行政院國家科學委員會專題研究計畫成果報告

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行政院國家科學委員會專題研究計畫成果報告

探討淡水生綠藻萃出物於延緩光照皮膚老化的應用及生物分子原理

研究成果報告(精簡版)

計畫類別：個別型

計畫編號： NSC 97-2320-B-041-004-

執行期間： 97 年 08 月 01 日至 98 年 07 月 31 日執行單位：嘉南藥理科技大學藥學系

計畫主持人：施美份

共同主持人：程中玉、沈家瑞、陳莉螢計畫參與人員：此計畫無其他參與人員

處理方式：本計畫可公開查詢

中華民國 98 年 10 月 16 日

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! " # $% &' ( ) * + ,- . ,/ 0 ,1 2 ,

3 4 5 6 78 9 : # ; < = > ? @A " # $% Bphotoaging^C^{DE <} FG H IBextracellular matrix^CJ ^K ^LM ^N Ô ^P ^QR Matrix metalloproteinases^BMMPs^CS ^TQU ^H ÎBextracellular matrix^CV ^W Bremodeling^CX ^Y ^Z ^[ ^V ^\ ^] ³ ^{^_} ^` â ^b ^c ^d ê ^fg ^h Î@type Iⁱ ^j g h Btype I collagens^C^{^k} ^l ^mQU ^no ⁾ ^p ^Dtype Iⁱ ^j ^g ^h ^q

\ Fr s Qt Bfibroblast^Cuv ^w ^x ^y ^z ^y ^{ ^L| ^} ^! ^~ ⁱ ^j ^g ^h Bprocollagen^C= ô ^M ^N ^S ^{ ^L ^} ⁱ ^j ^g ^h ^s ^QDi ^j ^g h J ) e BVerzijl et al., 2000^CÂ û ^w ^Li j g h , # ,L 6 $% Fe f" # $% BFisher et al., 1996; 1997; Talwar et al., 1995^C^D

F e A ¡ ¢ D£¤ | M ¥ &

UVC (<290nm), UVB (290-320nm), UVA2 (320-340nm), and UVA1 (340-400nm)^D

^_ UVB^u ¦ ^l ^q ^\ ^F" ^# ^§ ^¨ UVA2 ^© ^ª ^l ^«

¬UVA1ⁿ ^; ^F " ^# ^q ^\ ^£¤ e D : # ®

? Fe f¯ # ° r ±# ² protein kinase C^BPKC^C±PKC

±³ ´ µ ¶ · ¸ Interstitial collagenase^B¹MMP-1^{CJ K} ^w ^Li ^j ^g ^h

M N BBerneburg et al, 1999^C^DMMPs^M ^N ^` type I IIIⁱ ^j ^g ^h ^s QDMMP-3 (stromelysin-1) ^S ^Fº ^» ^M ^N ^¼ ^g ^h proteoglycans^,fibronectin ^½ type IIIⁱ ^j ^BGiambernardi et al, 1998; Kuroda & Shinkai 1997^C^D¾ ^¿ ^_ ^| ^Àm MMPs^±Á ^Â ^P ^Ã ^IS ^Ftissue inhibitor of metalloproteinase^BTIMP^C^D

Ä z w LMMPs^±³ ^´ TIMPs^±Å ^Æ ^Ç ^z

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: # ¹È É ,- . Ê J BTalwar et al., 1995^C^D

" # Ë 3 ' ( - . ¤ Ì u Í Î / Ï ; < =

> ? @A " # $% Bphotoaging^C^{DÐ Ñ} ^Ò ^Ó ^Ô ^Õ ^A ^Ö ^{Ì B}UV irradiation^Cu " # $ % m H I Bextracellular matrix^{C ¹ i} ^j ^g ^h Bcollagen^C^{, g} ^{h B}elastin^C^,proteoglycans^¾ ^L× ^: ^FØ ^N ⁾ ^{p B}Fisher et al, 1997; Krutmann, 2001;Varani et al, 2001^C^{D8 9} ^× ^: ⁼ ^> ^@mÙ ^Ú ^Û ^Ü ^Ý Þ ß à Bsignal transduction pathways^C ^±# ^á ^â ^ã ^Btranscription factor^C

ä H Btarget gene^C^BFisher et al, 1998^C^DUV^Ì ^±# MAP kinase^Û ^Ü ^ß à å [ # á â ã AP1^æ MMP^Ø ^N ^ç ^Ä ^H ^è ^$ ^é ^v ^w ⁸ 9 Ø N ç Ä å [ ê $M N H IBextracellular matrix^Cµ ^{¶ B}Fisher et al., 1996; 2000^C^D

Ð Ñ Ò Ó Ô Õ A Ö Ì BUV irradiationCu " # $% m H IBextracellular matrix^C^¹i ^j ^g ^{h B}collagen^C^{, g} ^{h B}elastin^C^,proteoglycans

¾ L× : FØ N ) p BFisher et al, 1997; Krutmann, 2001;Varani et al, 2001^CD 8 9 × : = > @mÙ Ú Û Ü Ý Þ ß à Bsignal transduction pathways^C ^±

# á â ã Btranscription factor^Cä ^H ^Btarget gene^C) ^p ^BFisher et al, 1998^C^DUV^Ì ^±# MAP kinase^Û ^Ü ^ß ^à ^å ^[ ^# ^á ^â ^ã AP1^æ MMP^Ø ^N ^ç ^Ä ^H ^è ^$ ^ë ^a ^é ^v ^w ⁸ ⁹ ^Ø ^N ^ç ^Ä ^å ^[ ^ê ^$M ^N

H IBextracellular matrix^Cµ ^¶ ^BFisher et al., 1996; 2000^C^DUVA ^ì ^í

" # î ï ° r ³ ´ MMP1^Ø ^N ^H ^IBextracellular matrix^Ccollagen^® ^? mPCK^,MAP kinase^,AP1 ^á ^â ^{ã B}transcription factors^{Cß à} ⁾ ^{p B}Watanabe et al., 2004^C^Dß ^à ^_ ^ð ^Àmigration inhibitory factor (MIF)transforming growth factor-β^BTGF-β^Cuñ ^m] ³ ^ms ^Qt UVA^Ì ^u³ ^´ MMP

Á p b @ò ó BWatanabe et al., 2004^ôYin et al., 2003^C^DTGF-β^Fe ^fõ ^ö

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÷ TÑ k l Ú Ä Bcytokine^Cø ^ù ^ú ^û ^³ ^J ^½M ^# ^,¾ ^¿ ^V ^W Btissue remodeling^C¾ ^¿ ^ü ^U ^7k ^{l B}Massague,1998^C^DTGF-β^y ^m TGF-β type II receptor^BTbRII^Cµ ^K ^æ ^mTbRI^{ ^Lý ^K ^þ ^å ^[ # á â ã Smad2

½Smad3^æ ^mSmad4^µ ^K ^á ^² ^¸ ^ä ^H ^è ^$Type I i j g h H è $FTGF-β^uú ^N ^{^_} ^e ^fH ^BGhosh et al., 2000; Chen et al., 2000; Akagi et al., 1996; Roberts et al., 1990; Slack et al., 1993^C^ôT TbRII

{ TGF-β^Fõ ^mTbRI BWrana et al., 1992^C^DUV^Ö ^Ì z w L TbRII ^g ^h ^I mRNA ^è ^$ ^BQuan et al., 200^ôGambichler et al., 2007^C8 ⁹ F r Smad3 mRNA ^è ^$BKreuter et al., 200ô Gambichler et al., 2007^CD æ ´ TGF-β1 ^| ^À³ ^´ TbRII^a MMP1 è $Fr UV^Ö ^Ì ³ ´ TGF-β1 ^ê ^Û ^Ü ^Ý ^Þ ^È ^ú ù Á BYin et al., 2 0 0 2 ^C^DUVA Õ X = Ô Õ z ³ ´ TGF-β^; ^Å z w L ² Û TGF-β^{m^å} TGF-β type II receptor^BTbRII^C

µ K z BMori et al., 1998^C^{D^} ^Ò ^Ó ^! ^; ^" ^# ^µ ^¶ ^$

´ TGF-β^$% MMP1 ^è $& ' Fª î Smad3 Smad4 ^u ì AP1^ß ^BYuan & Varga, 2004^C^D TGF-β1 ^; ⁼ ^$melastin^è ^$) ^p BGibson et al., 1995^CDTGF-β1 type II receptor ⁼ ^> ^@Fm Cysteine-rich 61 BCYR61^{C) p}CYR61^F() ^mú ^û ⁱ ^j ^g ^h ^* ⁺ ⁾ ^p ^g ^h ^IM ^{ã B}Lau &

Lam, 1999^C^D , FE < " # FA Ì " # z ) ¬n CYR61 H è $- . [ TGF-β1 type II receptor ^ú ^û ^$% ^Bdown regulation^C

@ TGF-β1 type II receptor ^m TGF-β1 ^Û ^Ü ^Ý ^Þ ⁾ ^/ ^$p ⁰ ^; ^z ^m extracellular matrixⁱ ^j ^g ^h ⁾ ^{p B}Quan et al., 2006^CD1 " # mCYR61

p 0 ; = > @FmMAP kinase^ß ^à ^±# AP1⁾ ^{p B}Han et al., 2002ô3 4 5 6 et al., 2003^ôQuan et al., 2006^C^A ^Ì ^" ^# ^$% ^z ^w ^LMAP kinase^ß ^à ^±#

AP1^±# ^$% ^BFisher et al., 199ô1998^C^D

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Macrophage migration inhibitory factor (MIF)^j 7 r 8 9 u ê e f lymphokine^$% ^: ^; ^ù ⁾ ^n« ^±# ^{; =} ^> ^@m
u ^< ⁼ ) Á p BBloom and Bennett, 1966^ôDavid, 1966^CD >; = $z

8 fÚ Ä BShimizu et al., 1966^C ^TUVA^Ì ^z ⁼ ^Ù ^Ú ^r ^s ^Q t ê >MIF^| ^À# MAP kinase ^³ ^´ AP1^mDNA^µ ^K ^$

% ³ ´ MMP1^³ ^{J ³} ^J ^$% ^| ^À= MIF^Â ^þ ^? PKC^& ^' ^@ ^uA ^B BWatanabe et al., 2004^C^D

UVB ^Ö ^Ì C | l § # $% m D fç Ä ú û ) p &

orinithine decarboxylase^BODC^Ccyclooxygenase^BCOX^C^BAuvinen et al., 1992;

Herschman et a; 1995^C^D , Fþ ² þ E F Ô Õ UVB^Ì ^z ^³ ^´ ODC Ê J BRosen, et al., 1990^ôCôTG" H ÀTNFα^Ù ^Ú ODC^±F ^r COX^Ê ^J ^BVerma et al., 1977^ôYamamoto et al., 1992^C^æ ^T ^þ ^S I Á

BArnold et al., 1992^C^DSoriani^{7 B}1999^CÀ ^@ ^a UVB^Ì

] I s Qt ; J $COX-2^H ^è ^$³ ^´ODC H è $S QR : DCOX ^M ^N ^K ^J ^¥ ^L ^Barachidonic acid^C@! ^M ^Ä BprostaglandinsPGs^C^{^_} PGE2 = Ô Õ Ts Qt X | À ì Ñ fMMP

è $BPentland et al., 1995; Wahl and Lampel, 1987; Mauviel et al., 1994^C^DN

PGE2^; ^Fe ^f® ^? ⁱ ^j ^g ^h ^K ^L& ' Ã IBVarga et al., 1987^ô Clark et al., 1982^C^DPGE2⁼ ^> ^@FT" ^# ^î ^ï ^_ ^ì MMPs^±¸ ^H Ig h IM N X Y Z O fV \ ] 3 D1 ? P Q COX2& ' @ B¹NS-398^C S | À) R & ' COX2^,PGE2MMP1^g ^h ^Iè ^$BHan et al., 2004^C^DCOX mRNA^è ^$F ^r transcription factor NFκB^uS ^{' ^±ð} IkB

# ú S D8 T ß à U V W ¹ &

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Cytoplasm

Nucleus

NF-kB COX-2 iNOS

COX-2

PGE₂

INOS NO ONOO^-

O2.-

LBP

LPS CD14 LPS

IKK

P P

IB P50 P65

TNF-,IL-1,IL-6

LPS M D -2 T LR 4

UV^Ö ^Ì TX Y Z Ã [ Å ù ) ±# NFκB^{^uú} ^û ^H ^BBender et al., 1997; Herrlich et al., 1997^C^D MAPKsNFκB^ß ^à ^ñ ^m\ IL-1β^$ MMP1^è ^$ú ^{û B}Fan et al., 2006^C^Ds ^Qt ^TUVA^FUVB^Ö ^Ì z r NFκB^ú ^û IL-6^H ^ë ^a IL-6^Ô ^Õ IL-6^z MMP1

è $ BKock et al, 1990^ôWlaschek et al, 1994^ôKondo et al, 1997;

Scharffetter-Kochanek et al, 1997^ôBrenneisen et al, 1999^ôWenk et al., 2004^C^DN

IL-6^ Ä B¹IL-8^,TNFα^CFJ ^¤ ^{ã B}CSF^,PDGF^C

F IL-1α^ú ^û ^s ^Qt ^J ^Ê ^H ^IBextracellular matrix^C

] å E < k l BElisa & Lentz, 1990^ôShroder et al., 1990^ô Last-Barney et al., 1988^ôZucali et al., 1986^ôRaines et al., 1989^C^D) ^ F&UV Ö Ì í Fª î IL-1α autocrine/paracrine _ ` É ±# NFκB BBender et al., 1998^C^DMonocyte chemoattractant proyein-1^BMCP-1^{C æ} ^Fh a b ±# Å u e fm C ) p Ú Ä MCP-1 ^c ⁼ ^Ô ^Õ ^T ^d ^Ù ^Ú

z r s Qt u BYoshimara et al., 1989^C^DTë H e ¡ ¾ ¿

$ MCP-1 ^z ^Ù ^Ú ⁱ ^j ^g ^h ^H ^è ^{$ 8} ⁾ ^{$ %} ^S ^F] ^å ^r ^f ^X ^ú ^g

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Bup-regulation^CTGF-β^H ^è ^{$uh B}Gharaee-Kermaniet al., 1996^C^DMCP-1 Ts Qt _ i = Ô Õ z IL-1α^K ^j ^³ ^´ MMP1^H ^è ^{$ $%}

= > @Fe fautocrine loop^BYamamoto et al., 2000^C^D

Ò Ó k l m n $7 Ò Ó o y ! u $&Ø } p qr ê Ã ù ) & ' TPA B@ PKC ^±# ^Ã ^IC UVB MMP1 ^è ^$k ^R ^BShih & Cherng, 2008NSC 91-2626-B-041-001^C ^e s Ò Ó Ø } p qr ê Ã & ' MMP M ã ß à m ) R Ø } p qr ê LM t % # u v X d | À

w A Ê v Dk l m x y Ø } p q r ê Ã Fz $%

transforming growth factor-β^BTGF-β^Cmigration inhibitory factor^BMIF^C) ^u

^_ ð À! Ï { \ | } ~ @r UVA^u ^ì ½ ´ TGF-β

$% MMP1 ^Á TGF-β type II receptor^BTbRII^CmRNA^è ^$

; e j ´ Àx y D® ? UVA^z ^r PCK^,MAP kinase^,AP1 transcription factors Bc-Fos & c-Jun^CMMP1ⁱ ^j ^g ^h ^è ^$ ^Ø ^} ^p ^qr ^ê ^Ã ^F z | ) R ß à & ' MMP1 ^k R ; m (j T Ò Ó _ x y Dr % PGE2^® ^? ^z ^r NFκB ^ß ^à ^ª ^î COX-2MMP1^±
H ^Ig ^h IBextracellular matrix^Cv ^w ^Ú ^Ä ^B¹ IL-1^,IL-6^,TNFα^7Cz T X å NFκB^± ^Ø ^} ^p ^qr ^ê ^Ã ^Fz ^$% NFκB^ß ^à ù ) Á m F1 e ) x y ä D

1. TGF-β^a ^| ^À? ELISA kits^É " D

2.1 TbRII mRNA 217bp (RT-PCR) see ref: TRbII primer for PCR condition 2.1.1 Sense primer: 5’TCA TGG CAA ACT GTC TCT AGT GTT A3’

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2.1.2 Antisense primer: 5’CGG TTA ATA ACG ACA TGA TAG TCA C3’

2.2 B-actin mRNA 295 bp

2.2.1 Senese Primer : 5’ TCA CCC ACA CTG TGC CCA TCT A3’

2.2.2 Antisense primer: 5’CAG CGG AAC CGC TCA TTG CCA A3’

2.2.3 3. CYR61 mRN

3.1 Senese Primer: 5’ TCA AAG ACC TGT GGA ACT GGT ATC-3’

3.2 Antisense primer: 5’-CAC AAA TCC GGG TTT CTT TCA-3’

4.1 c-FosmRNA^B332bp^Cè ^$—RT-PCR

Sense primer: 5’-GGA GAA TCC GAA GGA AAG G-3’

Antisense primer: 5’-5’GCT TGG GCT CAG GGT CAT TG-3’

4.2 c-JunmRNA^B196bp^Cè ^$—RT-PCR

Sense primer: 5’-GGA TCA AGG CGG AGA GGA AG-3’

Antisense primer: 5’-GCG TTA GCA TGA GTT GGA AC-3’

PCR conditions are 30 cycles of 94 for 50 seconds55 for 50 seconds and 72 for 90 seconds followed by a 7-mintue extension at 72.

5. MCP-1 ^o ^è ^$| ^À? ELISA kits^É ^" ^D

TGF-β^Fe ^fõ ^ö ^÷ ^TÑ ^k ^l ^Ú ^{Ä B}cytokine^C^{ø ù} ^ú ^û ^³ J ½M # ,¾ ¿ V W Btissue remodeling^C¾ ^¿ ^ü ^U ^7k ^l ^BMassague,1998^C^D

f UV^£¤ $% TGF-β^# ^õ ^e ⁱ ^s ^Qt ^x ^y ^ê

>FTGF-β^F ^TTbRII receptoruÀd 8 9 £

^RNA^è ^$J ⁾ UVB^ù ⁾ & ' Ø p S | À×

UVB TbRII RNA TGF-β TGF-β type II receptor

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BTbRII^{Cµ K} ^r ^e | Àú û Type I ⁱ ^j ^g ^h ^H ^è ^$BGhosh et al., 2000; Chen et al., 2000; Akagi et al., 1996; Roberts et al., 1990; Slack et al., 1993^Celastin^è ^$) ^p ^BGibson et al., 1995^C^D

Cysteine-rich 61^BCYR61^CF() ^mú ^û ⁱ ^j ^g ^h ^* ⁺ ⁾ ^p ^g ^h ^IM ^ã BLau & Lam., 1999^C^{D ,} ^FE ^< ^" ^# ^FA ^Ì ^" ^# ^z ⁾ ^¬n

CYR61^H ^è $- . [ TGF-β1 type II receptor ^ú ^û ^$% ^Bdown

regulation^C^{Õ F} ^µ ^¶ c Ô Õ fUV^£ù ⁾ f X ú û CYR61^H

FUVA^ú ^û ^{^} 2^fUV^£¬ ^DØ ^p ^| ^Àm ^î ^o ^è ^$

CYR61^H ^& ' D K X Ø p TÂ UV^" ^# ^$% ^| ^l ^F@ ^{^} _ e ) ¦ á k R D

Monocyte chemoattractant proyein-1^BMCP-1^{C æ} ^Fh â ^b ^±# ^Å û ê ^f m C ) p Ú Ä MCP-1^c ⁼ ^Ô ^Õ ^T ^d ^Ù ^Ú ^z ^r ^s ^Qt

u BYoshimara et al., 1989^C^DMCP-1^Ê ^a ^TUVA^±UVB⁸ ^b ë Ê J Å ] e æ IL-6^TUV⁸ ^J ⁾ UVB^ù ⁾ ^Ù ^Ú UVA TÕ F a ] ù : # DØ } p qBØ p C$% , FUVA^FUVB

ì MCP-1^ù ⁾ ^& ^' ^F$% IL-6 ^F´ ^nÏ ^Ù ^Ú # FmØ p mú û < = k R ) p µ ¶ F K DMCP-1^Ts Qt _ = Ô Õ z IL-1α^K ^j ^³ ^´ MMP1^H ^è ^{$ $%} ⁼ ^> ^@Fe fautocrine loop^BYamamoto et al., 2000^C^D@Ø ^p ^ù ^& ^' IL-6 ^uÀ y ! 7 Õ F FÔ Õ Ø p ù ) & ' MMP-1 ^| ^l ^J ^F& ^' ^T$% MCP-1

MMP-1^è ^$X ^D

1. TGF-β

, FÀUVUVUVC^Ì ^ù ⁾ ^s ^Qt TGF-β

B¹ è uU CD

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2. TNF-α

, FÀUVUVUVC^Ì ^ù ⁾ ^s ^Qt TNF−α

B¹ è uU n=3^C^D

3. MCP-1

À UV^Ì ^µ ^¶ ^$UVB^TÌ 4^GÅ MCP-1 ^a ^m

Ì ¾ ) b ë ¡ ¢ P Q Ì Å ] d ä £ ¤ ¥ v Å ] Dd s Qt ´ ) ¦ } p qB¦ p C0.40.2mg/ml^@ ^a ^Å UVB^Ì 4^G Å MCP-1 ^a ^§ ^¨ ^© ^ë ^ª ^æ ^« ^F´ ⁾ ^QJ ^Ä EC^; ^| À¨ © & ' MCP-1 ^{F^&} ^' ^µ ^¶ ^{© æ} ^¹¦ ^p ^R ^¶ ^D ^Ø ^} ^p qBcgf^CT ^! ^k ^l ^_ ⁿ ^$MMP^±mRNA^è ^$) ^& ^' cgf

; T = E ^k R µ ¶ $cgf^B1 mg/ml^F2 mg/ml^C$MCP-1^& ^' k R ¬ F QJ Ä EC^¬ ^Bµ ^¶ ^® ^W n=6^C^D

UVB 4h

blank UVB 0.4 0.2 vit C vit E 2 1 0

25 50 75 100 125 150

--- UVB ---

MCP-1 % of blank

À UV^Ì ^µ ^¶ ^$UVA^TÌ 8^GÅMCP-1 ^a ^m

Ì ¾ ) b ë ¡ ¢ P Q Ì Å ] d ä £ ¤ ¥ v Å ] Dr

$ £¤ $MCP-1 ^Ù ^Ú ^F ^D

d s Qt ´ ) ¦ } p qB¦ p C0.40.2mg/ml^@ ^a ^ÅUVA

Ì 8 ^GÅ MCP-1 ^a ^§ ^¨ ^© ^ë ^ª ^æ ^« ^F´ ⁾ ^QJ ^Ä E

C^; ^| ^À¨ ^© ^& ^' MCP-1 ^{F^&} ^' ^µ ^¶ ^© ^æ ^¹¦ ^p ^R ^¶ ^D Ø } p qBcgf^CT ^! ^k ^l ^_ ⁿ ^$MMP^±mRNA^è ^$) ^& ^'

cgf^; ^T ⁼ ^E ^{^k} ^R ^µ ^¶ ^$cgf 1 mg/ml^$MCP-1^& ^' ^k ^R ^mQ J Ä E C ^T& ^' UVA^$ MPC-1 ^Ù ^Ú ^S ^R ^¶ ^¬Á ^d cgf 2 mg/ml^$MCP-1^& ^' ^k ^R ^S ^¬QJ ^Ä EC ^\ ^{n9 Bµ} ^¶ ^® ^W n=6^C^D

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UVA 8h

blank UVA 0.4 0.2 vit C vit E 2 1 0

25 50 75 100 125

--- UVA ---

MCP-1 % of blank

4. IL-6

£¤ UVAUVB^$s ^Qt IL-6^Å ^] ^p ⁰ ^¹ ^W ^uU ^D

UVA^$IL-6 ^¯ ^m° ⁾ ^Ì û IL-6â ê UVA

õ ± ² é $IL-6^E ^{F Dæ}UVB^S ^TÌ 12^GÅ ⁾ ^¨ ^© ^³ ^´ IL-6

a QR 24^GÅ ^D IL-6^Õ ^F ^³ ^P ^Q ^Ì 24^GÅ ^¤ ^¥ ^Dd s Qt ´ ) ¦ } p qB¦ p C0.40.2mg/ml^@ ^a ^Å UVB^Ì 24^GÅ IL-6 ^a ^§ ^¨ ^© ^{æ
«} ^F´ ⁾ ^QJ ^Ä EC^S ^& ^' IL-6

· ¨ © D Ø } p qBØ p C$IL-6^S ⁾ ^Ù ^Ú ^Bµ ^¶ ^® ^W ^C^D

UVB 24h

blank UVB 2 1 0.4 0.2 vit C vit E 0

50 100 150 200 250 300

***

###

#

IL 6 level % of blank

(12)

mRNA data

s Qt ÀUVAUVB^Ì 24^GÅ ^¤ ^¥ ^´ ^É ^µ ^¡ RNA ÀPCR^á ^LDNA ^é ^Õ ^F ^D

figure of gel density

5. TbRII RNA

UVB^$TbR II mRNA ⁾ ^¶ ^· ^¨ ^© ^& ^' ^¬¸ ^@ ^a ^Ø ^p ^Bcgf^C

¹ ) & ' ¦ p Blipo^C½QJ ^Ä C ^ù ^º ^Â UVB^& ^' TbR II mRNA

k R D

TbRII

basal UVA UVB UVC

05 1015 2025 3035 4045 5055 6065 7075 8085 9095 100105

relative TbRII mRNA/β-Actin (to control %)

TbRII

basal UVB 3 2 1 0.4 0.2 0.1 vit C 0

25 50 75 100 125

relative TbRII mRNA

/β-Actin (to control %)

(13)

6. CYR61 mRNA

, FUV, F » $ »¼ ½ ¾ ½ ¿ ) Ù Ú F »

Ù Ú ¬nDØ p BÀÁ Â C,¦ p BÃ Ä Å Æ C,QJ Ä » ) CYR61 mRNA

CYR 61

basal UVB 3 2 1 0.4 0.2 0.1 vit C 0

25 50 75 100 125

relativeCYR 61 mRNA/β-Actin (to control %)

á â ã è $&

7. c-FOS mRNA

á â ã AP-1^Fc-Fos^½c-Jun^¾ ^KUVC^$c-Fos ^m$c-Jun FÁ Ç DØ p BÀÁ Â C,¦ p BÃ Ä Å Æ C,QJ Ä » # ; F¥ _ T ÀÈÉ Æ

Ê ÀÈË 4 5 D« m »¼ ½ ¾ ½ ¿ ¬# »¼ ½ ¾ ½ ¿ è $F ÀÈÉ Æ

¾ ½ ¿ Ê ÀÈË 4 5 D

FOS

basal UVB 3 2 1 0.4 0.2 0.1 vit C 0

10 20 30 40 50 60 70 80 90 100 110 120

relative c-Fos mRNA/β-Actin (to control %)

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8. c-Jun

JUN

basal UVB 3 2 1 0.4 0.2 0.1 vit C 0

10 20 30 40 50 60 70 80 90 100 110 120 130 140

relative c-Jun mRNA/β-Actin (to control %)

ñ Ì Í Î

Akagi Y, Isaka Y, Arai M, Kaneko T, Takenaka M, Moriyama T, Kaneda Y, Ando A, Orita Y, Kamada T, Ueda N, Imai E: Inhibition of TGF-β1 expression by antisense oligonucleotides suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 1996, 50:148–155

Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J (1999) Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J. Biol. Chem.

274:15345-15349

Bloom, B. R., and Bennett, B. (1966) Science 153, 80–82

Chen S-J, Yuan W, Lo S, Trojanowska M, Varga J: Interaction of Smad3 with a proximal Smad-binding element of the human 12(I) procollagen gene promoter required for transcriptional activation of TGF-β. J Cell Physiology 2000, 183:381–392

David, J. R. (1966) Proc. Natl. Acad. Sci. U. S. A. 56, 72–77

Elisa JA and Lentz V (1990) IL-1 and TNF synergistically stimulate fibroblast IL-6 production and stabolize IL-6 messenger RNA. J. Immunol. 145: 161-166

Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, Voorhees JJ: Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature 1996, 379:335–339

Fisher GJ,Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ: Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J M ed 337:1419-1428,

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1997

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