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대한화상학회지제 15 권제 2 호 96 Journal of Korean Burn Society Vol. 15, No. 2, 96-101, 2012 화상흉터개선을위한돼지태반추출물의효과 김홍신 1 이정희 1 윤천재 1,2 이정숙 1 1 베스티안중앙연구소, 2 베스티안부천병원응급의학과 The Therapeutic Effect of Porcine Placenta Extract for Improvement Sequelae of Burn Hong Shin Kim, M.S. 1, Jung Hee Lee, M.S. 1, Cheon-Jae Yeon, M.D. 1,2 and Jung-Suk Lee, Ph.D. 1 1 Bestian Research Center, Deajeon, 2 Department of Emergency Medicine, Bestian Bucheon Hospital, Bucheon, Korea Purpose: Burn injury cause pruritis, pain, psychological and functional sequelae. The one of burn injury sequelae is the hypertrophic scar. It is difficult to control devastating fibrotic condition for hypertrophic scar. The objective of this study was to investigated the therapeutic effect on burn hypertrophic scar and wound healing for sequelae of burn injury by Porcine placenta extract (PPE). Methods: To investigate the effect of PPE, we performed in vitro cell cytotoxity test (MTT assay), antioxidant activity assay (SOD like activity), melanin content assay, cell migration asssay and RT-PCR. Results: As a result of cell cytotoxity test (MTT assay), PPE showed above 80% cell viability. From Antioxidant activity assay (SOD like activity), this effect was similar to vitamin C. In the melanin content assay, melanin synthesis was inhibited 23% on PPE treatment than control. PPE enhanced cell migration on human fibroblast and decreased the expression of hypertropic scar related gene (a-sma and P311). Conclusion: Our data showed anti-oxidant effect, diminution of melanin and decrease of the expression of hypertropic scar related gene on the treatment of PPE. These results may provide the insight into the potential use of porcine placenta extract as support to control skin fibrosis related to burn hypertrophic scar and alternative medicine for burn sequelae. (J Korean Burn Soc 2012;15:96-101) Key Words: Porcine placenta extract (PPE), Wound healing, Melanin synthesis inhibition, SOD like activity, Hypertropic scar 책임저자 : 이정숙, 대전시동구신안동 293-1 300-060, 베스티안중앙연구소 Tel: 070-7603-7422, Fax: 042-633-7055 E-mail: nostalgie@daum.net 서론화상은심한경우치료후에도기능적장애및많은후유증을가져온다. 집중력장애, 우울증, 가려움증, 통증, 불안, 관절구축, 색소침착, 저색소증및비후성반흔이발생된다. 2도및 3도이상의화상피부에서나타나는비후성반흔 (Hypertrophic scar) 은피부가두꺼워지고소양감및건조함과일상생활장애를유발한다 1-4). 비후성반흔조직에서 a-sma (alpha smooth muscle actin) 를표현하는과도한근섬유세포증가및콜라겐과섬유결합소의과도한합성이관찰되어지고, 형질전환인자 (TGF-β1, Transforming growth factor), PDGF (Platelet derived growth factor) 등의성장인자와 IL-16 (Interleukin-16), IL-13 (Interleukin-13) 등의사이토카인이증가한다 5). 그외비후성반흔및섬유증 (Fibrosis) 형성에 TGF-β1 와 P311 유전자가깊이관여하고 6) 발현이증가되는것으로보고되었다. TGF-β1은활성화된혈소판, 대식세포및림프구에서분비되며 7) 근섬유세포에의한콜라겐과섬유소결합생성을증가시킨다. P311은근섬유세포분화와섬유증을촉진하고, TGF-β1와콜라겐 type1 의 mrna 발현을유도하고상처표면수축 (Contraction) 에관여하는것으로실험결과가보고되었다 7,8). 일반적인비후성반흔치료는스테로이드의병변내주사, 압박요법외과적절제술, IFN-α 주입및 TGF-β1에대한항체요법등이시도되고있다 5,6). 태반은혈액융모막으로구성되어있는태아와모체의자궁을연결해주는기관으로태아에게산소와영양분을공급해주고, 노폐물을제거하는역할을한다 9). 태반추출물은싸이토카인, 호르몬, 활성펩타이드, 효소, 성장인자, 인, 철, 지방, 핵산단백질이포함된것으로알려져있다 10). 그중돼지태반추출물은비교적안전하고인간과면역효과가유사한것으로알려졌으며, 대식세포활성억제로접촉성피부염 (Contact hypersensitivity) 진행을억제시켜염증반응줄여주는것이실험적으로확인되었다 10,11). 또한섬유아세포 (NIH3T3 cell, fibroblast) 에 30 mg/ml의돼지태반추출물을처리하였을때, 4.17배정도의세포성장이증

김홍신등 : 화상흉터개선을위한돼지태반추출물의효과 97 가하였고, Rat에화상을유도후 30 mg/ml의돼지태반추출물을피부에처리하여 1, 5, 10, 15일에 bfgf (basic fibroblast growth factor) 발현을확인한결과, 5일째부터 bfgf 발현이양이증가하는것이보고되었다 12). 화상피부재활치료에있어서비후성반흔치료와관련하여돼지태반추출물의효능은거의알려지지않았다. 본연구는비후성반흔과색소침착등의돼지태반추출물의효과를분자생물학적인기법을이용하여확인하고자하였다. 대상및방법 1. 재료본연구에사용한돼지태반추출물 (Porcine placenta extract) 은일본호루스 (Horus, ホルス ) 사의 SPF 돼지태반으로부터효소를이용한추출과동결건조방법의동결효소추출법 13) 을이용하여추출하였다. 2. 세포배양 1) 섬유아세포배양및각질형성세포배양사람의각질형성세포 (HaCaT cell) 및섬유아세포 (Human fibroblast) 를 Dulbecco's modified eagle media (DMEM), Bovine calf serum (BCS) 와 Penicilline-streptomycin이함유된배지에서 37 o C, 5% CO 2 의조건으로배양시켰다. 배양된섬유아세포및각질형성세포는 2 10 5 개로나누어 6 well cell culture dish에넣고돼지태반추출물을 24시간동안처리하였다. 2) 멜라닌세포배양사람의멜라닌세포 (Human melanoma cell: MNT-1 cell) 를 Dulbecco's modified eagle media (DMEM), fetal bovine serum (FBS), Non-essential amino acid, 1 mm Sodium pyrubate, Penicilline-streptomycin 배지에서 37 o C, 5% CO 2 의조건으로배양시켰다. 3. 세포생존율시험세포생존율시험은 6 well에섬유아세포 (Human fibroblast) 및인체각질형성세포 (HaCaT cell) 를 24시간배양후돼지태반추출물을 1.25 10% 농도로처리하여 24시간동안다시배양하였다. 여기에 MTT (3-(4,5 dimethylothiazol-2-yl)-2.5-diphenyltetrazolium bromide, sigma) 5 mg/ ml를첨가하여 37 o C, 5% CO 2 의조건으로 3시간동안배양후 DMSO로녹이고, 96 well plate로옮겨 595 nm에서 ELISA reader로흡광도를측정하였다. 4. 항산화활성측정시험 (SOD like activity) 14) 항산화활성측정시험은 SOD (Superoxide dismutase) 유사활성으로측정하였다. SOD 유사활성은활성산소종을과산화수소 (H 2O 2) 로전환시키는반응을촉매하는 pyrogallol의생성량을측정하여나타내었다. 각시료용액 0.2 ml에 Tris-HCl의완충용액 (50 mm Tris, 10 mm EDTA, ph 8.5) 2.6 ml와 7.2 ml pyrogallol 0.2 ml (Sigma) 를가하여 25 o C에서 10분간반응시킨후 1.0 M HCl 0.1 ml을가하여반응을정지시키고, 반응액중산화된 pyrogallol의양을 420 nm에서 ELISA reader로흡광도를측정하였다. SOD 유사활성은시료용액의대조군과처리군의흡광도감소율로나타내었다. 5. 멜라닌함유량측정시험 15,16) 인간의멜라닌세포 (Human melanoma cell, MNT-1 cell) 를 6 well에접종하여 24시간배양후돼지태반추출물 2, 5% 를처리하였다. 48시간추가배양하여세포를수확한후멜라닌양을측정하기위해 NaOH를처리, 가열및용해시켜 475 nm에서 ELISA reader로흡광도를측정하였다. 6. 세포창상치유능시험 (Wound healing assay, Cell migration assay) 17) 섬유아세포 (Human fibroblast) 를 12 well plate에접종한후 24시간동안배양하여세포가 plate에 80% 이상되도록하였다. Yellow tip을이용하여배양접시바닥을십자로그어균일한너비로선을만들었다. PBS로세척한후돼지태반추출물 10% 를포함한배지를처리하였다. 돼지태반추출물을처리하지않은대조군과처리군을비교하였고, 세포창상치유능시험은현미경배율 100으로 3, 12, 24, 48 시간에서사진을찍어비교하였다. 7. Total RNA 분리및 cdna 합성 수확된세포에 easy-blue TM Total RNA Extraction Kit (Intron Biotechnology Inc, Korea) 를잘풀어준후 0.2 ml의클로로포름을첨가하여교반해주었다. 이혼합물을 4 o C에서 13,000 rpm으로 10분간원심분리후상등액을취한후, 동량의 2-propanol을첨가한다. 4 o C에서 10분이상을두었다가 13,000 rpm으로 10분간원심분리하였다. 튜브의상층액을제거하고얻은침전물에 80% 에탄올을넣어 13,000 rpm으로원심분리후에탄올은제거해준다. 실온에서건조후 0.1% ddepc water 30μl에용해시켜 RNA를얻었다. 2μg의 RNA에전체량이 9.5μl가되도록멸균수와혼합후 oligo dt 1μl를 tube에첨가하고 75 o C에서 5분간반응시킨다. Ice에적어도 1분이상처리하고, 10 U/μl

98 대한화상학회지 Vol. 15, No. 2, 2012 Fig. 1. Cell cytotoxity test (MTT assay) of porcine placenta extract. Cell cytotoxity test showed cell viability in treatment group on human fibroblast cell (A) and on HaCaT cell (B). *P<0.05, student s t-test. RNase inhibitor 1μl, 5x RT buffer 4μl, 10 mm dntp 2μ l, 0.1 M DTT 2μl, AMV RT enzyme 0.5μl를넣고 42 o C에서 60분간반응시켜 cdna를합성하였다. 8. 역전사중합효소연쇄반응 (Reverse transcription polymerase chain reaction, RT-PCR) 실험에사용된모든 PCR primer는 Gene bank data base 를기본으로 Primer 3 software를이용하여합성하였고, initial denaturation으로 95 o C에서 5분, 95 o C에서 30초 (denaturation), 58 o C에서 30초 (annealing), 72 o C에서 30초 (extension), 72 o C에서 5분 (final extension) 으로, 유전자에따라 25 32 cycle을증폭시킨후 1.2% agarose (BIO-Rad, USA) gel 에 reaction mixture 5μl씩전기영동하고발현정도를확인하였다. 결과 1. 세포생존율시험 (MTT assay) 돼지태반추출물의세포생존에미치는영향을확인하기위하여추출물을 1.25, 2.5, 5, 10% 의농도로배지에첨가하여세포생존율을 MTT assay로측정하였다. 섬유아세포 (Human fibroblast) 는 2.5, 5, 10% 의농도에서 100% 이상의세포생존율을보였으며, 각질형성세포 (HaCaT) 에서는 80 90% 의세포생존율을확인할수있었다 (Fig. 1). 2. 항산화시험 (SOD like assay) 돼지태반추출물항산화효능검정을 SOD (Superoxide dismutase) 유사활성으로검정하였다. SOD (Superoxide dismutase) 유사활성을가지는물질은 Superoxide로부터 Fig. 2. SOD (superoxide dismutase) like activity assay of porcine placenta extract. Antioxidant effect using SOD like activity, antioxidant effect was a similar effect compared to the control group: Vitamin C. *P<0.05, student s t-test, PPE = porcine placenta extract. 생체를보호하며, 인체내의 Superoxide를제거함으로써노화억제와산화적장애방어효과를가지는것으로알려져있다 13). 돼지태반추출물의 SOD 유사활성은 19% 로, Vitamin C 1 mm 보다높고, Vitamin C 5 mm와비슷하였다 (Fig. 2). 3. 멜라닌함유량측정시험 멜라닌세포 (Human melanoma cell- MNT-1 cell) 에돼지태반추출물을 2, 5% 처리하여 Melanin 함유량저해능력을확인한결과, 돼지태반추출물 2% 처리하였을경우 19%, 5% 처리하였을경우 23% 의 Melanin 함유량저해능력을확인하였다 (Fig. 3).

김홍신등 : 화상흉터개선을위한돼지태반추출물의효과 99 Fig. 3. Melanin contents assay by porcine placenta extract. After 48 hours, PPE treated group were decreased the amount of melanin compared to the non-treated group. *P<0.05, student s t-test, PPE = porcine placenta extract. 4. 세포상처치유능시험섬유아세포 (Human fibroblast) 를이용하여세포이동성시험 (Cell migration assay) 통해돼지태반추출물 10% 처리한군에서처리하지않은군보다빠른이동과증식이확인되어양단간의간격이좁아졌다. 돼지태반추출물을처리한후 12시간대까지는차이가거의없으나 24시간이후부터간격이서서히좁아지는것을확인되었고, 48시간에서대조군에비해간격이많이좁아진양상이확인되었다 (Fig. 4). 5. 역전사중합효소연쇄반응각질형성세포 (HaCaT cell) 에돼지태반추출물 5, 10% 를처리한후 24시간후에 Cell을회수하여역전사중합효소연쇄반응 (RT-PCR) 을통하여세포부착단백질 (Cell adhesion protein) 인 JUP (Plakoglobin), DSP (Desmoplakin) 발현을확인한결과, 유전자발현변화는없었다. 그외에스트레스및콜라겐합성인자로알려진 HSP47 (Heat shock protein 47) 은발현의차이를보이지않았지만, 열등의스트레스에의해발생하는 HSP70 (Heat shock protein 70) 는대조군과비교하여돼지태반추출물을처리하였을때발현이감소됨을확인하였다. 섬유아세포를동일한방법으로근섬유세포의확인표지자로쓰이는 a-sma와근섬유세포분화촉진및수축에관여하는것으로알려진 P311발현을확인한결과, 처리하지않은것과비교하였을때, 돼지태반추출물 5, 10% 를처리한군에서 a-sma와 P311 발현이농도의존적으로감소되는경향을관찰하였다 (Fig. 5). Fig. 4. Wound healing assay by porcine placenta extract ( 100). Cell were treated with PPE after wounding. The wound width gradually were decreased in PPE group after 48 hours. PPE = porcine placenta extract. 고 본연구에서는화상치료후에빈번하게발생하는비후성반흔과색소침착등의문제를개선하기위해돼지태반추출물 (Porcine placenta extract) 의효능을피부세포를이용하여분자생물학적기법을통해분석하였다. 돼지태반추출물은피부미용과관련하여엘라스타아제 (Elastase) 20 30% 의저해활성을보이며주름개선효과가있는것으로보고되었다 10). 또한간손상을유발한백서에서돼지태반가수분해물을투여하였을때, 혈청 AST와 ALT 농도증가를억제시키고간조직형태변형을감소시켜손상억제효과가있는것으로확인되었다 9). 화상과관련하여, Rat 에화상을유도한후돼지태반추출물을처리하였을때, 유도된피부에서 bfgf와 TGF-β1의발현이피부도포후 5일째부터발현이증가하였고상처치유기간이 50% 정도빠르게진행됨이보고되었다 12). 본연구에서는세포독성시험결과, 섬유아세포에서는 찰

100 대한화상학회지 Vol. 15, No. 2, 2012 Fig. 5. RT-PCR analysis of porcine placenta extract treated on HaCaT and human fibroblast. (A) JUP, DSP and HSP47 were not changed on HaCaT. But HSP70 gene expression were decreased than control by treatment of PPE. (B) a-sma and P311 gene expression were decreased in dose dependent on human fibroblast. JUP = plakoglobin, DSP = desmoplakin, HSP47 = heat shcok protein 47, HSP70 = heat shock protein 70, a-sma = alpha smooth muscle actin. 100% 이상의세포생존율을보였고각질형성세포에서는 80% 이상의생존율을보여세포독성이없는것으로나타났다. SOD like activity를이용한항산화효능확인시험에서는대조군인 Vitamin C 1 mm보다높고, 5 mm과유사한것을확인하였다. 항산화작용은화상에서 Mitochondria integrity를유지하는데도움을준다고보고되었고 18), 세포손상을억제하는것으로알려져있다. 이와같은결과로 Vitamin C 5 mm과유사한항산화효능을갖고있어, Mitochondria integrity 유지및세포손상억제에도움을줄수있다고추측할수있다. 화상치유과정은지혈 (hemostasis), 염증반응 (Inflammation), 면역반응 (Immune response), 상피화 (re-epithelization), 혈관생성 (Angiogenesis), 재형성 (Remodelling) 의과정을거치게된다. 치유과정마지막단계, 화상후 3개월 6 개월사이결합조직침착및상처표면수축이나타난다 19). 과도한결합조직침착및상처표면수축 (contraction) 은비후성반흔의특징이며, 근섬유세포증가가주요원인으로알려져있다. 이와관련하여돼지태반추출물의효능을확인하기위하여섬유아세포에처리한후근섬유세포분화와수축에관련되는유전자로보고된 a-sma와 P311 발현을역전사중합효소연쇄반응을통하여확인하였다. 결과적으로돼지태반추출물 5, 10% 을처리하였을때, a-sma와 P311 발현이농도의존적으로감소하는경향이확인되었다. 이러한결과는돼지태반추출물이재형성과정에서비후성반흔으로인한흉터개선에효능이있다는것을의미하기도한다. 각질형성세포에돼지태반추출물을처리한후 RT-PCR 을통하여세포부착 (Cell adhesion) 및세포접합 (Cell Junction) 유전자인 JUP (Plakoglobin), DSP (Desmoplakin) 20) 발현을확인하였지만변화를관찰할수없었다. 그외에섬유화과정에서콜라겐생성을증가시키는역할로알려진 HSP47 (Heat shock protein 47) 21,22) 발현은큰변화를확인할수없었으나, 열과산화스트레스 (Stress) 지표이며방어기작지표인 HSP70 23) (Heat shcok protein 70) 은처리군에서대조군과비교하였을때유전자발현이감소되는양상을관찰할수있었다. 이를통하여돼지태반추출물은세포부착과콜라겐합성증가에는영향을주지않고, 열과산화스트레스등을억제하는효과가있음을확인할수있었다. 또한세포창상치유능시험을통해, 돼지태반추출물이섬유아세포의이동을좀더원활하게함으로써상처치유시간을줄여줄것으로볼수있었다. 화상후유증중색소침착과관련된멜라닌함유량측정시험에서는대조군대비 23% 멜라닌합성억제효능을나타내어돼지태반추출물이멜라닌합성을억제함으로써색소침착을완화시키는데도움을줄것으로생각한다. 결론태반추출물은상처치유또는화상에대한여러효능이보고되어왔다. 성장호르몬, 싸이토카인등의발현증가로상처치유를촉진하고접촉성피부염등의질환에도효과가있다고알려져있다. 하지만, 본연구에서는화상을입은후오게되는문제점중비후성반흔등의피부이상증상과관련하여돼지태반의효능이비교적알려지지않아이를분자생물학적으로증명해보고자하였다. 피부세포를이용하여분석한결과, 항산화효과, 멜라닌합성억제, 창상치유효능및 a-sma와 P311 유전자발현이감소되는현상을확

김홍신등 : 화상흉터개선을위한돼지태반추출물의효과 101 인하였다. 이는돼지태반추출물이화상치유과정중재형성과정에서비후성반흔과관련된피부이상현상을억제하는효능과화상후피부개선을위한유효성이있음을말해준다. 화상피부에발생하는비후성반흔등의증상에돼지태반추출물을사용할경우임상적인효과를볼수있을것으로기대한다. REFERENCES 1) Agha RA, Agha M. Skin stretiching for burn scar excision a critically appraised topic. AMS. 2012;1:1-9. 2) Curran TA, Ghahary A. Fibrocyte and keratinocyte like and their role in burn wound healing. Surgery. 2012;2:106-110. 3) Seo SH, Jang KU. Multidisciplinary team for burn injury rehabilitation. J Korean Burn Soc. 2005;8:19-26. 4) Roh YS, Cho H, Oh JO, Yoon CJ. The effectiveness of skin rehabilitation nursing on skin status and depression of burn patients. J Korean Burn Soc. 2005;8:27-33. 5) Han YJ, Jeong Y, Whang KK. Treatment of hypertrophic scars and keloids using intense pulsed light. Korean J Dermatol. 2009;47:395-402. 6) Penn JW, Grobbelaar AO, Rolfe KJ. The role of the TGF-β family in wound healing, burns and scarring: a review. Int J Burn Trauma. 2012;2:18-28. 7) Kim JH, Han HH, Ahn JD, Lee IK, Kim EJ, Hwang MY, et al. The effect of antibody and gene therapy for transforming growth factor β1 on scar formation. Korean J Pathol. 2001;35:424-432. 8) Tan J, Peng X, Luo G, Ma B, Cao C, He W, et al. Investigating the role of P311 in the hypertrophic scar. PLos One. 2010; 5:1-6. 9) Park SY, Kim DS, Kang S, Park S. Hepatoprotective effect of pig placental hydrolysate on liver damage induced rat by injecting carbon tetrachloride. J Appl Biol Chem. 2012;55: 109-115. 10) Kim BY, Kim T, Kang WY, Baek HH, Cheon Y, Kim D. Functional cosmetic effect of porcine placenta. Korean Chem Eng Res. 2010;48:327-331. 11) Jash A, Kwon HK, Sahoo A, Lee CG, So JS, Kim JH, et al. Topical application of porcine placenta extract inhibit the progression of experimental contact hypersensitivity. J Ethnopharmacol. 2011;133:654-662. 12) Wu CH, Chang CY, Chang WC, Hsu CT, Chen RS. Wound healing effect of porcine placental extracts on rat with thermal injury. Br J Dermatol. 2003;148:236-245. 13) Kentaro Y. For useful guide for those seeking placenta based remedies. Placenta Power. 2004;2:2-121. 14) Kang DH, Kim HS. Functionally analysis of Korean medicine fermented by lactobacillus strains. Korean J Microbiol Biotechnol. 2011;3:259-265. 15) Ko JY, Kim YC. Effective of sciripi rhizoma ethanol extract on skin whitening using in vitro test. J Environ Toxicol. 2010;25:69-77. 16) Senthilkumar KJ, Yang JC, Chu FH, Chang ST, Wang SY. Lucidone, a novel melanin inhibitor from the fruit of lundera erythrocarpa makino. Phytother Res. 2010;24:1158-1165. 17) Kim CH, Moon SK, Bae JH, Lee JH, Choi EC. Effect of HGF in proliferation, dispersion and migration of hypopharyngeal squamous cell carcinoma. Korean J Otolaryngol. 2005;48:208-215. 18) Zang Q, Maass DL, White J, Horton JW. Cardiac mitochondrial damage and loss of ROS defense after burn injury: the beneficial effect of antioxidnat therapy. J Appl Physiol. 2007;102:103-112. 19) Shin JC, Seo CH, Jang KU, Jung KY. Scar quality and hand fuction after moist exposed burn ointment and skin graft treatment in full thickness hand burn. J Korean Acd Rehab Med. 2007;31:582-589. 20) Wang TW, Sheng J, Huang YC, Chin HSI, Wu HC, Chen LT, et al. Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR. Biomaterial. 2007;27:5059-5068. 21) Chun KW, Park JH, Jung JC, Kim DJ, Park SG, Kim JS, et al. Expression of E-cadherin, heat shock protein 47, transforming growth factor β1 and C4d in chronic allograft nephropathy. J Korean Soc Translpant. 2010;24:298-305. 22) Milar N, Murrell G. Heat shock protein in tendinopathy: novel molecular regulators. Mediat Inflamm. 2012;2012:1-7. 23) Shin YO, Han MK, Lee JB, Um BH. The effect of supplementary selenium on leukocytes and HSP70 expression after half-body immersion. Korean J Nutr. 2011;44:378-383.