대한근전도 전기진단의학회지 9(1):69~74, 2007. 인체지방유래세포의근육세포분화가능성 연세대학교의과대학재활의학교실, 근육병재활연구소, 휴림바이오셀, 연세성형외과 박윤길 문재호 이은영 도병록 김지향 김균태 김동수 Abstract Myogenic Potential of Human Adipose-Tissue-Derived Cells Yoon Ghil Park, M.D., Ph.D.*, Jae Ho Moon, M.D.*, Eun Young Lee, B.A.*, Byung Rok Do, Ph.D. **, Ji Hyang Kim, Ph.D. **, Kyun Tae Kim, M.D., Ph.D.***, Dong Soo Kim, M.D. Department of Rehabilitation Medicine and Rehabilitation Institute of Muscular Disease, Yonsei University College of Medicine*, HurimBiocell** & Yonsei Plastic Surgery Clinic*** Objectives: Cell therapy has been extensively studied as a gene complementation approach in such genetic diseases as Duchenne muscular dystrophy (DMD). Adipose tissue has recently been identified as an alternative, uniquely abundant and accessible source of pluripotent cells. In the present work we investigated myogenic potentials of adipose-tissue-derived cells (ATDCs) as mesenchymal stem cells (MSCs). Methods: Human ATDCs were obtained by liposuction and cultured in three different media; control, myogenic and conditioned media. The following observation was made to evaluate differentiation with using immunoflurescence study and western blotting. Results: Conversion of ATDCs to a myogenic phenotype is observed by indirect immunoflurescence study of MyoD and Myf-5 in regardless of media type. However, secondary myogenic regulatory factors (Myf-6 and myogenin) and desmin are negative in all different culture condition. Conclusion: Our findings suggest that human adipose-tissue-derived cells might have a mesencymal stem cell population and myogenic potentials. Since human adipose tissue is plentiful, easily harvested in large quantity under local anesthesia with little patient discomfort, it may be a good source of an alternative cell therapy for DMD patients. Key Words: Cell therapy, Duchenne muscular dystrophy, Adipose-tissue-derived cells, Mesenchymal stem cells 서 론 근육질환중두시엔느 (Duchenne) 형근디스트로피는점차적인근력약화를일으키는질환으로성염색체에의해열성유전되며 X 염색체의단완 (Xp21) 에위치한디스트로핀 (dystrophin) 유전자의변이에의해출생 남아 3,500명중 1명의빈도로발생한다. 1-3 현재까지는근본원인을치료할수있는방법이확립되지않았으며병의진행과합병증을최소화하는물리치료, 운동치료및호흡재활치료등에그치고있다. 4 그러나최근약물치료를비롯한유전자치료및세포치료등의실험적치료법이시도되고있으며이중정상세포가환자의근육 Address reprint requests to Yoon Ghil Park, M.D., Ph.D. Department of Rehabilitation Medicine & Rehabilitation Institute of Muscular Disease, Yonsei University College of Medicine, 146-92 Dogok-dong, Gangnam-gu, Seoul, 135-270, Korea Tel : 82-2-2019-3493, Fax : 82-2-3465-7585, E-mail : drtlc@yumc.yonsei.ac.kr 본연구는연세대학교의과대학 2004년도장기해외교수연구비에의하여이루어졌음 ( 과제번호 : 6-2004-1119). 69
박윤길 문재호 이은영 도병록 김지향 김균태 김동수 세포와융합되어디스트로핀단백질을생성하도록유도하는근육모세포 (myoblast) 이식법이개발되어서임상실험이진행되었으나몇몇제한점이밝혀지면서효과에대해서는논란이되고있다. 5-8 근육모세포이식의제한요소중하나인면역거부반응을극복하기위해줄기세포를이용하는방법이연구되고있으나아직근육줄기세포의순수분리와배양이쉽지않으며분리된줄기세포가근육모세포를거쳐근관세포 (myotube) 와근육조직으로분화가일어나도록하는기전과조절물질이명확히밝혀지지않아서실험실내배양과동물실험단계에만머무르고있으며, 임상실험까지는많은문제점이남아있는상태이다. 이전부터근육생성줄기세포를제대혈, 9,10 골수, 11,12 근육 13 및지방 14 등과같은여러조직에서부터얻으려고하는시도가이루어졌으며특히지방조직의경우지방흡입술시행중나오는것을사용할수있기때문에공여자의부담이적고대량의조직을얻을수있는장점이있다. 그러나지방조직에서유래된줄기세포는대부분중간엽성으로뼈, 연골, 근육등다양한조직으로분화가될수있으므로근육세포로만분화를유도하는것이지방조직에서줄기세포의추출과더불어배양에중요한단계이다. 근육세포로분화되는과정에는발생학적으로도여러단계에걸쳐다양한인자들이관여하는것으로알려져있지만아직모든과정이명확히밝혀지지는않았다. 그러나근육생성조절인자 (myogenic regulator factors, MRFs) 로알려진 basic helix-loop-helix (bhlh) 전사인자 (transcription factor) 의일부분이중요한역할을하는것으로보고되었으며이중일차근육생성조절인자로분류되는 MyoD와 Myf-5는증식된체세포를근육모세포로유도하는역할을하며이차근육생성조절인자인 myogenin과 Myf-6는근육모세포를근관세포로분화시키는역할을한다. 15,16 따라서줄기세포가근육세포로분화되는것을형태학적인변화와더불어근육생성조절인자의발현을관찰하는방법으로확인할수있다. 본연구의목적은인체지방유래세포를이용하여배양상태를달리한후근육세포로분화되는과정의차이를관찰하고이러한변화를근육생성조절인자와근육세포막단백질의발현을통해확인함으로써적절한분화유도조건을알아보고향후근육병환자의세포치료에대한기초자료를확립하고자한다. 연구대상및방법 1. 연구대상 성형외과에서지방흡입수술중분리된지방세포 (lipoaspirate cell) 를이용해서일차배양을하였다. 모든공여자는수술전에신체검진과혈액검사를통해건강상태를확인하였다. 지방세포흡입술은일반적으로시행되는방법과같이피하조직을부분절개하여흡입관을삽입한후지방조직을흡입하는것이며이때출혈을감소를위해혈관수축제인 epinephrine 을생리식염수와같이사용하였다. 2. 연구방법 1) 세포분리및일차배양 (Primary Culture) 채취된지방조직을 phosphate buffered saline (PBS; GIBCO BRL, Grand Island, NY, USA) 에넣어 2 회세척하여혈액등을제거하고 0.075% collagenase (Sigma, St. Louis, MO, USA) 를지방조직과동량의 PBS 에첨가하여 37 에서 30 분동안처리하여세포외간질 (extracellular matrix) 을분해하였다. 이후분해용액의작용을약화시키기위해 10% fetal bovine serum (FBS; GIBCO) 을포함한 Dulbeco s modified Eagles medium (DMEM; GIBCO) 을넣은후, 250 g 에서 10 분간원심분리하여세포를침전시켰다. 침전된세포를 10% FBS-DMEM 세포배양액에서풀고적혈구를분해하기위해 0.16 M NH 4 Cl 를첨가하여상온에서 10 분간대기한후다시 250g 에서 10 분간원심분리하였다. 침전물을다시 10% FBS-DMEM 에풀어서 75 cm 2 배양접시 (culture flask)(nunc, Rosklde, Denmark) 에 1 10 6 /ml 의세포수로접종하였다. 배양은 37, 5% CO 2 배양기에서유지하였으며배양액은일주일경과한후교환하였다. 배양접시에서세포가차지하는비율 (confluence) 이 70% 이상이되면 0.05% trypsin-0.53 mm EDTA 로세포를배양접시에서분리시키고원심분리하여계대배양을시행하였다. 이후배양액은 3 일마다교체하였다. 2) 근육생성줄기세포분화유도및분석일차배양된세포군을나누어제 1 군은대조군으로일차배양에사용된 10% FBS-DMEM 에 1% penicillin 과 streptomycin 을첨가한배양액을계속사용하였으며제 2 군은이전연구들 14,17 에서분화배양액으로알려진것을이용하였는데이는대조군배양액에 5% horse serum 과 50 um hydrocortisone (Sigma) 을추가한것으로근육생성배양액 (myogenic media) 으로분류하였다. 제 3 군은근육모세포를 1 주일이상배양하였던배양액을사용하였으며조건배양액 (conditioned media) 으로분류하였다. 세군모두 37, 5% CO 2 배양기에서 1 주, 3 주, 6 주간배양후근육생성조절인자와근막단백질인 desmin 의발현을면역형광염색법 (immuno- 70
인체지방유래세포의근육세포분화가능성 fluorescence stain) 으로관찰하였으며 3 주이내의초기변화를관찰하기위해서는웨스턴블로팅 (western blotting) 을이용하여일차근육생성조절인자인 MyoD 와 Myf-5 에대한결과를분석하였다. (1) 면역형광염색법 (Immunofluorescence Stain) 근육생성조절인자 (myogenic regulatory factor) 인 MyoD, Myf-5, Myf-6, myogenin 와근막단백질인 desmin 에대한면역형광염색을다음과같이시행하였다. 배양된세포는슬라이드에서 1:1 Acetone/Methanol 로고정후 tris-buffered saline (TBS; Sigma) 으로세척하였다. 이후 30 분간 2% bovine-serum albumin (Sigma) 와 5% goat serum (Sigma) 이첨가된용액으로고정시켰다. 이후 MyoD, myogenin, Myf-5, Myf-6 및 desmin 에대한일차항체 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 를사용하여실온에서 1 시간동안반응시킨다음 biotin 이부착된 Anti-mouse IgG 를이용하여 30 분간실온에서처치하고다시 TBS 로세척하였다. 이후 Avidin-FITC 로 30 분간반응시켜발색을유도하여형광현미경으로관찰하였다. (2) 웨스턴블로팅 (Western Blotting) 배양초기에발현되는 MyoD 와 Myf-5 의상태를검 사하기위하여대조군과근육생성배양액군에서만 western blot 을통해 1 주, 2 주, 3 주배양상태에아래와같은방법으로시행하였다. 배양한근세포를 tris base 125 mm ph 6.8 (Sigma aldrich Co, St. Louis, MO, USA), sodium dodecyl sulfate 4% (SDS; Amresco Co, Solon, OH, USA), glycerol 10% (Amresco Co.), mercaptoethanol 5% (Sigma aldrich Co.), dithiothreitol 100 mm (DTT; Amresco Co.), phenylmethylsulfonyl fluoride 5 mm (PMSF; Amresco Co.) 가함유된버퍼에넣고용해시킨후원심분리하여상층액을얻고이를같은양으로만들었다. 얻어진동량의단백질을 bromophenol blue (Sigma aldrich Co.) 를첨가한후 Biorad miniprotean II cell (Bio-rad, Hercules, CA, USA) 을이용하여 acryl-amide gel (Bio-rad, Hercules, CA, USA) 에전기영동하고 polyvinylidene fluoride (PVDF; Millipore, Bedford, MA, USA) membranes 으로이동시켰다. 이동된 membranes 을 5% skim milk (Becton Dickson, Sparks, MD, USA), 0.05% tween 20 (Amresco Co.)-tris 버퍼용액에실온에서 1 시간고정시킨후 MyoD 와 Myf-5 에대한일차항체 (Santa cruz Biotechnology) 와실온에서 3 시간동안반응시키고세척하였다. 세척후 peroxidase conjugated anti mouse IgG (Amersham, Arlington Fig. 1. Differentiation of adipose-tissue-derived cells into myogenic cells. MyoD was expressed in all different media groups from 3 weeks ( 100). CM: control media, MM: myogenic media, CdM: conditioned media 71
박윤길 문재호 이은영 도병록 김지향 김균태 김동수 Heights, IL, USA) 와실온에서 1 시간반응시키고다시세척하였으며이후발색반응은 enhanced chemiluminescence (ECL; Amersham) 를이용하여필름에감광시키고비교하였다. 역형광염색에서 3 주와 6 주에서양성반응을보였다 (Fig. 1). 웨스턴블로팅에서도대조군과분화군모두에서배양 2 주부터양성반응을보였는데제 2 군에서시간이지날수록더뚜렷하게나타났다 (Fig. 3). 결 과 2. Myf-5 1. MyoD 대조군을포함하여세군모두에서 MyoD 에대한면 면역화학염색에서모든군에서배양 1 주부터양성반응을보이기시작하여 3 주와 6 주에서도강한양성반응을보였다 (Fig. 2). 웨스턴블로팅에서도제 1 군과제 2 Fig. 2. Differentiation of adipose-tissue-derived cells into myogenic cells. Myf-5 was expressed in all different media groups from 1 week ( 400) to 6 weeks ( 100). CM: control media, MM: myogenic media, CdM: conditioned media Fig. 3. Myogenic differentiation of adipose-tissue-derived cells as determined by western blot for expression of MyoD and Myf-5 from 1 week to 3 weeks. Lipoaspirate cells were cultured in control (CM) and myogenic medium (MM). 72
인체지방유래세포의근육세포분화가능성 군모두에서배양 1 주부터양성반응을보였으며시간이지날수록더뚜렷하게나타났다 (Fig. 3). 3. Myf-6, Myogenin 및 Desmin 세군모두에서전배양기간을통해서면역화학염색에반응을나타내지않았다. 고 찰 최근두시엔느형근디스트로피를포함한근육병환자의치료를위해유전자치료와줄기세포 (stem cell) 이식및조직배양을이용한치료등이활발히연구되고있다. 이중지방세포에서유래된줄기세포를이용해서근육생성줄기세포를분리배양하는방법이개발되고있는데아직은한정된기관에서만진행이되고있는상태이다. 포유류의지방조직은지방세포와콜라겐섬유질이혼재되어있으며간질혈관부분 (stromal vascular fraction) 도포함되어있다. 여러조직으로분화가가능한것으로알려진지방전구세포와서로다른세포군과섞여있는지방조직은골수와마찬가지로배양조건에따라다양한조직으로분화할수있는것으로밝혀지고있다. 14,18,19 최근 Rocco 등 17 은근육모세포와지방유래세포를같이배양 (co-culture) 하였을때지방세포가배양조건에관계없이자연적으로근육세포로분화되는것을보고한바있는데본연구에서도세가지배양액의조건에영향을받지않고시간이지나면서일차근육생성조절인자의발현반응이양성으로나타났다. 이렇게지방세포가근육세포로전환되거나분화되는이유는척추동물의발생과정에서골격근세포, 골세포, 연골세포및지방세포가같은중배엽성기원을하며각조직으로분화할때조절과정과인자가공유되는점이있기때문이다. 20 또한 Asakura 등 21 과 Csete 등 22 은근육모세포가골세포, 지방세포로분화할수있다는연구결과를발표하였는데이러한연구결과로보면지방세포와골격근세포간에는발생단계부터많은유사성이공존하며분화과정에서여러인자가공통으로작용하는것으로추정할수있다. 그러나현재이러한조절인자나분화과정을제어할수있는조건이밝혀진것이없으며향후연구과제이기도하다. 본연구에서는배양액의조건에관계없이지방세포가근육세포로전환되는것이확인이되었으며배양초기부터근육생성에관계되는인자들이나타나기시작하였다. 그러나근육모세포로분화는단계중일차근육생성조절인자인 MyoD와 Myf-5는양성반응을보였으나이차근육생성조절인자인 Myf-6나 myogenin 등은배 양시간이지나도계속음성반응을보여근육모세포에서근관세포로분화되는단계를이어가지못하는현상이나타났다. 이는 Rocco 등 17 이주장한중배엽성줄기세포가골격근으로분화하기위해서는근육세포와직접접촉이필요하다는것과유사한결과이다. 본연구의제한점으로는배양에이용된지방조직에어떤세포들이혼재되어있는지확인이되지않은상태에서분화를시도한것이므로일차배양전에세포의성분분석을시행하고이후근육세포의표식인자를이용한세포분리를통해서배양실험을한다면좀더정확한정보를얻을것으로생각된다. 또한전체세포중근육모세포로분화된비율이어느정도인지파악을한다면분화배양방법의효율을평가할수있을것으로사료된다. 따라서앞으로지방세포가근육모세포로분화되고이후근관세포를거쳐근육조직까지분화되는데필요한최적의배양조건을밝혀서향후근육세포치료의임상실험에기초자료로사용되도록지속적인연구가필요할것으로생각된다. 결 론 지방조직에서유래된세포들을이용하여근육세포로분화를유도한결과일부근육생성조절인자가나타나근육세포로분화되는것을확인할수있었으나이후이차근육생성조절인자나근막단백질의발현은확인할수없었다. 따라서향후근관세포까지분화를진행시키는연구와더불어분화된조직에대한근육기능에대한평가가필요한상태이다. 참고문헌 01. Hoffman EP, Brown J, Kunkel LM: Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987: 51: 919-928. 02. Monaco AP, Neve RL, Colletti-Feener C, Bertlson CJ, Kurnit DM, Kunkel LM: Isolation of candidate cdnas for portion of the Duchenne muscular dystrophy gene. Nature 1986: 323: 646-650. 03. Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes AH, Belfall B, Klamut HJ, et al.: The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 1988: 333: 466-469. 04. Kilmer DD. Myopathy. In: DeLisa JA, Gans BM, eds. Rehabilitation medicine: principles and practice. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2005, pp 73
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