J Korean Surg Soc 2010;79:S7-15 DOI: 10.4174/jkss.2010.79.Suppl1.S7 원 저 분화유도된근육줄기세포의내막증식억제효과 1 서울대학교의과대학외과학교실, 2 서울대학교보라매병원외과, 3 분당서울대학교병원외과, 4 서울대학교병원외과, 5 삼성서울병원외과 정인목 1,2 ㆍ한소리 1,3 ㆍ최금희 1,3 ㆍ권유진 1,3 ㆍ이태승 1,3 민승기 1,4 ㆍ박양진 5 ㆍ정중기 1,2 ㆍ하종원 1,4 ㆍ김상준 1,4 Differentiated Muscle-derived Stem Cells Attenuate Intimal Hyperplasia after Carotid Balloon Injury in Rat In Mok Jung, M.D. 1,2, So Rhee Han 1,3, Keum Hee Choi 1,3, Yujin Kwon, M.D. 1,3, Taeseung Lee, M.D. 1,3, Seung-Kee Min, M.D. 1,4, Yang Jin Park, M.D. 5, Jung Kee Chung, M.D. 1,2, Jongwon Ha, M.D. 1,4, Sang Joon Kim, M.D. 1,4 Department of Surgery, 1 Seoul National University College of Medicine, 2 Seoul National University Boramae Hospital, Seoul, 3 Seoul National University Bundang Hospital, Seongnam, 4 Seoul National University Hospital, 5 Samsung Medical Center, Seoul, Korea Purpose: Although progenitor cells may contribute to intimal hyperplasia (IH) after arterial injury, positive contribution of IH is variable with type of injury or cells. This study was designed to examine whether differentiated muscle derived stem cells (MDSC) attenuate IH in rat. Methods: MDSCs were retrieved using preplate techniques from rat calf muscle and MDSCs (preplate 6th culture fraction, pp6) were exposed to VEGF (50 ng/ml) for endothelial differentiation prior to injection. Male rats were divided into two groups (cell treated vs. control) and underwent carotid balloon injury with 2-Fr catheter. The virus containing Green fluorescent protein (GFP) gene was transfected into cells for monitoring. Cells (5 10 6 ) were indwelled into carotid artery for 30 minutes after injury and then blood flow was restored. Arteries were harvested at various intervals (1, 2 and 4 weeks) after injury. The intima to media thickness ratio (IMTR) was calculated with morphometric analysis. Results: Endothelial surface markers such as VE-CADHERIN were strongly expressed on differentiated MDSCs. At 4 weeks after injury, IH was predominantly observed in control group compared to cell treated group. The intensity of GFP was strongly observed at 1 week and declined at 4 weeks in carotid artery wall at MDSC group. CD31(+) endothelial cells were observed at MDSC group compared to control. The mean IMTR in cell treated groups were significantly lower than control at 2 weeks (P=0.005) and 4 weeks (P 0.001). Conclusion: Our study demonstrates that MDSCs therapy promotes re-endothelialization and leads to attenuation of IH after balloon injury in rat. (J Korean Surg Soc 2010;79:S7-15) Key Words: Intimal hyperplasia, Stem cell therapy, Muscle derived stem cell 중심단어 : 내막증식증, 줄기세포치료, 근육유래줄기세포 책임저자 : 이태승, 경기도성남시분당구구미동 300 463-707, 분당서울대학교병원외과 Tel: 031-787-7092, Fax: 031-787-4055 E-mail: tslee@snubh.org 접수일 :2010 년 4 월 29 일, 게재승인일 :2010 년 7 월 28 일이논문은 2006 년정부의재원으로한국학술진흥재단의지원을받아수행된연구임 (KFR-2006-331-E00155). 서론 동맥폐색질환환자들에서시행되는많은동맥우회술혹은혈관내치료들이초기의우수한성공률에도불구하 S7
