2002-2003 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Hung, LK

Leung, PC

Wan, KC

Wu, HT

PolyU Institutional Repository

·??·????:???????????????(? ? 、??? 、???);??????????????(???)?????????????????????????????胡 棠　熊良俭　梁秉中　温冠清　　【??】　??　????????????????????????????????????? ,??????????????????? ,?????????????。??　?????????????????? ,?? 8 ????????????????????????:??????? 、????? 、???????? 、???? 、??? ,???????????。?37℃?? 4 h? , ???????????????????????????????。 ??　???????????????????????????? , ???????? 、??????????????(P<0.05), ????????????????????????????? ,????????????????? ,???????? 、????。??　?????? 5 ???????????????????? ,????????????? ,???????????????????????? ,???????????。【???】　?? ,???;　?????;　????;　???The effects of antioxidants on pyridinoline cross linkage formation in human fibroblasts culture fromhypertrophic scars　　WU Haytong＊, HUNG Leungkim , LEUNG Pingchung , WAN Koonching.＊Departmentof Orthopaedics &Traumatology , Chinese University of Hong Kong , Hong Kong , China【Abstract】　Objective　To investigate the effects of antioxidants on the pyridinoline crosslink formation inhuman fibroblasts culture from hypertrophic scars.The role of these antioxidants in reducing the level ofpyridinoline crosslinks in-vitro without damaging the cells has important implications.These antioxidants can beapplied for the treatment of hypertrophic scars.Methods　9 cases of hyprotrophic scar biopsies were taken fromburn patients.The specimens were explant culture for 7 days.Insolated fibroblasts from these explant cultures weresubcultured for 8 passages.At the last passage , the cultures were subjected to a free radical generating system andwere added different antioxidants.The supernatant and cells were collected after 4 hrs of incubation and theconcentration of pyridinoline crosslinks was determined using Pyrilink kit.Results　When the fibroblast cultureswere subjected to free radicals and antioxidants , all samples showed a decrease in pyridinoline crosslinksconcentration.The more potent antioxidants found in this study were catalase , superoxide dismutase anddesferrioxamine.Particularly catalase which did not damage the cells.Conclusion　The antioxidants used in thisstudy were found to be effective in reducing the concentration of pyridinoline crosslinks.The best agent is catalase ,which did not damage the cells and could be considered for clinical use.【Key words】　Cicatrix , hypertrophic;　Fibroblasts;　Antioxidants;　Pyridines　　?????????????????????。????????????? ,??????? ,??????????????????????????????[ 1-4] ,??????????????????????????[ 5 ,6] ,???????????????????[ 7-10] 。???????????? ,??????????????????????????? ,?????????????????????????。?????? 、??1.???????:?????????????????????? ,? 9? 。???? 4? ,?? 5? ,?? 4 ～ 39? 。????????????? ,???????? 1?? ,?? 5? 。????????????????????? ,????????????????????????·590· ?????? 2002? 5? 10?? 82?? 9?　Natl Med J China , May 10, 2002 ,Vol 82 , No.9?? 。??????????????????????。2.????:???????(superoxidedismutase)、?????(catalase)、????????(glutathione peroxidase)、????(glutathione)、???(desferrioxamine)、????(ascorbic acid)???????(EDTA)??? Sigma ???? ,????(hydrogen peroxide)??? Riedel???? ,FeCl2-6H2O??? Merck ???? , DMEM ??? 、?????????????? GIBCO??? ,???????????Metra Biosystems???? 。? 、??1.???????:(1)???????:?????????????????????????? 。?????? ,????????? ,????????1 mm3?? ,??????? 60 mm ???? ,??????????? 20 min ,???????????? ,?? 3 ml? 10%?????DMEM??? ,? 37℃? 5%CO2 ???????? ,????????? 2? 。(2)???????:?? 7 d? ,????????????????? 。?????????????????????????? ,??????? 0.25%?????? 1 min ,???????????????(????????????)????????? ,?37℃?? 。(3)????:??????????70%～ 80%?????????? , ????0.25%???????? ,?????? ,???????????? ,??? 10%????? DMEM???? 37℃?? 。?? 2 ～ 3 d ???? 1? 。????? 8?? ,???????????? 6??? 35 mm ????? ,???????? 3×105 。? 37℃?? 24 h ??????????DMEM????? 48 h , ???????? 。2.????:??????????????????????? 。(1)???:5???????????5???????? , ?????????????????:58 μg/ml??????? 、67 μg/ml????? 、1 mmol/L ???? 、0.38 mg???????? 、1 mmol/L ??? ,?????????????(?????????????0.1 mmol/L Fe3+ 、0.1 mmol/L EDTA 、1.7 mmol/L ???? 、1 mmol/L H2O2)。(2)???:???????????? 。9 ????? ,???? 6 ???(5????? 1????),????????。?????? 37℃?? 4 h ? ,????????????? ,??????????????????????。3.?????????:(1)???????:? 1 ml 6 mol/L HCl????????????????????? ,? 110℃?? 。(2)????????:?????????? ,??????????? 10 ??? ,??????????。50 μl????????????????? ,???100μl?????? 4℃??3 h ,?????? 250μl? 3? ,??? 150 μl?? P-??? ,????60 min??? 100 μl 1 mol/L NaOH ,????。??? Biorad???? 2550? EIA ???????? ,?? 405 nm。4.????:????????? 9?????????????????。????????SPSS??? t????????? , P <0.05????????。? ?? 、???????????????????????????????????????? 6 ～ 7?(? 1)。????????????????????????????? ,??????????????????????????? ,???????? 、?????????????(P<0.05)。?????????????????? ,??????????????????(P >0.05)。? 1　?????????( x±s , nmol/L)?? ?? ???? ??????1???(?????) 9 55±17 389±482???????? 9 56±26 272±40＊3?????? 9 38±13 288±42＊4????? 9 68±25 287±545????????? 9 49±24 308±606???? 9 48±17 274±42＊　　?:＊?????? P<0.05? 、????????????????????????????(? 1)??????? ,???????????????????????????????? ,?????????????????? ———·591·?????? 2002? 5? 10?? 82?? 9?　Nat l Med J China , May 10 , 2002 , Vol 82 , No.9?:①???(?????)②???????? ③?????? ④????? ⑤????????? ⑥????? 1　????????? 4 h?????????? ,???????????????????????? ,??④????????????? ,????? ,????(????)　×100????? 、????。? ??????????????????????????? ,??????????。???????????????????? ,??????????????????? ,????????DNA?? 。?????????????????????????? ,??????????????????????????????????????? ,?????????????;??????????????? ,???????????? 。?? ,????????????????????????????????????????? ,????????????。??????? ,???????????????[ 11 ,12] ;????????????????????????????????????????? ,??????????? ,?????????????????? ,?????????? ,????????????? ,???????????????? ,??????????????????? ,??????????????????? 。? ? ? ?1 Castagnoli C , Trombotto C, Ondei S , et al.Characterization of T-cellsubsets infiltrating post-burn hypertrophic scar tissues.Burns , 1997 ,23:565-572.2 Rosen GM , Pou S , Ramos CL , et al.Free radicals and phagocyticcells.FASEB J ,1995 , 9:200-209.3 Pansarasa O , Bertorelli L , Vecchiet J , et al.Age-dependent changes ofantioxidant activities and markers of free radical damage in humanskeletal muscle.Free Radic Biol Med ,1999 , 27:617-622.4 Babior BM.Oxygen-dependent microbial killing by phagocytes.N Engl JMed , 1978, 298:659-668.5 Wan KC , Evans JH.Free radical involvement in hypertrophic scarformation.Free Radic Biol Med ,1999 , 26:603-608.6 Wan KC , Lewis WH.Study of free iron and pyridinoline in hypertrophicscars and normal skin.Br J Biomed Sci ,1996 , 53:196-203.7 Inalsingh CH.An experience in t reating five hundred and one patientswith keloids.Johns Hopkins Med J , 1974 , 134:284-290.8 Moriguchi T , Fujimoto D.Crosslink of collagen in hypertrophic scar.JInvest Dermatol , 1979 , 72:143-145.9 Linares HA.From wound to scar.Burns , 1996, 22:339-352.10 Wan KC , Chow TC.Age-related changes in the concentration ofpyridinoline cross-links in human skin.Ann Clin Biochem ,1999 , 36:666-668.11 Shigeki S , Murakami T , Yata N , et al.Treatment of keloid andhypert rophic scars by iontophoretic transdermal delivery of t ranilast.Scand J Plast Reconst r Surg Hand Surg , 1997 , 31:151-158.12 Zhao L , Hung LK , Choy TT.Delivery of medication by iontophoresis tot reat post-burn hypertrophic scars:investigation of a new electronictechnique.Burns , 1997 , 23(Suppl1):S27-29.(????:2001-10-15)(????:???)·592· ?????? 2002? 5? 10?? 82?? 9?　Natl Med J China , May 10, 2002 ,Vol 82 , No.9

The effects of antioxidants on pyridinoline cross linkage formation in human fibroblasts culture from hypertrophic scars

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