대한화상학회지제 12 권제 1 호 38 Journal of Korean Burn Society Vol. 12, No. 1, 38-42, 2009 CD34 인체제대혈유래단핵구세포가피부창상치유촉진에미치는영향 박상은ㆍ유대현ㆍ탁관철ㆍ나동균 연세대학교의과대학성형외과학교실, 인체조직복원연구소 Acceleration of Wound Regeneration by Implantation of CD34 Mononuclear Cell from Human Cord Blood Sang Eun Park, M.D., Dae Hyun Lew, M.D., Kwan Chul Tark, M.D. and Dong Kyun Rah, M.D. Department of Plastic and Reconstructive Surgery, Yonsei University College of Medicine, Institute for Human Tissue Restoration, Seoul, Korea 책임저자 : 유대현, 서울시서대문구신촌동 134 120-752, 연세대학교의과대학성형외과학교실 Tel: 02-2228-2217, Fax: 02-393-6947 E-mail: dhlew@yuhs.ac Purpose: Cord blood stem cells are easily obtainable and have characteristics of higher proliferative potential and lower immunogenicity and with benefits in allogenic transplantation for wound treatment. To provide treatment evidence of cord blood stem cell on burn injury, CD34+ (hematopoietic lineage) and CD34 cells (mesenchymal stem cells lineage) were isolated and applied to full thickness skin wounds for evaluation of their effect. Methods: Mononuclear cell layer was separated by Ficoll- Paque from human cord blood, followed by selection of CD34 +, cells. Two full-thickness wounds were made on dorsal area of ICR mice with 8 mm punch. CD 34+, cell suspension was injected into the wound sites and wound healing rate was measured every 3 days with analysis of neovascularization. Immunohistochemical study for stem cell survival and TGF-β1 was performed. Results: The wound healing rate of CD34, CD34+ and control group was 34.73%, 9.24%, 5.132% on day 3 and 82.60%, 64.45% 76.20% on day 9 respectively. The CD34 group showed a significantly higher wound healing rate (p <0.01). The CD34 group revealed a relatively well organized epidermis and showed a remarkably increased neo-vascularization. Anti-human nuclei was detected on CD 34 group. TGF-β1 was stained all groups with no differences. Conclusion: CD34 mononuclear cells group showed increased wound healing than the control and CD34+ mononuclear cells treated group. CD34 mononuclear cells enhanced epithelization and neo-vascularization by means of differentiation into endothelial cells, myofibroblast and so on. We can conclude that isolated CD34 mononuclear cells can be helpful for burn wound. (Journal of Korean Burn Society 2009;12:38-42) Key Words: Cord blood stem cell transplantation, Wound healing, Leukocytes, Mononuclear 서 오늘날줄기세포의창상치유에대한많은연구들이진행중이다. 이중에서인체의제대혈에서유래한줄기세포는골수에서얻은줄기세포에비해미성숙하고높은증식력을가진다 1-4). 게다가제공자에게서위험없이풍부한양을얻을수있고 5), 면역반응이경미하므로이식에용이하다 6). 이러한제대혈유래줄기세포는재생의학의치료방법으로서많은장점을가지고있다. 인체제대혈은조혈모세포뿐만아니라조직재생의과정에서중요한역할을하는비조혈모세포로도분화할수있다는연구들이발표되고있다 7-9). 많은연구자들은줄기세포가창상치유를향상시킨다고보고하고있는데, Badiavas 등 10) 은상처를입은후 21일간의조직치유과정중에골수세포들이 perifollicular cells, blood vessel cells, perisebaceous gland cells로바뀔수있다고보고하였고몇몇의임상적연구에서는조직허혈, 혈관염등으로인한만성피부궤양을치료하는데골수의세포를투여함으로써좋은효과를얻을수있다는것을보여주었다 11,12). Valbonesi 등 13) 은기초적인수준의연구에서제대혈모세포가염증과숙주대조직반응없이피부조직의상처치유를향상시킨다는보고를하였고, Yamaguchi 등 14) 은투여한골수세포가상처조직에서근섬유아세포 (myofibroblasts) 로분화를일으켜조직의치유를증가시킨다고주장하였다. 우리는이번연구에서인체제대혈유래단핵구세포, 특히조혈모세포계열로알려진 CD34+ 단핵구세포가신생혈관생성을증가시킴으로써, 피부창상치유를향상시킬 론
박상은등 :CD34 인체제대혈유래단핵구세포가피부창상치유촉진에미치는영향 39 수있을것이라가정하여인체유래제대혈단핵구세포가창상의크기를줄이는정도와신생혈관생성정도를객관적으로살펴보았다. 특히창상에국소적으로투여된인체제대혈유래단핵구세포가창상의주변조직에서살아남아창상치유와신생혈관생성을촉진시킨다고알려진 TGF-β1 의분비량을증가시킬것이라가정하였다. 재료및방법 1. 제대혈로부터 CD34+/ CD34 세포의분리인체제대혈의수득은어머니의동의를받고연세의료원분만실에서출산직후이루어졌다. 태반이제거된후, 탯줄로부터무균, 24.5 ml의헤파린처리가된 PVC백으로제대혈을 250 cc 정도모은후 24시간내에모든실험과정을시행하였다. 제대혈을저밀도분획 (Ficoll-Paque plus; 1.077 g/l; Amersham Biosciences, UK) 을이용하여단핵구세포를분리하고세척하였다. 이후 CD34 전구세포분리킷 (CD 34 progenitor cell isolation kit, Miltenyi Biotech, Germany) 을사용하여단핵구세포를 CD 34+, 세포로분리하였다. 실험군으로약 4 10 7 제대혈유래 CD 34+ & 단핵구세포를각각 0.2 ml PBS (phosphate buffered saline) 용액에넣고, 대조군으로는 0.2 ml PBS 용액만을준비하였다. 2. 동물실험모델에서의창상형성모든동물실험은 Guide for the Care and Use of Laboratory Animals (National Institutes of Health [NIH] publication No. 86-23) 에따라시행되었으며연세대학교실험동물위원회의승인을받았다. 동물실험모델로는중앙실험동물 (Central Lab animal, Inc., Korea) 에서공급받고 AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) 인증된연세대학교산하실험동물부사육실에서키워진 ICR 쥐를사용하였다. 창상형성을위해복강마취 (ketamine 30 mg/kg, xylazine 3 mg/kg) 하에생후 4주된 10마리의 ICR 쥐의등쪽두부에서미부까지의털을제거한후, 직경 8 mm의피부조직검사용펀치 (Biopsy punch, Kai medical, Japan) 를이용, panniculus carnosus 근육층을포함한 2개의원형전층피부결손을만들었다. 생성된피부결손은 Tegaderm R (3M Health Care, St Paul, MN, USA) 를이용하여창상이마르지않고조직액이고이도록 24시간유지시켰다. 이후실험군으로 CD 34+와 34 세포액을국소적으로각각 5마리의 ICR 쥐의두피부결손중한쪽에투여하고, 대조군으로 0.2 ml PBS 용액만을 10마리의남은한쪽의피부결손에 투여하였다. 3. 창상의평가 1) 상피화정도를통한창상치유속도측정줄기세포투여후 3일간격으로 12일까지창상의상피화및수축정도를사진을통해측정하였다. 개방창의크기는 computer planimetry (Scion-image R ; NIH-Scion corporation, USA) 을이용하여면적을산출하였다. 2) 조직검사를통한혈행화정도측정실험 3, 6, 9, 12일째창상부위조직 (10 mm 인접부위 ) 을채취하여 H&E 염색을실시하고무작위로 10군데를선택하여고배율시야 (HPF 400, Olympus DP12- PC, Tokyo, Japan) 당존재하는신생혈관의개수를특정하였다. 3) 면역조직화학염색을통한창상내투여세포발현유무와 TGF-β1 발현정도실험 12일째창상조직을채취하여창상조직에서제대혈유래줄기세포의생존여부를확인하기위해 Anti human nuclei Ab (MAB1281, Chemicon International Inc., Massachusetts, USA) 를일차항원으로사용하였고,α-SMA (smooth muscle actin) 과수축력, 신생혈관생성을증가시킨다고알려진 TGF-β의분비를증가시키는지여부를확인하기 Anti mouse & human TGF-β1 Ab (Santa cruz, sc- 146, CA, USA) 를일차항원으로사용하였다. 4) 통계분석모든자료의통계처리는 Excel 2003 (The office, Microsoft, USA) 를이용하여수행하였으며, 창상치유율의분석을위해 Student s T-test를이용하여유효성여부를검증하였다. 모든값은평균 ± 표준편차 (mean±sd) 로표시하였으며, p값이 0.01 미만인경우를통계적으로유의한것으로평가하였다. 결과 1. 창상의크기변화 10마리의쥐를대상으로한실험에서실험시작후 12일까지제대혈유래 CD34 단핵구세포를투여한군에서 CD34+ 단핵구세포를투여한군이나대조군과비교하여통계적으로유의하게높은창상치유율을보였다 (Fig. 1, p-value=0.005<0.01). 특히, 실험후 3일째까지 CD34 단핵구투여군은 CD34+나대조군에비해높은창상치유증가속도를보였다. CD34+ 단핵구투여군과대조군간의유의한차이는없었다 (p-value=0.172>0.01).
40 대한화상학회지 Vol. 12, No. 1, 2009 였다. CD34 단핵구 투여군은 고배율 시야(HPF 400)당 2. 조직학적 변화 평균 8.48개의 신생 혈관 개수를 보였으나 CD34 단핵구 실험 3, 6, 9, 12일째의 조직으로부터 상피층의 형태학적 투여군과 대조군은 각각 2.78, 3.64개의 신생 혈관 개수만을 변화와 신생혈관 생성 정도를 조사하였다. 6일째 H/E 염색 보였다. 에서 CD34 단핵구 투여군은 CD34 단핵구 투여군이나 대조군에 비해 상대적으로 빠르고 잘 조직화된 상피층을 3. 면역조직화학염색 보였다(Fig. 2). 실험 6일째, 창상 부위의 조직에서 생존하고 있는 인체 실험 12일째 조직의 진피층에서 신생 혈관의 개수를 측 제대혈 유래 단핵구 세포를 확인하기 위해 Anti-human 정하였다(Fig. 3). 신생 혈관 개수는 H/E 조직염색의 고배 nuclei Ab에 대한 면역조직화학염색을 시행하였다(Fig. 4). 율 시야(HPF 400)상에서 무작위로 선정된 신생 진피층 부 CD34 단핵구 투여군에서는 염색 후 갈색의 DAB (dia- 위의 사진 10장에서 얻어진 혈관 개수의 평균으로 측정하 minobenzidine) 양성 반응을 보였으나 대조군에서는 양성 반응을 보이지 않았다(Fig. 4). Fig. 1. Wound healing rate (%) after transplantation of human CB-derived CD34 / mononuclear cells in mice. The CD34 CB-derived mononuclear cells group has higher wound healing rate than CD34 and control group. Especially, until 3 days CD34 mononuclear cell group revealed high acceleration. Fig. 3. 12 days after transplantation, the numbers of newly formed vessels per high-power field ( 400). Neovascularization was examined as mean value counted in 10 sectional areas acquired from wound lesion. Fig. 2. Epithelialization on day 6 after transplantation. CD34 mononuclear cell group (left) had relatively well organized epidermis faster than control group (right).
박상은등 :CD34 인체제대혈유래단핵구세포가피부창상치유촉진에미치는영향 41 Fig. 4. On day 6, the specimen of wound lesion analysed with immunohistochemistry. Anti-human nuclei Ab as a specific marker for human CB mononuclear cells was applied. Immunohistochemstry of CD34 mononuclear cells treated group (left, 40) showed brown colored DAB+ in the wound lesion. But, control group did t (right, 40). 인체제대혈유래단핵구세포를투여가 TGF-β1 분비량차이에미치는영향을알아보기위해 Anti mouse & human TGF-β1 Ab을이용한면역조직화학염색을시행하였다. 면역조직화학염색상, CD34 단핵구투여군과대조군모두갈색으로염색된 DAB 양성반응을보이며유의한차이를보이지않았다. 고찰이번연구에서저자들은인체제대혈유래단핵구세포중 CD34+ 단핵구세포가신생혈관생성을증가시킴으로써피부창상치유를향상시킬것이라는가정하에인체제대혈에서 CD34+ 단핵구와 CD34 단핵구세포를분리하였다. CD34+ 단핵구세포는적혈구등으로분화하는것으로알려진조혈모세포계열이다. CD34+ 단핵구세포가신생혈관생성을증가시키는혈관내피전구세포로분화를함으로써, CD34+ 단핵구투여군이 CD34 단핵구투여군이나대조군보다향상된창상치유율을보일것이라기대하였으나실제로는 CD34 단핵구투여군이 CD34+ 단핵구투여군이나대조군보다현저하게향상된창상치유율을보였다. CD34 단핵구투여군은다른군에비해더빨리상피화 (epithelialization) 가진행되었고신생혈관생성이증가되었으며, 또한투여한세포가창상부위에서생존하고있었다. Volk 등 15) 은골수유래단핵구세포가근섬유아세포활성을증가시키는 CTGF (connective tissue growth factor), TGF-β1 등의여러성장인자를분비하고혈관내피세포동원을향상시키며혈관의재형성을조절하는 VEGF나 CTGF (connective tissue growth factor) 등의여러 chemokine을생성할수있다고발표하였다. 게다가골수유래단핵구세포는각질세포 (keratinocyte) 의증식과이동에중요한여러성장인자 (Heparin-binding EGF-like growth factor, TGFα, EGF) 를생산할수있고골수유래단핵구세포에서분리한중간엽줄기세포는비슷한방식을통해골수유래단핵구세포보다더강한창상치유능력을보인다는것을밝혀냈다. 이러한연구결과를통해인체제대혈유래 CD34 단핵구세포는상피화와신생혈관생성을향상시킴으로써피부창상치유를촉진하고있는데이는인체제대혈유래 CD34 단핵구세포계열에속해있는중간엽줄기세포가이러한작용을하기때문이라유추할수있다. 이것은 2가지방법으로설명될수있는데, 첫번째로 CD34 줄기세포가창상치유를향상시키는상피세포, 내피세포전구세포, 근섬유아세포로직접분화하여작용한결과라생각할수있고, 두번째로 CD34 줄기세포에서분비되는 TGF-β1 등의성장인자나 cytokines 같은체액인자 (humoral factor) 가창상치유를촉진하는것으로생각할수있다. 전자의경우, 창상에투여된인체제대혈유래줄기세포가재상피화 (integrin α3), 혈관내피전구세포 (endoglin-cd105), 근섬유아세포 (α-smooth muscle actin) 에대한특이표지가증가된양상을보이는지여부를통해판단할수있다. 후자의논리는제대혈유래줄기세포가창상치유를증가시키는체액인자의분비량을증가시키는지여부를확인함으로써뒷받침될수있다. 본실험의경우에서실험군과대조군의 TGF-β1 의분비가차이가없는것으로나왔으나이비교결과는실험후 6일째시행되었기때문에초기 3일까지의창상치유에중요한인
42 대한화상학회지 Vol. 12, No. 1, 2009 자로인식되는 TGF-β1 등의체액인자가큰영향을미치지않는다고생각하기어렵다. 따라서추후 integrin α3, endoglin-cd105, α-smooth muscle actin, 초기 (3일) TGF-β1 발현량등의면밀한실험을통해서창상치유의기전을밝혀야할것으로사료된다. 인체제대혈유래단핵구세포는제대혈유래중간엽줄기세포에비해배양의과정을거치지않기때문에감염 ( 배양액에포함되어있는소태아혈청 (fetal bovine serum) 으로부터인수공통감염등 ) 의가능성이상대적으로적어안전하며빠른적용이가능하므로창상치료에도움이될것으로여겨진다. 결 CD34 단핵구세포는 CD34+ 군에비해높은창상치유율을나타내는것으로보아, 피부창상치유에조혈모세포계열로여겨지는 CD34+ 단핵구세포는큰영향을주지않고중간엽줄기세포를포함하고있는 CD34 단핵구세포가상피화와신생혈관생성을통해창상치유를향상시키는것으로추론할수있다. 론 REFERENCES 1) Szilvassy SJ, Meyerrose TE, Ragland PL, et al. Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood and fetal liver. Blood. 2001;98:2108-2115. 2) Vaziri H, Dragowska W, Allsopp RC, et al. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA. 1994; 91:9857-9860. 3) Migliaccio G, Migliaccio AR, Petti S, et al. Human embryonic hemopoiesis. Kinetics of progenitors and precursors underlying the yolk sac-liver transition. J Clin Invest. 1986;78:51-60. 4) Zanjani ED, Ascensao JL, Tavassoli M, et al. Liver derived fetal hematopoietic stem cells selectively and preferentially home to the fetal bone marrow. Blood. 1993;81:399-404. 5)Rubinstein P, Rosenfield RE, Adamson JW, et al. Stored placental blood for unrelated bone marrow reconstitution. Blood. 1993;81:1679-1690. 6) Rocha V, Wagner Jr JE, Sobocinski KA, et al. Graft-versushost disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. N Engl J Med. 2000;342(25):1846-1854. 7) Lee OK, Kuo TK, Chen WM, et al. Isolation of multi-potent mesenchymal stem cells from umbilical cord blood. Blood. 2004;103:1669-1675. 8) Mareschi K, Biasin E, Piacibello W, et al. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica. 2001;86(10):1099-1100. 9) Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol. 2000;109(1): 235-242. 10) Badiavas EV, Abedi M, Butmarc J, et al. Participation of bone marrow derived cells in cutaneous wound healing. J Cell Physiol. 2003;196:245-250. 11) Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrow-derived cells. Arch Dermatol. 2003;139:510-516. 12) Yamaguchi Y, Yoshida S, Sumikawa Y, et al. Rapid healing of intractable diabetic foot ulcers with exposed bones following a novel therapy of exposing bone marrow cells and then grafting epidermal sheets. Br J Dermatol. 2004;151:1019-1028. 13) Valbonesi M, Giannini G, Migliori F, et al. Cord blood stem cells for wound repair preliminary report of 2 cases. Transfus Apher Sci. 2004;30:153-156. 14) Yamaguchi Y, Kubo T, Murakami T, et al. Bone marrow cells differentiate into wound myofibroblasts and accelerate the healing of wounds with exposed bones when combined with an occlusive dressing. B J Dermato. 2005;152:616-622. 15) Volk SW, Antoneta R, Zhang L, et al. Stromal progenitor cell therapy corrects the wound healing defect in the ischemic rabbit ear model of chronic wound repair. Wound Rep Reg. 2007;15:736-747.