S8 J Korean Surg Soc. Vol. 79, Suppl. 1 고장기간추적관찰시재협착혹은폐색을흔히보인다. 주요한폐색원인으로알려진내막증식증내막손상후혈관평활근세포의내막이동및증식과결체조직의침착으로혈관내강의협착이초래되어발생하며상기치료들의실패요인으로이해되었다.(1) 그러나최근에혈관전구세포의발견및관련된많은연구를통해서내막증식증을초래하는병태생리개념이새롭게대두되었다. 즉혈중, 조직, 혹은골수내에존재하는전구세포들이혈관손상부위로원격이동하여내막증식을초래하는주세포원이라는주장이제기되고있다. 그러나전구세포들에의한내막증식연구는손상모델에따라서혹은전구세포들의종류에따라서내막증식의억제혹은촉진등의다양한결과를나타내고있다.(2) 저자들은근육내존재하는줄기세포를내피세포로의유도후백서경동맥손상부위에투여하여내막증식억제여부를분석하고그기전을규명하고자본연구를시행하였다. 방법 1) 근육줄기세포분리, 표식자주입및내피세포분화유도 Sprawley-Drew (SD) male rat (250 g) 가자미근에서채취한조직을잘게썰어 triple enzyme을처리하여근육세포를분리한후세포들이플라스틱에붙는접촉정도의차이를이용하여일정시간간격으로콜라겐을코팅한플라스크를사용하는전플라스크방법 (pre-plate technique) 을사용하였다. 근육내존재하는세포군들이 collagen 부착플라스크에붙는친화성차이를이용하여 1시간배양후부유세포들을새로운플라스크에계속옮겨가면서 6번째플라스크에서최종배양된세포들 (pp6) 을이용한다. 이세포들을 1주일간분리, 배양후 MiniMACS R (Miltenyi Biotec Inc., Auburn, CA, USA) 를이용하여 CD34(+) 인세포를추출하여면역염색을통해근육줄기세포의특성을확인하였다. 분리된근육줄기세포는 green fluorescent protein (GFP) 을발현하는 lentivirus (Microgen, Seoul, Korea) 를감염시킨후세포의이동및생존여부를형광현미경을이용하여확인하였다. 내피세포로의분화유도를위해서 VEGF 50 ng/ml (Sigma, St. Louis, Mo, USA) 배양한후내피세포특이표식자인 VE-cadherin primer를이용하여중합효소연쇄반응후과발현여부를측정하였다. 2) 실험동물및경동맥손상체중 550 600 g Sprague-Dawley rat을대상으로세포투여군 (n=15) 과식염수투여군 (n=15) 으로나누었고수술 1주, 2주, 4주후각각의경동맥조직을회수하였다. 백서를 Forane을사용한흡입마취하에우측경동맥을노출시킨후외경동맥을통하여 2 Fr. Fogarty catheter (Edwards Lifescience, Irvine, CA, USA) 를삽입하여 3 cm 정도의총경동맥부위를 3회에걸쳐풍선내막손상을가하였다. 손상부위를생리식염수로세척후위에준비한 MDSCs (5 10 6, 300 ul suspension with normal saline) 를동맥내강으로주입하여 30분간배양시킨후혈류를복원하였다. 3) 조직검사및형태학적계측 (morphometric analysis) 마취후경동맥을박리하고심장을통해 3% 완충 paraformaldehyde 25 ml를주입하여생체내고정하였다. 약 45분후에경동맥조직을채취하여 4 o C 고정액에 5시간동안더고정하였다. 조직은파라핀포매후위치에따라 2등분하여 5μm 두께로잘라조직검사를실시하였다. Hematoxylin- Eosin (H&E) 염색해서 Leica application suite (Leica Microsystems, Heerbrugg, Switzerland) 를이용하여내막-중막비를측정하였다. 내막-중막비는한조직절편에서 100배현미경시야의 12시방향을기준으로시계반대방향으로돌아가며 60 o 간격으로측정한후평균한값을사용하였다. 4) 세포및조직염색세포에대한면역형광염색은일차항체인 SCA-1 (1:100 Abcam, Cambridge, United Kingdom), CD34 (1:100 Abcam), CD45 (1:100 Abcam), Desmin (1:100 Abcam) 을이차항체는 rodamin이붙은 goat antibody (1:100, Abcam) 를이용하였다. 회수된조직을 4μm 박절표본을만든뒤면역조직화학염색을위해 silane 코팅에부착된박절표본을 37 o C 세포배양기에하룻밤방치한후 10분간 3번 histoclear 용액에담가파라핀을제거하고 100%, 95%, 90% 알코올과증류수에단계적으로함수하였다. 그후포르말린고정으로조직내감추어진항원을노출시키기위하여 ph 9.0 Tris/EDTA 완충액에담가 decloaking chamber에서 125 o C에서 3분간가열하였다. 실온에서 20분가량식힌뒤흐르는물에수세하였다. 조직내에존재하는내인성페록시다아제를차단하기위해메탄올과과산화수소가 7:3 (3% H 2O 2) 의비율로섞인용액에서 10분간처리하고흐르는물에수세후 TBS
In Mok Jung, et al:differentiated MDSCs Attenuate IH S9 Fig. 1. Muscle-derived stem cells (pp6) using preplate technique from rat calf muscle show positive staining with anti-sca-1 antibody (A), anti-cd34 (B), indeterminate staining with anti-desmin (C) and negative staining with anti-cd45 (D) with immunohistochemical staining ( 400). Tween-20 (Tris- buffered saline and tween 20: ph 7.6) 에수세하였다. 일차항체로 CD31 (1:100) 을실온에서 1시간반응시켰다. TBS Tween-20으로 5분간 3회수세후이차항체로 anti-mouse peroxidase (DAKO K4001, Produktionsvej, Denmark) 를가하여 30분간반응시킨후 TBS Tween 20으로수세하고 DAB (3,3 -diaminobenzidine tetrahydrochloride) 로 3 10분간실온에서발색시키고 Mayer s hematoxylin으로대조염색을실시하여광학현미경에서관찰하였다. 음성대조군으로는일차항체대신에 TBS를사용하여동일한방법으로면역조직화학염색을시행하였다. 5) 통계처리 모든값은평균값 ± 표준편차로표시하였다. 두군간평균값의비교는 Mann-Whitney U test를사용하였다. 통계적계산은 SPSS for Windows version 15.0 (SPSS Inc., Chicago, IL, USA) 프로그램을사용하였으며, P값이 0.05 미만인경우통계적으로유의하다고판단하였다. Fig. 2. MDSCs (pp6) are exposed to VEGF for endothelial differentiation. VE-cadherin is up-regulated in dose dependent manner. Values are the mean±sd of three separated experiments.
S10 J Korean Surg Soc. Vol. 79, Suppl. 1 Fig. 3. Endothelial denudation and intimal hyperplasia are observed in difference following rat carotid balloon injury; Intimal denudation is confirmed in immediately harvested tissue in H&E staining ( 100)(A), and in anti-cd31 imunohistochemical staining ( 400)(B). Endothelial cell coverage rate in intima is less than 10% in control (C) and 50% in cell-treated group at 1week (D), 30 40% in control (E) and more than 80% in cell-treated group (F) at 2 weeks, 50 70% in control (G) and 90 100% in cell-treated group (H) at 4 weeks after carotid balloon injury. Arrows indicate CD31(+) endothelial cells.
In Mok Jung, et al:differentiated MDSCs Attenuate IH S11 결과 1) 근육줄기세포의특성및내피세포분화 전플라스크방법으로분리배양된최종세포 (pp6) 들을대상으로면역세포화학염색상에서줄기세포표지자 sca-1 (+), 내피전구세포표지자 CD34 (+), hematogenous marker CD45 ( ), 근육성표지자 Desmin (±) 를보였다 (Fig. 1). 내피세포의분화유도를위한 VEGF투여시내피세포의특이표지자인 VE-cadherin 발현이농도에따라증가하였다 (Fig. 2). Fig. 4. Contribution of MDSC to intimal hyperplasia. MDSCs infected with lenti-hcmv- GFP were delivered into carotid artery lumen for 30 minutes after carotid balloon injury. MDSCs with GFP (green fluorescent protein) are strongly expressed at 1 week (B) and reduced at 4 weeks in carotid artery (D) compared to controls (A, B). Arrows indicate GFP(+) endothelial cells. MDSCs with CD31 are weakly expressed at 1 week (F) and strongly expressed at 4 weeks in the intima of carotid artery (H) compared to controls (E, G). All sections were viewed at 100.
S12 J Korean Surg Soc. Vol. 79, Suppl. 1 2) 경동맥내피세포의재생 경동맥내막손상후바로채취된조직을대상으로 Hematoxylin-Eosin염색및 CD31 면역조직화학염색에서내피세포의탈락을관찰하였다 (Fig. 3A, B). 손상후 1주에서시행된면역조직화학검사에서대조군에서는내피세포가드물게관찰되었으나세포치료군에서내막내내피세포부착율이 50% 로관찰되었다 (Fig. 3C, D). 2주후대조군에서는 30 40% 로관찰되었으며세포치료군에서는 80% 이상으로관찰되었다 (Fig. 3E, F). 4주에대조군에서는 50 70% 정도인반면치료군에서는 90 100% 로관찰되었다 (Fig. 3G, H). 3) Lenti-hCMV-GFP 바이러스에감염된근육유래줄기세포 Lenti-hCMV-GFP 바이러스에감염된근육유래줄기세포를동맥손상후내피세포탈락부위에 30분간배양시켰다. 1주군과 4주대조군에서는 GFP 발현줄기세포의형광발현을전혀관찰할수없었으나 (Fig. 4A, C), 세포치료군에서는경동맥내막에서발현이강하게관찰되었고 (Fig. 4B), 4주군에서는 GFP발현정도가감소되었다 (Fig. 4D). 내피세포특이항원인 CD31 염색시세포치료군에서는 1주에신생내막에서약하게발현되고있었으나 (Fig. 4F), 4주에서는 CD31의강한발현이관찰되었다 (Fig. 4H). 반면에, 대조군에서는 CD31(+) 내피세포의발현이 1주군에서는거의없 Fig. 5. The intima to media thickness ratio (IMTR) is calculated with morphometric analysis. The mean IMTR in cell treated groups are significantly lower than control at 2 weeks (P=0.005) and 4 weeks (P 0.001). Black bar indicates control and white bar indicates cell treated group. Values are the mean±sd of five separated experiments. 었고 (Fig. 4E), 4주군에서는약하게관찰되었다 (Fig. 4G). 4) 광학현미경을이용한형태학적계측실험군의모든표본에서내측탄력소층 (internal elastic lamina) 이잘유지되고있어동맥손상깊이가일정함을보여주었다. 1주대조군및치료군에서는내막증식의정도가약하게관찰되었으며내막과중막의두께비에있어서두군간에차이는없었다 (0.59±0.16 vs. 0.56±0.19, P=0.65)(Fig. 5). 2주대조군과비교해서세포치료군에서는의미있게내막증식의억제를관찰할수있었으며 (1.66±1.0 vs. 0.89±0.52, P=0.005)(Fig. 5), 4주군에서도뚜렷한내막증식억제효과를관찰할수있었다 (2.28±0.35 vs. 1.18±0.35)(Fig. 6). 고찰 내막증식증은동맥폐색성질환환자를대상으로시행되는혈관내치료 ( 풍선확장술, 스텐트삽입술 ) 및외과적치료 ( 내막절제술, 동맥우회술 ) 시동반되는혈관벽의기계적자극이나혈역학적변화 ( 허혈및재관류, 탄성부조화 ) 등에의해발생되는손상에대한혈관조직의창상치유과정으로이해할수있다. 따라서혈관벽에받는손상정도에따라내막증식기전차이가예상되며 5/0 nylon loop를이용하여경동맥중막층의손상없이내피세포의손상을가한군과본실험과같이 2 Fr. Fogarty catheter를이용한풍선확장술및내피세포탈락을동반한군으로비교하였을때내피손상군에서는평활근세포의증식이미미하였으나풍선군에서는심한평활근증식반응을보고하였다. 또한양군에서내피세포손상에따른혈소판부착및활성화인자인 platelet-derived growth factor (PDGF) 및 PDGF receptor mrna 발현정도는차이가없이양군에서높이관찰되었다.(3) 이는내막증식증이초래되는기전은단순한내피세포의손상외에도중막층의손상정도가관여함을암시한다. 반면손상된내막의내피세포가조기에복구되면중막층손상으로초래되는평활근세포의증식반응은급격히완화되는것으로보고하였다.(1) Asahara 등 (4) 은시간에따른전자현미경추적관찰을이용한혈관손상실험에서혈관손상표면에많이붙어있는세포들중 CD34(+) 세포가간헐적으로붙어있는것을관찰하고이러한세포는손상변연부주위의내피세포로부터자라오는것이아니라골수에서나와서말초혈액내로순환하는내피전구세포에서유래되었을가능성을제시하였
In Mok Jung, et al:differentiated MDSCs Attenuate IH S13 Fig. 6. Intimal hyperplasia is inhibited in cell-treated group compared to control group; Intimal hyperplasia is minimally observed in both celltreated (A) and control group (B) at 1 week. Suppression of intimal hyperplasia is more prominent in cell-treated group (D) than control group (C) at 2 weeks and 4 weeks (E, F). All sections were viewed at 100. 다. 이후로골수유래전구세포가동맥손상후내막증식증유발에직, 간접으로기여한다는연구가보고되고있다.(5,6) 또한간엽줄기세포가혈관손상부위로이동하여유사내피세포혹은혈관평활근세포로분화되는것이보고되고있다.(7) 내피세포는혈관활성물질인 prostacyclin (PGI2) 과 nitric oxide (NO) 를분비하여혈소판의활성, 유착및응집을억제하는기능이있다. 혈관손상부위에활성화되는혈소판및염증세포등이분비하는 granulocyte-colony stimulating factor (G-CSF), stromal derived-1, VEGF, PDGF 등과같은다양한사이토카인등이강력한 mitogen으로작용하여전구세포나평활근세포의활성을초래하게한다. 따라서손상된내막을조기복구시키기위한여러연구가진행되어특정유전자를넣은내피전구세포, 혹은내피전구세포를골수에서혈중으로유도하는 G-CSF 혹은 statin을투여하여조기 내막을복구하여내막증식을억제하는결과가보고되고있다.(8-10) 근육유래줄기세포 (muscle-derived stem cell, MDSC) 의분리는 Qu-Petersen 등 (11) 이백서의근육내존재하는다양한세포군들에서 preplate technique을이용하여 sca-1(+), CD34(+), CD4( ) 표면항원을보이는특정세포군을분리하였고이세포군이 200번의계대배양후에도같은세포형태및증식능력을보유하며심근, 신경, 내피세포, 골조직등다양한세포조직으로체내및체외에서분화됨을보고하였다.(12) MDSC의특징은근육성이지만내피세포의특성을갖고있어간엽계세포로서 hematopoietic cell maker인 CD34(+) 를발현한다. Okada 등 (13) 은인체의근육에서 myogenic endothelial cell을분리하여심근경색부위로주사하여심근기능향상및혈관신생을보고하였다. 그러나저자들의선행연구 (unpublished data) 에서는 naïve MDSC를주
S14 J Korean Surg Soc. Vol. 79, Suppl. 1 입하였을때쥐의뒷다리허혈모델에서혈관신생정도는뚜렷하게관찰되지않았다. 본실험에서는 naïve MDSC에 VEGF를투여했을때 vwf및 VE-cadherin이과발현된유사내피세포로의분화를유도하였고이를사용하여증강된체외혈관신생능을입증하였다 ( 결과는기술안함 ). VGEF투여에의한다른간엽줄기세포의혈관내피세로의분화유도는이미보고된바가있다.(14,15) 내피세포에특이적인 CD31염색에서대조군에비해세포주입군에서강하게염색됨이관찰되는것은먼저부착된 MDSC의 paracrine effect 로내피세포의이동이나생착을촉진하는것으로추정할수있다. 한편술후 4주군에서는 MDSC의발현이매우약하게관찰되어초기부착되었던 MDSC의탈락이나사멸이추정되었고, 내막증식부위에 CD 31(+) 신생내피세포로대체되었다. 형태학적계측에서도대조군과비교해서 MDSC 투여군이 2주, 4주군에서통계적으로의미있게내막증식억제효과를보여서조기내막재생정도와밀접한연관을추정하게한다. 동물시험이아닌임상에서전구세포를뽑아서내막억제여부를수행한선행연구가있는데, 이들은 G-CSF를투여하여골수로부터말초혈액으로전구세포를동원한후세포를뽑아서관상동맥스텐트치료후동맥내로주입하였다. 이 MAGIC cell trial에서세포투여후심근경색이되었던심근기능이심장초음파에서기능적향상은발견하였으나추적검사상스텐트재협착률이높음을보고하였다.(16) 이들의연구와본연구와의차이는세포를직접병변부위에투여한치료전략은유사하지만본실험에서이용된 MDSC는정제된단일줄기세포군으로유사내피세포로분화시켜이용한장점이있다고생각되며, 내피세포가아닌평활근세포로분화된다면오히려내막증식을악화시킬수있는가능성이추정된다고하겠다. 본연구의단점은어떤기전에의해서투여된 MDSC가신생내피세포를유도하는지에의문과 MDSC의시간적, 기계학적 (mechanistic) 조망이부족하다는점이다. 다만투여된 MDSC가 4주까지도신장및간조직에발견되는것으로보아세포외기질이풍부한조직에서는상당기간생존하는것으로추정되었다 ( 결과는기술안함 ). 그동안내막증식을억제하기위한많은약물, 방사선, 유전자, 약물용출스텐트치료등 (17) 이대부분실패로돌아간현시점에전구세포를활용한치료전략에대한가능성을확인하였으며향후과감한연구가필요한시점이라고사료된다. 결 본연구는실험적내막손상후유사내피세포로의분화된근육유래줄기세포를이용하여내막손상부위에세포를주입하여내막증식을의미있게억제하였다. 중, 대동물을대상으로내막증식억제에대한추가연구가필요하리라생각된다. 론 REFERENCES 1) Clowes AW, Clowes MM, Fingerle J, Reidy MA. Regulation of smooth muscle cell growth in injured artery. J Cardiovasc Pharmacol 1989;14(Suppl 6):S12-5. 2) Tsai S, Butler J, Rafii S, Liu B, Kent KC. The role of progenitor cells in the development of intimal hyperplasia. J Vasc Surg 2009;49:502-10. 3) Fingerle J, Au YP, Clowes AW, Reidy MA. Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury. Arteriosclerosis 1990;10:1082-7. 4) Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964-7. 5) Sata M, Saiura A, Kunisato A, Tojo A, Okada S, Tokuhisa T, et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403-9. 6) Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 2003;93:783-90. 7) Wang CH, Cherng WJ, Yang NI, Kuo LT, Hsu CM, Yeh HI, et al. Late-outgrowth endothelial cells attenuate intimal hyperplasia contributed by mesenchymal stem cells after vascular injury. Arterioscler Thromb Vasc Biol 2008;28:54-60. 8) Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 2002;105:3017-24. 9) Kong D, Melo LG, Mangi AA, Zhang L, Lopez-Ilasaca M, Perrella MA, et al. Enhanced inhibition of neointimal hyperplasia by genetically engineered endothelial progenitor cells. Circulation 2004;109:1769-75. 10) Griese DP, Ehsan A, Melo LG, Kong D, Zhang L, Mann MJ, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation 2003;108:2710-5. 11) Qu-Petersen Z, Deasy B, Jankowski R, Ikezawa M, Cummins
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