, 그리고활액막조직상층액을 1:1 로혼합한용액을각각처리하여배양하였다. 각배양군에서세포증식반응및골형성전사인자 (Runx2, Dlx5, Osterix) 와제 1 형, 3 형, 5 형, 11 형교원질, 그리고 osteocalcin 에대한 mrna 발현을측정하였으며, Alkaline phosphatase 염색및 Western Blotting 을이용해서골형성표현형및 osteocalcin 발현검사를하였다. 한편, 활액막조직상층액대신우태아혈청이함유되지않은 DMEM/F-12 를동량처리한황색인대세포를이실험의대조군으로정하였다. 8. 통계 위와같은실험을 3 회에걸쳐서반복하여결과를얻었으며, 모든자료는대조군에대한백분율로서평균 ± 표 6. Alkaline phosphatase 염색 황색인대세포를 citrate buffered acetone (Sigma) 이들어있는고정액을사용하여 30 초동안고정시킨후, 증류수로 45 초동안씻어내고미리준비해둔 alkaline-dye mixture (Sigma) 를각 well 에적용하였다. 이후상온에서빛을차단한채 30 분동안반응시켰다. 반응시킨세포를 2 분동안증류수로씻어낸후 Mayer s Hematoxylin 용액에서 10 분간반응시켰으며, 반응후다시한번증류수로 1 분간씻어내어관찰하였다 15). 7. 실험군 Fig. 1. Alamarblue assay for cytotoxicity and cellular metabolism. Control (white round) denotes ligamentum flavum cell culture with culture media. Supernatant (black square) denotes ligamentum flavum cell culture with supernatant mixture from facet joint synovium culture. Table 2. Conditions of reverse transcription-polymerase chain reaction Primer Conditions Denaturation Annealing Polymerization Cycles Dlx5 94 C 05 sec 58 C 05 sec 72 C 30 sec 35 Runx2 94 C 05 sec 58 C 05 sec 72 C 30 sec 35 Osterix 94 C 05 sec 55 C 05 sec 72 C 30 sec 30 Osteocalcin 94 C 05 sec 60 C 05 sec 72 C 30 sec 30 Collagen type I 94 C 05 sec 48 C 05 sec 72 C 30 sec 25 Collagen type III 94 C 30 sec 54 C 30 sec 72 C 01 min 30 Collagen type V 94 C 30 sec 60 C 30 sec 72 C 01 min 35 Collagen type XI 94 C 30 sec 54 C 30 sec 72 C 01 min 40 Beta-actin 94 C 05 sec 53 C 05 sec 72 C 30 sec

활액막액이황색인대에미치는영향 이광일외 준편차로표시하였으며, SPSS (SPSS Inc. Chicago IL) 를이용하여처리하였다. One-way Analysis of variance 및 Fisher s protected LSD post-hoc test, power analysis 로실험군간비교하였다. 통계방법의유의수준은 p<0.05 로정하였다. 결 과 1.

5 활액막액이황색인대에미치는영향 이광일외 준편차로표시하였으며, SPSS (SPSS Inc. Chicago IL) 를이용하여처리하였다. One-way Analysis of variance 및 Fisher s protected LSD post-hoc test, power analysis 로실험군간비교하였다. 통계방법의유의수준은 p<0.05 로정하였다. 결 과 1. Alamarblue 를통한세포독성및세포증식 Alamarblue solution 을통해시간에따른세포의독성및증식을알아본결과, 활액막조직상층액을처리한황색인대세포는대조군보다약 10% 높은세포대사활성을보였다. 또한세포를배양한지각각 6 시간과 12 시간대, 그리고 24 시간과 48 시간대의사이에서세포의대사활성이급격하게증가하였다 (Fig. 1). Fig. 2. Reverse transcription-polymerase chain reaction products of osteogenic transcription factors collagens and osteocalcin. (A) Dlx5, Runx2, Osterix. (B) Collagen type I, III, V, XI and osteocalcin. Early osteogenic (Dlx5, Runx2, Osterix) and late osteogenic marker (osteocalcin) and various collagens (type I, III, V, XI) showed increase in expression with treatment of synovium supernatant from degenerated facet joint. Fig. 3. Densitometry of osteogenic mrna expression. (A) Dlx5, (B) Runx2, (C) Osterix, (D) Osteocalcin. Dlx5 and Runx2 mrna showed increase in expression in 6 to 12 hours after synovial supernatant treatment. Late osteogenic marker (osteocalcin mrna) showed upregulation 1 week after synovium supernatant treatment

대한척추외과학회지 Vol. 14, No. 1, 2007 2. 골형성전사인자인 Dlx5, Runx2, osterix, 그리고 osteocalcin 의 mrna 발현 황색인대조직의골화와관련하여대표적골형성전사인자인 Dlx5, Runx2, osterix 의초기 mrna 발현정도를 RT-PCR 에대한전기영동으로알아보았다.

6 대한척추외과학회지 Vol. 14, No. 1, 골형성전사인자인 Dlx5, Runx2, osterix, 그리고 osteocalcin 의 mrna 발현 황색인대조직의골화와관련하여대표적골형성전사인자인 Dlx5, Runx2, osterix 의초기 mrna 발현정도를 RT-PCR 에대한전기영동으로알아보았다. 각각의 mrna 발현을 densitometry 를통해분석한결과, 활액막조직상층액을처리한황색인대세포실험군은대조군에비해더높은발현증가를보였다. 특히배양한지 6 시간후의실험군의경우, 1 시간후에비해 Dlx5 는 300%, Runx2 는 340%, 그리고 osterix 는 123% 의발현증가율을보였다. 또한 6 시간이후에시간이지날수록대조군은골형성전사인자들의발현이줄어드는반면에, 실험군은꾸준한증가율을보였다. 역시골형성표현인자인 osteocalcin mrna 발현의경우도대조군에비해높은발현증가를하였으며, 1 주일째에는초기 1 시간배양에비해 300% 의발현증가를보였다 (Fig. 2, 3). 3. 제 1 형, 3 형, 5 형, 11 형교원질의 mrna 발현 황색인대조직의섬유화와관련하여세포의제 1 형, 3 형, 5 형, 그리고 11 형교원질 mrna 의발현정도를 densitometry 를통해분석한결과, 활액막조직상층액을처리한황색인대세포실험군은대조군에비해더높은발현증가를보였다. 제 1 형, 3 형교원질의경우대조군에 Fig. 5. Expression of osteocalcin protein in supernatant of ligamentum flavum cell culture with synovium supernatant (Sup) for 2 weeks. Expression was detected by Western blot analysis. Negative control denotes ligamentum flavum cell with culture media mixture and positive control denotes ligamentum flavum cell with BMP-2 (100 ng/ml). Fig. 4. Densitometry of collagen mrna expression. (A) Collagen type I, (B) Collagen type III, (C) Collagen type V, (D) Collagen type XI. Type I, III, V, XI mrna expressions were upregulated with course of time after treatment of synovium supernatant

활액막액이황색인대에미치는영향 이광일외 비해꾸준한발현증가를하였으며, 48 시간및 1 주일째에서활발한증가를보였다. 특히제 5 형교원질은 1 주일째에서 6200% 의가장높은증가율을보였으며, 제 11 형교원질은배양초기에꾸준한증가율을보이다가 1 주일째에는 570% 의발현증가를하였다 (Fig. 2, 4). 4. 활액막액처리에의한인간황색인대세포의 Osteocalcin 단백질발현 Osteocalcin 단백발현을검증하기위해배양한지 2 주일후에 Western Blot analysis 를시행하여본결과, 16 kda 의 osteocalcin 이검출되었다.

7 활액막액이황색인대에미치는영향 이광일외 비해꾸준한발현증가를하였으며, 48 시간및 1 주일째에서활발한증가를보였다. 특히제 5 형교원질은 1 주일째에서 6200% 의가장높은증가율을보였으며, 제 11 형교원질은배양초기에꾸준한증가율을보이다가 1 주일째에는 570% 의발현증가를하였다 (Fig. 2, 4). 4. 활액막액처리에의한인간황색인대세포의 Osteocalcin 단백질발현 Osteocalcin 단백발현을검증하기위해배양한지 2 주일후에 Western Blot analysis 를시행하여본결과, 16 kda 의 osteocalcin 이검출되었다. 인간황색인대세포에배지의혼합액만처리한배양군을음성대조군으로, 세포에활액막액상층액과배지의혼합액에 BMP-2 (100 ng/ml) 을처리한배양군을양성대조군으로설정하였다. 음성대조군에서 osteocalcin 발현이미미하게발견되었으나양성대조군및실험군에서는많은양의 osteocalcin 이검출되었다 (Fig. 5). 5. 활액막액처리에의한인간황색인대세포의 alkaline phosphatase 염색 황색인대세포에활액막액을처리한후 1 주일이지나초기골형성표현인자의생성여부를확인하고자 Alkaline phosphatase 염색을실시한결과, 발현됨을관찰할수있었다. 대조군에서는골형성인자가관찰되지않았으나활액막액을처리한세포배양군에서는명백한골형성표현인자가생성된것을확인할수있었다 (Fig. 6). 고 찰 척추황색인대의비후의기전에대해서여러가지실 Fig. 6. Alkaline phosphatase stain. Ligamentum flavum cell culture with synovium supernatant for 1 week showed positive alkaline phosphatase stain. 험적연구가있었다. 반복적인기계적신연자극에의한 transforming growth factor beta1 발현에의한세포증식및교원질발현증가 16) 노화및인대자체의퇴행성변화에의한비후 1) 등이제시되고있다. 본연구에서는척추후관절과인접한황색인대가척추후관절의퇴행성변화에의한염증성활액의작용으로비후혹은골화될수있다는가설하에실험적연구를진행하였다. 본연구에서인간척추황색인대에퇴행된활액막에서얻어진활액막액을처리한결과, 척추황색인대의골화및섬유화가되는것을실험적으로증명하였다. 퇴행된척추활액막으로부터얻은활액막액을처리한황색인대세포는초기골형성전사인자인 Dlx5, Runx2, Osterix 등의 mrna 발현이증가하였고, 후기골형성인자인 osteocalcin mrna 발현도증가하였다. 골형성표현형인 osteocalcin 의단백발현이검출되었으며, 골형성표현인자인 alkaline phosphatase 염색에서양성반응이있었음으로척추후관절에서기인한활액막액이척추황색인대의골화에영향을끼친다는것을확인할수있었다. 그리고섬유화와관련된제 1 형, 3 형, 5 형, 그리고 11 형의교원질의 mrna 발현이시간이지남에따라급격하게증가함을보면역시척추후관절활액막액이황색인대의섬유화에도영향을끼친다는것을알수있었다. 골형성의완전히증명하기위해서는장기배양후골형성결절을염색하는 von Kossa 염색이필요하나본연구에서는장기배양에서도의미있는염색결과를보이지않았는데이는활액막의골형성능이 bone morphogenetic protein-2 같은강력한골형성인자에비해서는아주약한골형성능력을보임으로실험기간중에의미있는골결절은형성하지않았다고추정된다. 본연구의결과로기존의반복적기계적신연에의한 transforming grwth factor beta1 발현 16), 퇴행된추간판조직에서발현하는염증성 cytokine 류에의한자극, 추간판간격감소로인한황색인대의겹침 (buckling) 현상등에추가하여퇴행된척추후관절에서분비되는염증성활액막액도척추황색인대의비후와골화에중요한기전임을알수있다. 이상의연구들과본연구의결과를종합하면척추황색인대비후및골화는단순히인대자체의변성퇴화, 일생에걸친반복적신연에의한성장인자 (transforming growth factor-beta1) 발현에의한비후, 퇴행된추간판의추간판간격감소로인한후방구조물황색인대의겹침현상, 퇴행된추간판조직에서발현되는염증성 cytokine 들에의한비후및골화, 그리고본연구에서증명된퇴행된척추후관절에서분비되는염증성활액막에의한비후및골화로종합될수있다. 기술된모든기전이황색인대의교원질형성을촉진시켜섬유화를이

8 대한척추외과학회지 Vol. 14, No. 1, 2007 루는최종과정을거치게되어있으므로치료적의미로국소적으로교원질화의방해를유도할수있다면노화및퇴행성변화로진행되는황색인대의비후를부분적으로막을수있을것이며이는침습적수술을피할수있는방법이될수도있다. 현재가장유망한항섬유화인자는 relaxin 17) 으로추정되며이와관련한연구가진행되어야할것이다. 본연구의의의는문헌상최초로퇴행된척추활액막으로부터나온활액막액이척추황색인대의골화및섬유화에영향을끼친다는사실을분자생물학적그리고조직면역학적기법을동원하여골성인자및교원질 mrna 의발현, 골성표현형의발현, 그리고 alkaline phosphatase 발현등을통해증명하였다는것이다. 그럼에도불구하고본연구의제한점도있는데이는척추후관절활액막액을활액막의조직배양에서추출하였음으로생체내의환경을반영할수없다는점과해부학적으로척추후관절과황색인대가인접한구조이기는하나적접적으로척추후관절활액막액이황색인대조직에전체적으로침투하여세포증식및섬유화를야기한다고실제조직에서증명하기가어렵다는점이다. 결 론 이상의결과로퇴행성척추협착증환자로부터추출한요추후관절의활액막조직의상층액은척추황색인대세포의세포증식및골형성인자 (osteocalcin, Dlx5, Runx2, osterix mrna 발현, alkaline phosphatase 염색반응, osteocalcin 단백질발현 ) 발현과교원질 ( 제 1 형, 3 형, 5 형, 11 형교원질 mrna) 발현의증가에영향을주었다. 따라서퇴행성척추후관절활액막액은황색인대의비대및골화에중요한영향을발휘한다고사료된다. 참고문헌 01) Lee HM: Pathophysiology of lumbar spinal stenosis. J Kor Spine Surg 2000; 7: ) Igarashi A, Kikuchi S, Konno S, Olmarker K: Inflammatory cytokines released from the facet joint tissue in degenerative lumbar spinal disorders. Spine 2004; 29: ) Keiseki K, Segami N, Sun W, Sato J, Fujimura K: Analysis of tumor necrosis factor-alpha, interleukin-6, interleukin-1beta, soluble tumor necrosis factor receptors I and II, interleukin-6 soluble receptor, interleukin-1 soluble receptor type II, interleukin-1 receptor antagonist, and protein in the synovial fluid of patients with temporomandibular joint disorders. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99: ) Lohmander LS, Atley LM, Pietka TA, Eyre DR: The release of crosslinked peptides from type II collagen into human synovial fluid is increased soon after joint injury and in osteoarthritise. Arthritis Rheum 2003; 48: ) Lettesjo H, Nordstrom E, Strom H, et al: Synovial fluid cytokines in patients with rheumatoid arthritis or other arthritic lesions. Scand J Immunol 1998; 48: ) Haynes MK, Hume EL, Smith JB: Phenotypic characterization of inflammatory cells from osteoarthritic synovium and synovial fluids. Clin Immunol 2002; 105: ) Specchia N, Pagnotta A, Gigante A, Logroscino G, Toesca A: Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis. J Orthop Res 2001; 19: ) Okuda T, Baba I, Fujimoto Y, et al: The pathology of ligamentum flavum in degenerative lumbar disease. Spine 2004; 29: ) Li H, Zou Z, Baatrup A, Lind M, Bunger C: Cytokine profiles in conditioned media from culture human intervertebral disc tissue. Acta Orthop 2005; 76: ) Nociari MM, Shalev A, Benias P, Russo C: A novel onestep, highly sensitive fluorometric assay to evaluate cellmediated cytotoxicity. J Immunol Methods 1998; 213: ) Derfoul A, Carlberg AL, Tuan RS, Hall DJ: Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells. Differentiation 2004; 72: ) Luppen CA, Leclerc N, Noh T, et al: Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without alteration of Runx2. J Biol Chem 2003; 278: ) Ryoo HM, Hoffmann HM, Beumer T, et al: Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol Endocrinol 1997; 11: ) Lee MH, Kwon TG, Park HS, John MW, Ryoo HM: BMP-2-induced osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 2003; 309:

9 활액막액이황색인대에미치는영향 이광일외 15) He J, Jiang J, Safavi KE, Spangberg LS, Zhu Q: Emdogain promotes osteoblast proliferation and proliferation and differentiation and stimulates osteoprotegerin expression. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 97: ) Nakatani T, Marui T, Hitora T, Doita M, Nishida K, Kurosaka M: Mechanical stretching force promotes collagen synthesis by cultured cells from human ligamentum flavum via transforming growth factor-beta1. J Orthop Res 2002; 20: ) Sherwood OD: Relaxin s physiological roles and other diverse actions. Endocrine Rev 2004; 25: 국문초록 연구계획 : 인간황색인대세포에퇴행성척추후관절로부터나온활액막액을처리한후배양한다. 연구목적 : 시간이지남에따라활액막액이황색인대세포의증식, 교원질생성및골형성에어떤영향을끼치는지에대하여알아보았다. 대상및방법 : 요추협착증으로수술한환자에게서황색인대조직을채취하여황색인대세포를배양하였으며척추후관절에서활액막을제거한후조직배양하였다. 활액막조직을 96시간동안조직배양하여모은상층액을황색인대세포에처리하여배양하였으며, 시간에따른세포의대사활성및생존력을확인하기위해 alamarblue assay를실시하였고, 전사수준에서의골형성관련유전자및 osteocalcin, 그리고교원질유전자의변화를확인하고자 RT-PCR 을통한densitometry를분석하였다. 또한골형성표현형확인을위해 alkaline phosphatase staining을실시하였고, osteocalcin 단백질발현정도를알아보고자 western blotting을실시하였다. 결과 : 활액막조직상층액을처리한황색인대세포의실험군에 Alamarblue assay를한결과, 대조군보다활발한대사활성을보임으로써상층액이무독성인것을확인하였으며, 세포들은일정시간에서급격한대사활성의증가를보였다. 또한대표적인골형성전사인자인 Dlx5, Runx2, 그리고 osterix들의 1시간에서 72시간간의발현이대조군에비해유의하게증가하였으며, osteocalcin과교원질유전자의 1시간에서 1주일간의발현역시후반부에서급격한증가를보였고, alkaline phosphatase staining을통해골형성분화가진행되는것을확인할수있었다. 또한 western blotting 을통해서 osteocalcin 단백질의발현이시간이지남에따라대조군에비해유의하게증가하였다. 결론 : 이상의결과로퇴행성척추협착증이있는환자로부터추출된퇴행성요추후관절내의활액막액은황색인대세포의증식및골형성인자 (osteocalcin, Dlx5, Runx2, osterix mrna expressions, positive alkaline phsphatase stain, osteocalcin protein) 발현을증가시켰으며, 제 1형, 3형, 5형, 11형의교원질의발현도증가시켰다. 따라서이전연구에서밝혀진퇴행된추간판과함께퇴행성요추후관절내의활액막액은황색인대의비후및골화에중요한영향을끼친다고사료된다. 색인단어 : 황색인대세포, 척추후관절, 활액막, 활액막액, 척추협착증 통신저자 : 문성환서울특별시서대문구신촌동 134 연세대학교의과대학정형외과학교실 Tel: Fax: shmoon@yumc.yonsei.ac.kr

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence