17-03김재윤

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성언 김
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1 대한응급의학회지제 24 권제 4 호 Volume 24, Number 4, August, 2013 원 저 피하층열상쥐모델에서 Polydeoxyribonucleotide (PDRN) 가봉합치료에미치는효과 순천향대학교부천병원응급의학과 김재윤 김호중 이종빈 성수동 조영순 Effectiveness of Polydeoxyribonucleotide (PDRN) Material on Murine Subcutaneous Laceration Wounds Jae-Yoon Kim, M.D., Ho-Jung Kim, M.D., Jong-Bin Lee, M.D., Soo-Dong Seong, M.D., Young-Soon Cho, M.D. group significantly increased compared to the Suture group (Suture: 11170±2475, Suture + PDRN: 27243±6621, p<0.05). Conclusion: The Suture+PDRN group can be used for the early treatment and histological healing of subcutaneous laceration wounds. Purpose: To evaluate the effectiveness of polydeoxyribonucleotide (PDRN) material on murine subcutaneous laceration wounds. Methods: Subcutaneous laceration wounds were made on the back of mice. The mice were divided into two groups according to method of PDRN applied: Group I (control, general dressing, and management) and Group II (PDRN injection). We evaluated gross findings and histological findings for the groups. Results: A total of 18 mice (9 in the Suture group and 9 with Suture+PDRN) were enrolled. In the mean results of gross finding (5-point Likert scale), the mean gross findings for wounds in the PDRN group were significantly higher than the suture groups on post-operative day 4 and 7 (for day 4, Suture: 2, Suture+PDRN: 3.2; for day 7, Suture: 2.7, Suture+PDRN: 4.2; p<0.05). In the histological analysis of wounds in the Suture+PDRN group after 10 days, reepithelization and granulation tissue formation were better than the Suture group. In terms of wound-healing grade, reepithelization and inflammation were not different; however, in the Suture+PDRN group, more granulation tissue formation was noted compared to the Suture group (p<0.05). In addition, the expression of VEGF in the Suture+PDRN 책임저자 : 김호중경기도부천시원미구중1동 1174 순천향대학교부천병원응급의학과 Tel: 032) , Fax: 032) lovelydr@schmc.ac.kr 접수일 : 2013년 7월 11일, 1차교정일 : 2013년 7월 20일게재승인일 : 2013년 7월 28일 453 Key Words: PDRN, Polydeoxyribonucleotide, Subcutaneous, Laceration, Wound Department of Emergency Medicine, Bucheon Hospital of Soonchunhyang University, Bucheon, Korea 서 피부열상에대한봉합술의방법은단순봉합술에서시작되어점차다양한방법들이동원되고있다. 이는환자들의피부상태에따른방법의전환이나추가일수있고상처의다양성에대한변화일수도있다. 그러나대부분소독과상처 dressing에대한기술과제품등에집중되어있고피부조직의재생에대한논의는거의없었다. 최근에국내에소개되고있는성장인자를이용하는 Polydeoxyribonucleotide (PDRN) 은 deoxyribonucleotide 중합체가포함된화합물로서 A2 purinergic receptor를자극하여특별한부작용없이화상이나만성창상등에효과적인것으로알려져있다 1). 본원에서는최근까지봉합술에대한많은연구와결과를발표하였는데특히 3년간본원응급실을내원한환자의 8% 에서단순봉합술을시행하였고외상에의해발생한피부열상치유를위해단순한드레싱이나봉합테입을통한봉합, 봉합사를통한봉합, 피부본드봉합술등이주로사용되었으며최근성장인자와같은다양한물질을이용한치료방법또한시도되고있다 2,3). 현재까지국내에서성장인자에대한소수의연구는있었지만응급실에서시행되는열상의봉합처치에대한연구는없었다. 따라서저자들은흰쥐의등에단순피하층열상을낸후봉합사를통한봉합을시행한후 PDRN을투여한군과투여하지않은군으로나누어 PDRN의열상치유효과를비교하고자하였다. 론

2 454 / 대한응급의학회지 : 제 24 권제 4 호 2013 대상과방법실험은순천향대학교부천병원동물실험윤리위원회의규정을준수하여시행하였다. 1. 실험재료 1) 실험동물생후 6주된 25~30 g ICR계 (CD-1 Mouse) 흰쥐 12 마리를생활조건 ( 습도온도빛등 ) 을같게하여사용하였고실험실에서마우스용사료와물로사육하여일주일간의적응기간을거친후실험을시작하였다. 쥐는 2그룹으로나누었다. 봉합사로봉합한군 9마리, 봉합사로봉합한쥐에 PDRN을투여한군 9마리로구분하였다. 2) 실험제재 a. PDRN (Placentex Integro, 5.625/3 ml, Mastelli, Garmaceutica, Italy) b. 4-0 nylon suture c. Zolazepam-tiletamine mixture (10 mg/kg, Zoletil Virbac, Carros, France) 2. 실험방법 1) 열상 ( 창상 ) 유도및처치 Zolazepam-tiletamine mixture (10 mg/kg, Zoletil Virbac, Carros, France) 마취제를이용하여 12마리쥐에게복강내주사하여마취를한후제모제를사용하여등에있는털을제거하고 10% povidone-iodine 및 70% ethanol로등을소독하고 11번 blade를이용하여피하층까지 2 cm vertical로상처를낸뒤 18마리는 4-0 nylon suture를이용하여 suture를하고그중실험군 9마리의피하층에 PDRN 3 ml를주입하였다. 2~3일간격으로 10% povidone-iodine 및 70% ethanol로 dressing을하였다. 실험을시행한쥐는각각 cage 하나에한마리씩넣고 10일간관찰하였으며관찰기간동안사료및물은무제한으로공급하였다. 2) 육안적검사열상유발후 4일째, 7일째, 10일째에열상의육안적소견을연구자 2인에의해관찰하였고사진촬영하였으며. 각각봉합사제거후벌어짐, 상처의균일성, 오염도 ( 붓기또는발적또는누액 ) 등을조사하였다. 모든육안적소견은실험이종료된이후실험과무관한응급의학과전공의 1인과전문의 1인이펼쳐놓은현장사진을통해전체적으로재평가하였다. 평가형식은 5점 Likert 척도 (1-가장 나쁨, 5-가장좋음 ) 로 2인의평균값으로정하였다. 3) 열상조직검사열상유발후 10일 쥐를희생시켜주변의열상조직과열상전체가포함되도록조직을채취하였다. 채취한조직을 10% 포르말린용액에 6시간이상고정한후헤마톡실린 (hematoxylin) 과에오신 (eosin) 염색과 VEGF 면역조직화학염색 (immunohistochemistry) 을시행하여광학현미경하에서관찰하여재상피화정도육아조직증식, 괴사및염증의정도및 VEGF 를측정하였다. VEGF 측정은 MetaMorph image analysis software (Universal Image Corp.) 를통하여분석하였다. 열상조직검사소견의정도에따라정량화하여비교하였다. 정량화는진피와표피의재생정도 (R), 육아조직증식정도 (G), 창상에나타나는괴사및염증정도 (I) 를정량화한수치로창상치유효과를비교하였다. 진피와표피의재생정도, 육아조직증식의형성정도를각각 0부터 3까지나누어수치화하였는데진피와표피의재생및육아조직증식이전혀형성되지않은경우를 0, 진피와표피의재생및육아조직증식이 1/3이하인경우 1, 1/3-2/3이면 2, 2/3이상인경우를 3으로수치화하였다. 열상에나타나는괴사및염증정도에따라열상의염증정도를 0부터 3까지나누어수치화하였으며정상피부의괴사및염증정도를 3으로정하여 neutrophil과같은염증세포가군락형성이없이세포형태로만부분적으로나타나면 2, 군락이뚜렷하지않고작은군락형태나세포형태로혼재되고있는상태를 1, 큰군락을이루고있는상태를 0으로하였다. 재상피화, 육아조직증식정도와열상의각질층및상피층에나타나는괴사및염증정도의정량화된수치를합산한값을 0에서 9로범위를정하였고값이클수록열상치유효과가좋은것으로가정하였다. 3. 통계모든측정치는평균표시하였다. 창상의육안적소견과조직검사결과는모수검정방법인 Student t-test를시행하였으며 p가 0.05미만일때유의한것으로평가하였다. 통계분석소프트웨어는 SPSS (SPSS for Windows 14.0, IBM corp. Chicago, IL, USA) 를이용하였다. 결과 1. 육안적소견열상의육안적소견을비교하였을때 4일째봉합군의 1 마리에서발적, 1마리에서붓기그리고 3마리에서발적과

김재윤외 : 피하층열상쥐모델에서 Polydeoxyribonucleotide (PDRN) 가봉합치료에미치는효과 / 455 POD #4 POD #7 POD #10 Fig. 1. Comparison of gross findings (Left: Suture group, Right: Suture+PDRN group).

PDRN: polydeoxyribonucleotide 붓기가관찰되었고봉합과 PDRN군에서는 3마리에서붓기, 1마리에서누액이관찰되었다. 7일째봉합사제거후상처를벌려보았을때봉합군의 3마리에서 1 mm이상의벌어짐소견이보였고봉합과 PDRN군에서는 1마리에서동일한소견이관찰되었다.

3 김재윤외 : 피하층열상쥐모델에서 Polydeoxyribonucleotide (PDRN) 가봉합치료에미치는효과 / 455 POD #4 POD #7 POD #10 Fig. 1. Comparison of gross findings (Left: Suture group, Right: Suture+PDRN group). Representative photographs of wound on dorsum of mouse at post-operatively day 4, 7 and 10. The wound condition in Suture PDRN group is better than suture group in course period. PDRN: polydeoxyribonucleotide 붓기가관찰되었고봉합과 PDRN군에서는 3마리에서붓기, 1마리에서누액이관찰되었다. 7일째봉합사제거후상처를벌려보았을때봉합군의 3마리에서 1 mm이상의벌어짐소견이보였고봉합과 PDRN군에서는 1마리에서동일한소견이관찰되었다. 10일째에서는모든군에서유의한차이가없었으나봉합과 PDRN군의 1마리에서누액이관찰되었으나다음날관찰시이상소견은없었다 (Fig. 1). 육안적소견에대한 5-Likert scale상결과는, 4일째봉합군 2.0점, 봉합과 PDRN군 3.2점이었고 7일째는봉합군 2.7점, 봉합과 PDRN군 4.2점으로유의한차이를보였다 (p<0.05). 10일째는봉합군 4.5점, 봉합과 PDRN군 4.4점으로차이가없었다 (p=0.297)(fig. 2). 2. 열상조직검사 *: p<0.05 Fig. 2. The mean results of gross finding (5-Likert scale). The mean gross findings of wound in PDRN group are significantly higher than suture groups on postoperative day 4 and 7 (p<0.05). 모든일자에시행하지않고 10일째창상을비교하였을때현미경적으로는진피와표피의재생과육아조직증식정도가봉합과 PDRN그룹에서높은것으로관찰되었다 (Fig. 3). 그러나정량적인수치상의결과에서는진피와표피의재생정도와괴사및염증정도는두군간의차이가없었으나육아조직증식정도에서유의한차이를보였다. (Reepithelization (R) Suture: 2, Suture+PDRN: 2, Inflammation (I) Suture: 0, Suture+PDRN: 0, granulation tissue formation (G) Suture: 1, Suture+PDRN: 2, p<0.05)(fig. 4). VEGF측정결과는현미경적소견에서봉합과 PDRN그룹에서 VEGF의발현 (expression) 이더증가한것으로관찰되었다보였다 (Fig. 5). MetaMorph image analysis software (Universal Image Corp.) 를통한분석결과는봉합군에서 VEGF발현측정치는 였고봉합과 PDRN군의발현측정치는 로유의하게높았다 (p<0.05)(fig. 6). 고찰 PDRN은창상치유, 피부재생에사용되는제재에포함되는활성성분으로하지궤양, 화상및함몰흉터등에효과적인것으로알려져있다 1,4). 저분자 deoxyribonucleotide (DNA) 일부분으로구성되어있고 purine과 pyrimidine nucleotide들이 phosphodiester결합에의해합쳐진형태의 50~2000개의염기로구성된 deoxyribonucleotides 의중합체로형성되어있어 deoxyribonucleotides, deoxyribonucleosides뿐만아니라 purine, pyrimidine 을공급하게된다 5). PDRN은주로 nucleotide에영향을주게되는데 nucleotides와 nucleosides는여러유형의세포성장을촉진시키며핵산의합성및창상치유를촉진하고가속화시키는것으로알려져있는데낮은에너지소비로핵산을생성하는 salvage pathway를촉진하고 A2 purinergic receptor를활성화하기때문인것으로보고되

456 / 대한응급의학회지 : 제 24 권제 4 호 2013 Suture Suture+PDRN Fig. 3. Representative Histological findings of the wound after 10 days (Hematoxylin and eosin stain, 100).

Re-epithelization (R) and Inflammation (I) were not different but, In Suture+PDRN group, more granulation tissue formation (G) was noted than Suture group (p<0.05). 고있다 6-9).

4 456 / 대한응급의학회지 : 제 24 권제 4 호 2013 Suture Suture+PDRN Fig. 3. Representative Histological findings of the wound after 10 days (Hematoxylin and eosin stain, 100). In Suture+PDRN group (Right), re-epithelization and granulation tissue formation were better than Suture group (Left). *: p<0.05 Fig. 4. Mean of wound-healing Grade. Re-epithelization (R) and Inflammation (I) were not different but, In Suture+PDRN group, more granulation tissue formation (G) was noted than Suture group (p<0.05). 고있다 6-9). 또한만성창상이나화상등에효과가있다고 보고되고있다 10). 창상의치유는재생피화, 수축, 육아조직 형성및교원질합성등을포함한복잡한과정을통해이루어지고피부전층결손창상에서는주로수축이중요한역할을하며피부부속기관이남아있는부분층결손창에서는수축보다는상피화가중요한역할을하게된다고알려져있다 11,12). 본연구는응급실기반창상에서의 PDRN의효과를보기위한것으로기본적인피하층열상에적용한동물실험이다. 결과적으로대상의차이는있지만기존의연구들과동일하게 PDRN을봉합술과같이적용하였을때단순봉합술에비해창상의치료효과가있는것을알수있다. 특히 10일째에는두군간의차이가없었지만열상의초기치료에서부작용이적고치료효과를증대시키는장점 을확인할수있었다. 그러나 10일째의조직검사상의결과에서는현미경적소견에서는상피조직의재생과육아조직생성이더낳은것으로보였고 VEGF상의결과에서도 PDRN을포함한치료가더좋은것으로관찰되었다. 현재까지 PDRN을통한보고에서도당뇨병의창상에도움이되고심부2도화상에서단순식염수를이용한습윤드레싱에비해창상의신생혈관생성을증가시켜치유시간을단축시킨다는보도등이있다 13,14). 국내에서도쥐를이용해등의표피를넓게제거한뒤일자별로단순드레싱과비교한연구에서 PDRN그룹이육안적소견과신생혈관생성촉진에서일반처치에비해모두좋은것으로보고되었다 1). 본연구의제한점으로는관찰자의수가적고응급의학과의사만으로진행하여육안적판단의전문성이부족할수있는것과창상치료에대한다양한치료방법을적용하지못하고피하열상의단순봉합과의비교를시행하여응급실내의다른열상치료에적용하기힘들다는것이다. 전자는오히려응급실내열상환자의치료를전문으로하는응급의학과의사의판단을신뢰해야하며오히려의료진의수가적지만열상에대한연구를전문으로하는의료진이기때문에이문제는극복이가능할것으로판단하였고후자와같은문제는향후피부접착제등과같은여러열상치료또는다양한창상모델에적용하는연구를진행함으로써해결할수있을것으로판단하였다. 결론 PDRN은응급실에내원한피하층열상환자의치료에

김재윤외 : 피하층열상쥐모델에서 Polydeoxyribonucleotide (PDRN) 가봉합치료에미치는효과 / 457 Suture Suture+PDRN Fig. 5. Microscopic image of VEGF immunohistochemistry ( 200).

The expression of VEGF in Suture+PDRN group was significantly increased compared to the Suture group (p<0.05). 봉합술과같이적용하였을경우창상의초기치료증진과조직학적치료에도움이될수있다. 참고문헌 *: p<0.05 01.

5 김재윤외 : 피하층열상쥐모델에서 Polydeoxyribonucleotide (PDRN) 가봉합치료에미치는효과 / 457 Suture Suture+PDRN Fig. 5. Microscopic image of VEGF immunohistochemistry ( 200). The expression of VEGF was increased in Suture+PDRN group (Right) than the Suture group (Left). Fig. 6. The quantitative analysis of VEGF expression. The expression of VEGF in Suture+PDRN group was significantly increased compared to the Suture group (p<0.05). 봉합술과같이적용하였을경우창상의초기치료증진과조직학적치료에도움이될수있다. 참고문헌 *: p< Kim YH, Lee JH, Min KH, Hong SH, Lee WM, Jun JH. The wound healing effect of PDRN (polydeoxyribonucleotide) material on full thickness skin defect in the mouse. J Korean Soc Plast Reconstr Surg. 2010;37: Koh CY, Kim HJ, Cho YS, Jun DH, Kim JW. Investigation of non-analgesic subcutaneous suture and skin bond in patients with deep facial lacerations. J Korean Soc Emerg Med. 2012;23: Gu BJ, Kim HJ, Cho YS, Lee MG, Yoo BD, Jun DH. Tissue adhesive effectiveness in laceration site. J Korean Soc Emerg Med. 2011;22: Rubegni P, De Aloe G, Mazzatenta C, Cattarini L, Finiani M. Clinical evaluation of the trophic effect of polydeoxyribonucleotide (PDRN) in patients undergoing skin explants. A piot study. Curr Med Res Opin. 2001;12: Cavallini M. Biorevitalization and cosmetic surgery of the face: symergies of action. J Appl Cosmetol. 2004;22: Victor-Vega C, Desai A, Montesinos MC, Cronstein BN. Adenosine A2A receptor agonists promote more rapid wound healing than recombinant human platelet-derived growth factor (Becaplermin gel). Inflammation. 2002; 26: Montesinos MC, Desai A, Chen JF, Yee H, Schwarzschild MA, Fink JS, et al. Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors. Am J Pathol. 2002;160: Yamaoka T, Kondo M, Honda S, Iwahana H, Moritani M, Ii S, et al. Amidophosphoribosyltransferase limits the rate of cell growth-linked de novo purine biosynthesis in the presence of constant capacity of salvage purine biosynthesis. Biol Chem. 1997;272: Abbracchio MP, Burnstock G. Purinergic signalling: pathophysiological roles. Jpn J Pharmacol. 1998;78: Valdatta L, Thione A, Mortarino C, Buoro M, Tuinder S. Evaluation of the efficacy of polydeoxyribonucleotides in the healing process of autologous skin graft donor sites: a pilot study. Curr Med Res Opin. 2004;20: Bothwell MA, Wilson WH, Shooter EM. The relationship between glandular kallikrein and growth factor-processing

6 458 / 대한응급의학회지 : 제 24 권제 4 호 2013 proteases of mouse submaxillary gland. J Biol Chem. 1979;254: Krawczyk WS. A pattern of epidermal cell migraion during wound healing. J Cell Biol. 1971;49: Galeano M, Bitto A, Altavilla D, Minutoli L, Polito F, Cal? M, et al. Polydeoxyribonucleotide stimulates angiogenesis and wound healing in the genetically diabetic mouse. Wound Repair Regen. 2008;16: Bitto A, Galeano M, Squadrito F, Minutoli L, Polito F, Dye JF, et al. Polydeoxyribonucleotide improves angiogenesis and wound healing in experimental thermal injury. Crit Care Med. 2008;36:

012임수진

012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar