Professional Documents
Culture Documents
Tiwari 2014
Tiwari 2014
14-14934
7DUJHWLQJWKHILEURQHFWLQW\SH,,,UHSHDWVLQ7HQDVFLQ&LQKLELWVHSLWKHOLDOPHVHQFK\PDO
WUDQVLWLRQLQWKHFRQWH[WRI3RVWHULRU&DSVXODU2SDFLILFDWLRQ
$QLO7LZDUL-DJDW5DPDQG0DQQL/XWKUD*XSWDVDUPD
)URPWKH'HSDUWPHQWVRI,PPXQRSDWKRORJ\DQG2SKWKDOPRORJ\
3RVWJUDGXDWH,QVWLWXWHRI0HGLFDO(GXFDWLRQDQG5HVHDUFK&KDQGLJDUK
5XQQLQJWLWOH7DUJHWLQJILEURWLFSDWKRORJLHV
7R ZKRP FRUUHVSRQGHQFH VKRXOG EH DGGUHVVHG 0DQQL /XWKUD*XSWDVDUPD 'HSDUWPHQW RI
,PPXQRSDWKRORJ\ 5HVHDUFK %ORFN $ 3RVWJUDGXDWH ,QVWLWXWH RI 0HGLFDO (GXFDWLRQ DQG
5HVHDUFK6HFWRU&KDQGLJDUKTel : 91-172-2755196; Fax : 91-172- 2744401;Email
: guptasarma.manni@pgimer.edu.in; mguptasarma@yahoo.com
.H\ZRUGV VF)Y ILEURVLV WHQDVFLQ ILEURQHFWLQOLNH UHSHDWV H[WUDFHOOXODU PDWUL[ SRVWHULRU FDSVXODU
RSDFLILFDWLRQ
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
$%675$&7
H[WUDFDSVXODUVXUJHU\DVVRFLDWHGZLWKILEURVLVRSDFLILFDWLRQDQGFRQWUDFWLRQRIWKHSRVWHULRUOHQV
FDSVXOH ,W LV FKDUDFWHUL]HG E\ LQFUHDVHG H[SUHVVLRQ RI H[WUDFHOOXODU PDWUL[ SURWHLQV VXFK DV
7HQDVFLQ& ILEURQHFWLQ FROODJHQV DQG SURWHRJO\FDQV 7HQDVFLQ& LV NQRZQ WR EH FULWLFDO IRU
LQMXU\LQGXFHG HSLWKHOLDOPHVHQFK\PDO WUDQVLWLRQ (07 LQ WKH OHQV HSLWKHOLXP :H DLPHG WR
FKDLQYDULDEOHIUDJPHQW DQWLERG\DQGHYDOXDWHLWVHIIHFWLYHQHVVLQWKHFRQWH[WRIOHQVHSLWKHOLDO
FHOOV
0HWKRGV 3KDJH GLVSOD\ OLEUDU\ VFUHHQLQJ ZDV XVHG WR JHQHUDWH DQ DQWLERG\ DJDLQVW WKH
71IQ,,,/HQVHSLWKHOLDOFHOOFXOWXUHVZHUHFXOWXUHGLQWKHSUHVHQFHRIWKHVF)YDQWLERGLHVWR
HYDOXDWH WKH HIIHFWV RQ FHOO SUROLIHUDWLRQ PLJUDWLRQ ILEURQHFWLQ SRO\PHUL]DWLRQ DQG GHSRVLWLRQ
PDUNHUV7KHHIIHFWRQ60$'GHSHQGHQWDQG60$'LQGHSHQGHQWSDWKZD\VZDVDOVRH[DPLQHG
5HVXOWV 7KH VF)Y 71 ZDV IRXQG WR EH HIIHFWLYH LQ UHJXODWLQJ WKH SUROLIHUDWLRQ PLJUDWLRQ
H[SUHVVLRQ RI 003 DQG 003 ILEURQHFWLQ SRO\PHUL]DWLRQ DQG GHSRVLWLRQ DV ZHOO DV
H[SUHVVLRQ RI (07 PDUNHUV 71 GLG QRW LQWHUIHUH ZLWK 60$' SKRVSKRU\ODWLRQ $OWHUHG
ORFDOL]DWLRQRIȕFDWHQLQDVZHOODVGRZQUHJXODWLRQRISKRVSKRU\ODWLRQ0$3NLQDVHVDQGIRFDO
DGKHVLRQNLQDVHZDVLQYROYHG
&RQFOXVLRQV 2XU GDWD VXJJHVWV WKDW WKH 71IQ,,, UHSHDWV SOD\ DQ LPSRUWDQW UROH LQ 3&2
SDWKRORJ\ 7KH LQKLELWLRQ RI (07 E\ 71 LV PHGLDWHG E\ 60$'LQGHSHQGHQW LQWHJULQȕ
2
FDWHQLQ)$. VLJQDOLQJ SDWKZD\ DQG LV WKHUHIRUH SURSRVHG DV D QRYHO DQWLILEURWLF WKHUDSHXWLF
FDQGLGDWH
,QWURGXFWLRQ
([WUDFDSVXODU FDWDUDFW VXUJHU\ LQYROYHV UHPRYDO RI WKH OHQV DQWHULRU FDSVXOH WR FUHDWH D
FDSVXODU EDJ IRUPHG E\ WKH UHPDLQLQJ DQWHULRU FDSVXOH DQG WKH HQWLUH SRVWHULRU FDSVXOH
IROORZHGE\SKDFRHPXOVLILFDWLRQRIWKHFDWDUDFWRXVOHQVDQGLPSODQWDWLRQRIDQLQWUDRFXODUOHQV
XQGHUJRLQJ D ZRXQG KHDOLQJ UHVSRQVH DLGHG E\ WKH SUHVHQFH RI F\WRNLQHV VXFK DV 7*)ȕ
SUHVHQW LQ KLJK FRQFHQWUDWLRQ LQ WKH DTXHRXV KXPRU HLWKHU OHDGLQJ WR IRUPDWLRQ RI
P\RILEUREODVWVRUE\GLIIHUHQWLDWLQJLQWROHQVILEHUFHOOVLQDQDWWHPSWWRUHJHQHUDWHWKHGDPDJHG
OHQV :KLOH WKH IRUPHU UHVSRQVH OHDGV WR WKH SUROLIHUDWLRQ PLJUDWLRQ DQG HSLWKHOLDO
XQGHUJR DQ DEHUUDQW RUJDQL]DWLRQ DQG VR WRJHWKHU WKH HIIHFWV RI (07 DQG ILEHU FHOO
GLIIHUHQWLDWLRQ UHVXOW LQ ORVV RI YLVXDO DFXLW\ DQG RSDFLILFDWLRQ RI WKH SRVWHULRU FDSVXOH DQGRU
3RVWHULRU FDSVXODU RSDFLILFDWLRQ 3&2 LV WKH PRVW FRPPRQ SUREOHP SRVWFDWDUDFW
VXUJHU\ $OWKRXJK LPSURYHPHQWV LQ LQWUDRFXODU OHQV PDWHULDO DQG VXUJLFDO WHFKQLTXHV KDYH
UHGXFHGWKHLQFLGHQFHRI3&2LWVWLOOUHPDLQVDVLJQLILFDQWSUREOHP7KHLQFLGHQFHUDWHRI3&2
LV DSSUR[LPDWHO\ LQ DGXOWV ZKLOH LQ FKLOGUHQ WKH UDWHV DUH KLJKHU &XUUHQWO\ WKH RQO\
HIIHFWLYH WUHDWPHQW LV QHRG\PLXP<$*FDSVXORWRP\ KRZHYHU LW LV DVVRFLDWHG ZLWK VHYHUDO
FRPSOLFDWLRQV OLNH UHWLQDO GHWDFKPHQW FRUQHDO HGHPD HWF DQG WKH SURFHGXUH LV QRW DOZD\V
3
DYDLODEOH LQ GHYHORSLQJ FRXQWULHV 7KHUHIRUH RWKHU PHWKRGV WR LQKLELW 3&2 VXFK DV
LPSURYHPHQWV LQ LQWUDRFXODU GHVLJQ DQG PDWHULDOV GHVLJQ RI EHWWHU SKDUPDFRORJLFDO
LQWHUYHQWLRQVDVZHOODVLPSURYHPHQWVLQVXUJLFDOSURFHGXUHVDUHEHLQJH[SORUHGLQDQHIIRUW
WR UHGXFH WKH LQFLGHQFH RI 3&2 7KH ODWWHU LQYROYHV XVH RI UHDJHQWV FDSDEOH RI VHOHFWLYHO\
GHSOHWLQJWKHUHVLGXDOOHQVHSLWKHOLDOFHOOV
5HPRGHOLQJ RI WKH H[WUDFHOOXODU PDWUL[ LV DQ LPSRUWDQW HYHQW LQ 3&2 SDWKRORJ\
,QFUHDVHGH[SUHVVLRQRI(&0SURWHLQVVXFKDVILEURQHFWLQWHQDVFLQDQGW\SH,FROODJHQKDVEHHQ
GRFXPHQWHGLQ/(&VSRVWHSLWKHOLDOPHVHQFK\PDOWUDQVLWLRQ ORVVRI7HQDVFLQ&KDVEHHQ
LQIDFWVKRZQWRDWWHQXDWH(07LQWKHOHQVHSLWKHOLXPSRVWLQMXU\)LEURQHFWLQDQG7HQDVFLQ&
DUH LPSRUWDQW (&0 SURWHLQV ZKLFK LQWHUDFW ZLWK HDFK RWKHU WKH W\SH ,,, PRGXOHV ± RQ
7HQDVFLQDUHNQRZQWRELQGWRDFU\SWLFVLWHLQWKH1WHUPLQDON'DGRPDLQRIILEURQHFWLQ
%RWK SURWHLQV PRGXODWHG WKH (&0 E\ LQWHUDFWLQJ ZLWK RWKHU (&0 SURWHLQV LQFOXGLQJ FROODJHQ
DQGRWKHUKHSDUDQVXOIDWHSURWHRJO\FDQV)LEURQHFWLQDQG7HQDVFLQ&DUHFRPSRVHGRIPXOWLSOH
UHSHDWLQJ GRPDLQV RI ILEURQHFWLQ W\SH ,,, UHSHDWV 71IQ,,, PDGH XS RI a DPLQR DFLGV ,W LV
QRZZHOOXQGHUVWRRGWKDWSURWHLQVZLWKVXFKUHSHDWVELQGWRDFWLYDWHGLQWHJULQVRQFHOOVXUIDFHV
OHDGLQJ WR FHOO VLJQDOLQJ HYHQWV WKHUHE\ LQIOXHQFLQJ WKH EHKDYLRU RI FHOOV ZLWK UHVSHFW WR FHOO
DGKHVLRQGLIIHUHQWLDWLRQJURZWKDQGPLJUDWLRQDQGFHOOPDWUL[FRPPXQLFDWLRQ
3UHYLRXV VWXGLHV KDYH VKRZQ WKDW WKH 71IQ,,, GRPDLQ DQG LWV VSOLFHG YDULDQW DUH RYHU
H[SUHVVHG XQGHU PDQ\ SDWKRORJLFDO FRQGLWLRQV )XUWKHU JLYHQ WKH IDFWV WKDW L WKHUH LV D
WKHUHLVLQFUHDVHGH[SUHVVLRQRI71&SRVW(07LQKXPDQ3&2ZHH[SORUHGWDUJHWLQJRIWKH
71IQ,,,GRPDLQXVLQJDQVF)YDQWLERG\WRDVVHVVWKHFRQWULEXWLRQRIWKLVGRPDLQLQFDXVDWLRQRI
WKH3&2SDWKRORJ\
4
+/(% FHOO OLQH DQG SULPDU\ FXOWXUH RI WKH OHQV HSLWKHOLDO FHOOV IURP H[SODQWV RI DQWHULRU
FDSVXOHV RI FDWDUDFW SDWLHQWV ZHUH XVHG WR VKRZ WKDW 71IQ,,, SOD\V DQLPSRUWDQWUROH EORFNLQJ
WKHGRPDLQZLWKDQVF)YDQWLERG\UHVXOWHGLQFRQWUROOLQJWKHNH\SURFHVVHVRI3&2,QDGGLWLRQ
FDQGLGDWHVLJQDOLQJSDWKZD\VDVVRFLDWHGZLWK7*)ȕLQGXFHG(07ZHUHHYDOXDWHG
0DWHULDODQG0HWKRGV
SDUWLFLSDQWVHQUROOHGLQDSURWRFRODSSURYHGE\WKH,QVWLWXWLRQDO(WKLFV&RPPLWWHHRI3*,0(5
WKHJXLGHOLQHVRIWKH'HFODUDWLRQRI+HOVLQNLZHUHREVHUYHG
Maintenance and culture of the cell lines: 7KH KXPDQ OHQV HSLWKHOLDO FHOO OLQH +/(% ZDV
)%67KHFHOOOLQHZDVXVHGHDFKWLPHDWWKHUGSDVVDJH
Culturing of anterior capsule explants derived from cataract patients: /HQV FDSVXOH RI WKH
FDWDUDFWSDWLHQWVZDVFROOHFWHGLQVWHULOHYLDOFRQWDLQLQJ0(0PHGLD&DSVXOHVZHUHDOORZHGWR
DWWDFKWRWKHZHOOSODWHSUHFRDWHGZLWKSRO\/O\VLQHLQWKHSUHVHQFHRIPLQLPDODPRXQWRI
FRPSOHWH PHGLD 0(0 )%6 IRU DERXW K IROORZHG E\ DGGLWLRQ RI FRPSOHWH PHGLD
DQGLQFXEDWHGDW&DQG&20HGLDZDVFKDQJHGDIWHUHYHU\GD\V&HOOVZHUHSDVVDJHG
DWFRQIOXHQF\DQGFHOOVZHUHXVHGDIWHUHYHU\UGSDVVDJH
LWV1WHUPLQXV7KHJHQHZDVDPSOLILHGXVLQJDVHWRISULPHUVZLWK6SK,VLWHLQFRUSRUDWHGLQWKH
IRUZDUGSULPHUDQG+LQG,,,VLWHLQWKHUHYHUVHSULPHU7KHDPSOLILHGJHQHZDVFORQHGLQWRYHFWRU
5
S4( IRU LQFRUSRUDWLQJ ; +LV WDJ DW LWV 1WHUPLQXV WR HDVH GRZQVWUHDP SXULILFDWLRQ 3&5
IRUZDUG¶*&***$7&*&$7*&7&$*$**7*7&7&&7&&&¶DQG
UHYHUVH¶&*&&$$*&77&7$$*7**$7*&&77&$&$7*7*&*¶
ZKLFKLQFOXGHWKH6SK,DQG+LQG,,,VLWHVUHVSHFWLYHO\7KH3&5FRQGLWLRQVXVHGIRUDPSOLILFDWLRQ
ZHUH DV IROORZV LQLWLDO GHQDWXUDWLRQ DW R& PLQ IROORZHG E\ F\FOHV RI GHQDWXUDWLRQ DW
R&PLQDQQHDOLQJDWR&PLQH[WHQVLRQDWR&PLQDQGDILQDOH[WHQVLRQDWR&IRU
OLJDWHGLQWRWKHFRUUHVSRQGLQJO\GLJHVWHGSODVPLGS4(YHFWRU)ROORZLQJWUDQVIRUPDWLRQLQWR
LQWR WKH YHFWRU ZDV GRQH E\ '1$ VHTXHQFLQJ ([SUHVVLRQ RI 71IQ,,, ZDV FDUULHG RXW E\
XQGHUQDWLYHFRQGLWLRQVE\1L17$FROXPQFKURPDWRJUDSK\
)URP WKH 71IQ,,, FRQVWUXFW WKH RWKHU YDULDQWV 71IQ,,, 71IQ,,, 71IQ,,, ZHUH
GHULYHGXVLQJDSSURSULDWHSULPHUVDOOWKHFRQVWUXFWVZHUHVLPLODUO\H[SUHVVHGDQGSXULILHG
Generation and purification of anti-TNfnIII 1-5 antibody: )LEURQHFWLQ W\SH ,,, OLNH UHSHDW RI
7HQDVFLQ& UHJLRQ 71IQ,,, ZDV XVHG DV DQ DQWLJHQ IRU VFUHHQLQJ WKH SKDJH GLVSOD\HG
DQWLERG\ OLEUDU\ 7RPOLQVRQ ,- 05& /RQGRQ E\ WKH XVXDO PHWKRGV RI ELRSDQQLQJ DQG
DPSOLILFDWLRQ7KUHHURXQGVRIELRSDQQLQJZHUHGRQHDJDLQVWLPPRELOL]HGDQWLJHQ71IQ,,,
(OXWHGSKDJHVDWWKHHQGRIWKHWKLUGURXQGZHUHWUDQVIRUPHGLQWRH[SRQHQWLDOO\JURZLQJ 2'
+% E.coli FHOOV WR SURGXFH VROXEOH VF)YV 7KH SKDJHPLG ZDV WUDQVIRUPHG LQWR E.coli
+% VWUDLQ D QRQVXSSUHVVRU VWUDLQ DQG WKH FHOOV ZHUH FXOWXUHG RYHUQLJKW DW & LQ WKH
6
SUHVHQFHRIJOXFRVHWRSURGXFHVROXEOHVF)YDQGSXULILFDWLRQRIWKHDQWLERG\ZDVFDUULHG
RXW DV GHVFULEHG HDUOLHU ,Q DOO H[SHULPHQWV VF)Y 2 JHQHUDWHG DJDLQVW SODWHOHW FHOO VXUIDFH
SURWHLQV ZDVXVHGDVDQLUUHOHYDQWQHJDWLYHFRQWUROVLQFHLWLVDVWUXFWXUDOO\PDWFKHGDQWLERG\WR
71
WKHVXSHUQDWDQWFRQWDLQLQJVROXEOHVF)YZDVXVHGDVSULPDU\DQWLERG\LQWKH(/,6$DVVD\$IWHU
ZDVKLQJ ZLWK ; 3%6 DQG LQFXEDWLRQ IRU K DW URRP WHPSHUDWXUH +53FRQMXJDWHG 3URWHLQ$
ZDVDGGHGWRWKHZHOOVDWDGLOXWLRQRIDQGLQFXEDWHGIRUK&RORUZDVGHYHORSHGZLWK
70% WKH UHDFWLRQ ZDV VWRSSHG ZLWK 1 VXOIXULF DFLG DQG WKH SODWHV ZHUH UHDG DW QP
6XSSOHPHQWDU\ )LJ $ 7KH VHOHFWHG VF)Y FORQH 71 ZDV IXUWKHU H[SUHVVHG DQG SXULILHG
XVLQJ1L17$DJDURVHFROXPQZHVWHUQEORWRI71XVLQJPRXVHDQWL+LV+53FRQILUPHGWKH
H[SUHVVLRQ RI WKH aN'D EDQG FRUUHVSRQGLQJ WR 71 H[SUHVVLRQ 6XSSOHPHQWDU\ )LJ %
:KHQ WKH YDULRXV 7HQDVFLQ YDULDQWV GHULYHG IURP 71IQ,,, ZHUH FKHFNHG IRU ELQGLQJ WR
71LWZDVREVHUYHGWKDWZKLOH71DQG71ERXQGWR71WKHUHZDVRQO\DYHU\ZHDN
71DJDLQVWPDWULFHOOXODUH[WUDFHOOXODUPDWUL[SURWHLQVILEURQHFWLQRVWHRSRQWLQDQGILEULQRJHQ
DOVR WHVWHG DORQJVLGH 7KH VF)Y 71 ZDV REVHUYHG WR ELQG WR 71IQ,,, DORQH DQG GLG QRW
ELQG WR DQ\ RI WKH RWKHU SURWHLQV WHVWHG 2 VKRZHG QR SRVLWLYLW\ ZLWK DQ\ SURWHLQ LQFOXGLQJ
71IQ,,, ,PPXQRIOXRUHVFHQFH VWDLQLQJ RI OHQV HSLWKHOLDO FHOOV JURZQ IURP SDWLHQW FDSVXOHV
VKRZHGWKDW71ZDVDEOHWRELQGWRWKHOHQVHSLWKHOLDOFHOOVFRORFDOL]LQJZLWKDQWLWHQDVFLQ&
DQWLERG\ 6LJPD &KHPLFDOV LQ DQRWKHU VXFK FDSVXOH 2 VKRZHG QR ELQGLQJ WR WKH OHQV
HSLWKHOLDOFHOOV 6XSSOHPHQWDU\)LJ
7
Western blotting:+/(%RUKXPDQOHQVHSLWKHOLXPSULPDU\FHOOVZHUHVHHGHGLQWKHSUHVHQFH
QJPO DQGLQFXEDWHGIRUK&HOOVZHUHZDVKHGZLWK3%6DQGFHOOO\VDWHZDVERLOHGLQ;
6'63$*(VDPSOHORDGLQJEXIIHUIROORZHGE\HOHFWURSKRUHVLVDQGZHVWHUQEORWWLQJ7KHVLJQDO
ZDVYLVXDOL]HGE\(&/UHJHQWDQGZDVFDSWXUHGRQ;5D\ILOPV
Immunofluorescence: +/(%FHOOVRUKXPDQOHQVHSLWKHOLXPSULPDU\FHOOVZHUHVHHGHGVLPLODU
WRWKHZHVWHUQEORWWLQJH[SHULPHQWV$WWKHHQGRIWKHSHULRGRIWUHDWPHQWZLWK7*)ȕDQGVF)Y
DQWLERGLHVFHOOV ZHUH IL[HG ZLWK SDUDIRUPDOGHK\GH SHUPHDELOL]HG XVLQJ WULWRQ;
DQGWZHHQDQGEORFNHGZLWK%6$LQ3%67KLVZDVIROORZHGE\LQFXEDWLRQZLWK
SULPDU\ DQWLERG\ DQG VHFRQGDU\ ),7&75,7&3(ODEHOHG DQWLERG\ IRU K HDFK ,PDJHV ZHUH
DFTXLUHGXVLQJ2O\PSXVFRQIRFDOPLFURVFRS\
ZHOO SODWH LQ FRPSOHWH PHGLD DQG LQFXEDWHGRYHUQLJKW &HOOVZHUH VHUXP VWDUYHG IRU K
IROORZHG E\ DGGLWLRQ RI WKH VF)Y DQWLERG\ LQ GRVHGHSHQGHQW PDQQHU DQG LQFXEDWHG IRU K
077 GLPHWK\OWKLD]RO\O GLSKHQ\OWHWUD]ROLXPEURPLGH DVVD\ ZDV FDUULHG RXW DV
GHVFULEHGHDUOLHU )RUSUROLIHUDWLRQ%UG8ZDVDGGHGDWDFRQFHQWUDWLRQRIX0SHUZHOOK
EHIRUHWKHHQGRIWKHLQFXEDWLRQIROORZLQJZKLFKPHGLDZDVGLVFDUGHGDQGWKHFHOOVZHUHIL[HG
XVLQJPHWKDQRO+IRUKGHQDWXUHGDQGSURFHVVHGDVGHVFULEHGHDUOLHU
FHOO PLJUDWLRQ ZRXQG KHDOLQJVFUDWFK DVVD\ ZDV SHUIRUPHG %ULHIO\ /(&V ZHUH FXOWXUHG WR
DFKLHYH QLQHW\ SHUFHQW FRQIOXHQF\ VHUXP VWDUYHG DQG D YHUWLFDO ZRXQG ZDV FUHDWHG XVLQJ D
8
QJPO ,PDJLQJ ZDV GRQH WR QRWH WKH VWDWXV DW K DQG K SHULRGV([WHQW RI PLJUDWLRQ ZDV
HYDOXDWHG E\ DQDO\]LQJ WKH LPDJHV XVLQJ WVFUDWFK VRIWZDUH 'DWD ZDV UHSUHVHQWHG DV SHUFHQW
003WKHFXOWXUHPHGLXPZDVFROOHFWHGDWWKHHQGRIWKHH[SHULPHQWVHWXSWRDVVD\IRUFHOO
PLJUDWLRQDQGH[SUHVVLRQRIWKH003ZDVHYDOXDWHGE\JHODWLQ]\PRJUDSK\XVLQJSUHYLRXVO\
GHVFULEHGSURWRFRO
DOC insolubility assay: 7KH H[WHQW RI GHSRVLWLRQ RI LQVROXEOH ILEURQHFWLQ LQ WKH (&0 ZDV
HYDOXDWHGE\SHUIRUPLQJ'2&LQVROXELOLW\DVVD\DVGHVFULEHGSUHYLRXVO\%ULHIO\[ FHOOV
ZHUHVHHGHGSHUZHOORIZHOOSODWHLQWKHSUHVHQFHRIFRPSOHWHPHGLDDQGLQFXEDWHGRYHUQLJKW
VHUXP VWDUYHG IRU K DQG WUHDWHG ZLWK WKH UHVSHFWLYH VF)Y DQWLERGLHV DORQJ ZLWK 7*)ȕ
QJPO DQG IXUWKHU LQFXEDWHG IRU K $W WKH HQG RI WKH LQFXEDWLRQ PHGLD ZDV UHPRYHG DQG
FHOOVZHUHO\VHGLQ'2&O\VLVEXIIHUDVSHUWKHGHVFULEHGSURWRFRO7KHEORWZDVGHYHORSHGE\
FKHPLOXPLQHVFHQFH %LR5DG DQG FDSWXUHG RQ ;UD\ ILOP ,PDJH- VRIWZDUH ZDV XVHG IRU
GHQVLWRPHWU\TXDQWLWDWLRQ7RHQVXUHHTXDOORDGLQJ'2&VROXEOHIUDFWLRQZDVVHSDUDWHGRQ
6'6 3$*( LQ VLPLODU IDVKLRQ DQG LPPXQREORWWHG ZLWK DQWL*$3'+ DQWLERG\ 6LJPD
&KHPLFDOV
FROODJHQW\SH,ȕLQWHJULQDQGĮ60$ZDVPHDVXUHGLQWKHSUHVHQFHDQGDEVHQFHRIWKHVF)Y
DQWLERGLHV WR HYDOXDWH WKH HIILFDF\ RI WKH DQWLERG\ )RU DVVHVVPHQW RI H[SUHVVLRQ RI Į60$
DQG1FDGKHULQOHQVHSLWKHOLDOFHOOVZHUHVHHGHGLQZHOOSODWHVRUFRYHUVOLSVDQGWKHVDPH
9
DQWL1FDGKHULQRUDQWLDOSKD60$DQWLERG\IROORZHGE\),7&FRQMXJDWHGVHFRQGDU\DQWLERG\
'$3,ZDVXVHGDVQXFOHDUG\H)OXRUHVFHQFHLQWHQVLW\ZDVTXDQWLILHGXVLQJ3HUNLQ(OPHU(/,6$
SODWH UHDGHU DQG '$3, ZDV XVHG WR HQVXUH HTXDO FHOO GHQVLW\ 'DWD ZDV SORWWHG DV SHUFHQW
IOXRUHVFHQFH LQWHQVLW\ )RU LPDJLQJ WKH FHOOV FRQIRFDO PLFURVFRS\ ZDV FDUULHG RXW XVLQJ WKH
2O\PSXV PLFURVFRSH )RU HYDOXDWLRQ RI OHYHOV RI FROODJHQ W\SH, DQG ȕ LQWHJULQ /(&V ZHUH
FXOWXUHGLQFRPSOHWH PHGLDIROORZHGE\VHUXPVWDUYDWLRQIRUWRK&HOOVZHUHWUHDWHGZLWK
DQG ZDVKHG ZLWK 3%6 )RU FROODJHQ W\SH, FHOOV ZHUH IL[HG SHUPHDELOL]HG EORFNHG DQG
LQFXEDWHG ZLWK PRXVH DQWLFROODJHQ W\SH, DQWLERG\ 6LJPD &KHPLFDOV )RU ȕ LQWHJULQ FHOOV
ZHUH LQFXEDWHG LQ EORFNLQJ EXIIHU IROORZHG E\ LQFXEDWLRQ ZLWKSULPDU\ PRXVH DQWLȕLQWHJULQ
DQWLERG\ 6LJPD &KHPLFDOV ),7& FRQMXJDWHG DQWLPRXVH DQWLERG\ ZDV XVHG DV VHFRQGDU\
DQWLERG\0HDQIOXRUHVFHQWLQWHQVLW\RIWKHFHOOVZDVDFTXLUHGRQ%')$&6&DQWRV\VWHP0),
RIWKHFRQWUROZDVWDNHQDV
DVVHVVHGE\LPPXQRIOXRUHVFHQFHDQGZHVWHUQEORWWLQJ,PPXQRIOXRUHVFHQFHZDVFDUULHGRXWDV
GHVFULEHG DERYH XVLQJ UDEELW DQWLȕFDWHQLQ DQWLERG\ 6LJPD &KHPLFDOV IROORZHG E\
PLFURVFRSH)RUZHVWHUQEORWWLQJF\WRVROLFDQGPHPEUDQHIUDFWLRQVZHUHSUHSDUHG)ROORZLQJ
WUDQVIHU RI SURWHLQV WR QLWURFHOOXORVH PHPEUDQH WKH PHPEUDQH ZDV EORFNHG LQFXEDWHG ZLWK
DQWLUDEELWDQWLERG\PHPEUDQHZDVGHYHORSHGXVLQJ%LR5DG(&/NLW
Evaluation of signaling pathways: /(&V ZHUH VHHGHG DW D GHQVLW\ RI [ FHOOV SHU ZHOO RI
ZHOO SODWH DQG LQFXEDWHG RYHUQLJKW &HOOV ZHUH VHUXP VWDUYHG DW FRQIOXHQF\ IROORZHG E\
10
WUHDWPHQW ZLWK WKH VF)Y DQWLERGLHV RU WKH UHVSHFWLYH LQKLELWRUV RI LQGLYLGXDO SDWKZD\V EHLQJ
VWXGLHG 60$' 6,6 (5. 3' S 6% DQG -1. 63 7*)ȕ
QJPO ZDVDGGHGDIWHUKDQGFHOOVZHUHIXUWKHULQFXEDWHGIRUWLPHSHULRGVDVVSHFLILHGLQWKH
UHVXOWVVHFWLRQ$WWKHHQGRIWKHLQFXEDWLRQFHOOVZHUHZDVKHGWZLFHZLWK3%6IROORZHGE\FHOO
O\VLV LQ XO LFH FROG 5,3$ EXIIHU P0 7ULV+&O S+ P0 1D&O P0 1D92
13 VRGLXP GHR[\FKRODWH 6'6 DORQJ ZLWK SURWHDVH LQKLELWRU FRFNWDLO &HOO
O\VDWH ZDV HOHFWURSKRUHVHG RQ 6'63$*( IROORZHG E\ WUDQVIHUULQJ RQ WKH QLWURFHOOXORVH
PHPEUDQH 7KH PHPEUDQH ZDV EORFNHG DQG SUREHG ZLWK SULPDU\ DQWLS)$. < 6LJPD
&KHPLFDOV DQWLERG\ RU DQWLS60$' < 6LJPD &KHPLFDOV RU DQWLS(5.
7< &HOO 6LJQDOLQJ RU S-1. 7< &HOO 6LJQDOLQJ RU ZLWK SS 7<
&HOO 6LJQDOLQJ DQG VHFRQGDU\ DQWLUDEELW+53 FRQMXJDWH 7KH PHPEUDQH ZDV
GHYHORSHGXVLQJ(&/7KHVDPHPHPEUDQHZDVUHVWULSSHGDQGSUREHGZLWKWKHFRUUHVSRQGLQJ
DQWLERGLHVIRUYLVXDOL]LQJ)$.60$'S-1.DQG(5.SURWHLQVIROORZHGE\SURELQJZLWK
PHPEUDQH ZDV GHYHORSHG XVLQJ (&/ NLW 0HPEUDQH ZDV DOVR SUREHG ZLWK DQWLEHWDDFWLQ
DQWLERG\DVDORDGLQJFRQWURO
,Q DGGLWLRQ WR ZHVWHUQ EORWWLQJ QXFOHDU SKRVSKRU\ODWLRQ RI 60$' ZDV DOVR H[DPLQHG E\
LPPXQRVWDLQLQJXVLQJFRQIRFDOPLFURVFRSH 2O\PSXV
Statistical Analysis: $OO GDWD DUH H[SUHVVHG DV PHDQ 6' XQOHVV RWKHUZLVH VWDWHG 6WDWLVWLFDO
DQDO\VLVZDVSHUIRUPHGE\WKHRQHZD\$QRYDWHVW$SYDOXHOHVVWKDQZDVFRQVLGHUHGWR
EHVLJQLILFDQW
5HVXOWV
11
TN64 reduces the proliferation of lens epithelial cells without significantly affecting the cell
viability:
$V GHVFULEHG LQ WKH 0HWKRGV VHFWLRQ 71 ZDV VHOHFWHG IRU ELQGLQJ DJDLQVW 71IQ,,, . 7KH
H[WHQW RI 077 PHWDEROLVP LQWR IRUPD]RQH FU\VWDOV ZDV XVHG WR DVVHVV WKH YLDELOLW\ RI WKH OHQV
HSLWKHOLDO FHOOV +/(% LQ WKH SUHVHQFH RI 71 7KHUH ZDV QR VLJQLILFDQW GHFUHDVH LQ WKH
YLDELOLW\ RI WKH FHOOV LQ WKH SUHVHQFH RI 71 LQ WKH UDQJH RI FRQFHQWUDWLRQ RI XJPO
6XSSOHPHQWDU\)LJ$ %UG8LQFRUSRUDWLRQDVVD\VKRZHGWKDWWKHUHZDVDVLJQLILFDQWGHFUHDVH
LQSUROLIHUDWLRQVWDUWLQJIURPDFRQFHQWUDWLRQRIXJPOLQWKHSUHVHQFHRI71QRVXFKHIIHFW
ZDVREVHUYHGLQWKHSUHVHQFHRIDQ\FRQFHQWUDWLRQRIWKHVF)Y2 LUUHOHYDQWFRQWURODQWLERG\
3UROLIHUDWLRQ RI WKH OHQV HSLWKHOLDO FHOOV LQ WKH DEVHQFH RI DQ\ VF)Y DQWLERG\ ZDV DVVLJQHG WKH
WKHUHVWRIWKHH[SHULPHQWV
The scFv TN64 has an inhibitory effect on the migration of lens epithelial cells:
6LJQLILFDQWUHGXFWLRQLQPLJUDWLRQRI+/(%FHOOOLQHDQGSULPDU\FXOWXUHVRI/(&VJURZQIURP
DQWHULRUOHQVFDSVXOHH[SODQWVIURPSDWLHQWVZDVREVHUYHGLQWKHSUHVHQFHRI71DVREVHUYHG
LH/(&VVHHGHGLQWKHSUHVHQFHRI7*)ȕDORQHFORVXUHRIWKHZRXQGGXHWRFHOOPLJUDWLRQ
ZDVDVVLJQHGDYDOXHRI,QWKHSUHVHQFHRI71WKHFORVXUHRIWKHZRXQGZDVDERXW
DQG XVLQJ +/(% FHOOV DQG SULPDU\ HSLWKHOLDO FHOOV UHVSHFWLYHO\ ZKLOH DQG
ZRXQG FORVXUH ZDV VHHQ LQ WKH SUHVHQFH RI WKH LUUHOHYDQW VF)Y 2 XVLQJ +/(% FHOOV DQG
SULPDU\+/(FHOOVUHVSHFWLYHO\ )LJ$DQG%UHVSHFWLYHO\
12
6LQFH WKH PLJUDWLRQ RI WKH FHOOV ZDV VLJQLILFDQWO\ UHGXFHG LQ WKH SUHVHQFH RI 71 ZH QH[W
HYDOXDWHG WKH HIIHFW RI WKLV DQWLERG\ RQ WKH H[SUHVVLRQ RI WKH 003 DQG 003 ZKLFK DUH
GLUHFWO\UHODWHGWRWKHPLJUDWLRQRIWKHFHOOV71ZDVDEOHWRUHGXFHWKHOHYHOVRIERWK003
FXOWXUHVRIWKHVHFHOOVWUHDWHGZLWKWKHLUUHOHYDQWVF)Y2 )LJ&
:KHQ+/(%FHOOVRUSULPDU\FXOWXUHVRIOHQVHSLWKHOLDOFHOOV FXOWXUHGIURPFDSVXOHH[SODQWV
REWDLQHG IURP SDWLHQWV XQGHUJRLQJ FDWDUDFW VXUJHU\ ZHUH FXOWXUHG LQ WKH SUHVHQFH RI 7*)ȕ
FRQWURO RU 2 LUUHOHYDQW FRQWURO DQWLERG\ H[WHQVLYH IRUPDWLRQ RI )DFWLQ VWUHVV ILEHUV ZDV
SDWWHUQDQGVLJQLILFDQWDWWHQXDWLRQRIDFWLQVWUHVVILEHUIRUPDWLRQ
3RVW FDWDUDFW VXUJHU\ WKH UHPDLQLQJ OHQV HSLWKHOLDO FHOOV XQGHUJR D ZRXQG KHDOLQJ UHVSRQVH
OHDGLQJ WR LQFUHDVHG H[SUHVVLRQ RI ĮVPRRWK PXVFOH DFWLQ Į60$ H[SUHVVLQJ P\RILEUREODVWV
DVZHOODVGHSRVLWLRQRI(&0SURWHLQVVXFKDVILEURQHFWLQ7HQDVFLQ&DVZHOODVĮ60$ȕ
LQWHJULQ 7KH ILEURQHFWLQ W\SH ,,, UHSHDWV ± RI 7HQDVFLQ& ELQG QRW RQO\ WR D PDWXUH
ILEURQHFWLQ PDWUL[ EXW DOVR WR QHZO\ V\QWKHVL]HG ILEURQHFWLQ ILEULOV )XUWKHU LW LV DOVR ZHOO
XQGHUVWRRG WKDW SRO\PHUL]HG ILEURQHFWLQ LV QHFHVVDU\ IRU WKH LQGXFWLRQ RI WKH P\RILEUREODVWLF
SKHQRW\SHVHHQLQ3&2&RQVLGHULQJDOORIWKHDERYHLWFDQEHLQIHUUHGWKDWLWLVSRVVLEOHIRUDQ
DQWLERG\ ZKLFK LV GLUHFWHG DJDLQVW WKH 71IQ,,, UHSHDWV WR EH DEOH WR EORFN WHQDVFLQ
WKHWUDQVIRUPDWLRQRIOHQVHSLWKHOLDOFHOOVWRDP\RILEUREODVWLFSKHQRW\SH)URP)LJXUH%LWLV
13
HYLGHQW WKDW 71 LV DEOH WR GHFUHDVH WKH ILEURQHFWLQ DVVHPEO\ ZKHQ FRPSDUHG ZLWK FHOOV
FXOWXUHG LQ WKH SUHVHQFH RI 7*)ȕ DORQH FRQWURO QR VF)Y DGGHG 7KLFN H[WHQVLYH DQG
FRQWLQXRXVILEURQHFWLQILEULOVDUHREVHUYHGLQWKHFRQWUROSRSXODWLRQDVZHOODVLQWKHSUHVHQFHRI
2DQWLERG\ZKLOHWKHILEULOIRUPDWLRQLVVLJQLILFDQWO\DIIHFWHGLQWKHSUHVHQFHRI717KH
H[WHQWRIILEURQHFWLQGHSRVLWLRQLQWKHDEVHQFHDQGSUHVHQFHRIWKHVF)YDQWLERGLHVZDVHYDOXDWHG
E\ '2& LQVROXELOLW\ DVVD\ )LJ & WKH OHYHO RI ILEURQHFWLQ GHSRVLWLRQ ZDV IRXQG WR EH
transition
)ORZ F\WRPHWU\ ZDV GRQH WR HYDOXDWH WKH FKDQJHV LQ H[SUHVVLRQ OHYHOV RI FROODJHQ DQG ȕ
DEVHQFH RI DQ\ VF)Y RU LQ WKH SUHVHQFH RI WKH LUUHOHYDQW DQWLERG\ 2 )LJXUH $ DQG %
)XUWKHU71ZDVHIIHFWLYHLQUHGXFLQJWKHH[SUHVVLRQRIERWK1FDGKHULQĮ60$DVZHOODV
%FHOOVDQGSULPDU\FXOWXUHVRI/(&VUHVSHFWLYHO\&RQWUROFXOWXUHVRIERWKW\SHVRIFHOOV ZLWK
QR VF)Y DGGHG ZHUH DVVLJQHG WKH YDOXH RI DQG WKHUH ZDV QR VLJQLILFDQW GHFUHDVH LQ WKH
OHYHOVRI1FDGKHULQDQGĮ60$LQWKHSUHVHQFHRIWKHLUUHOHYDQWVF)Y26LPLODUUHVXOWVZHUH
VHHQLQLPPXQRVWDLQLQJGDWDVKRZQIRUWKHWZRPDUNHUVLQ)LJXUH(
14
6LQFHWKHȕFDWHQLQVLJQDOLQJSDWKZD\LVLQYROYHGLQWKHSDWKRJHQHVLVRIVHYHUDOILEURWLFGLVHDVHV
WKURXJK LWV UROH LQ WKH LQGXFWLRQ RI (07 ZH H[SORUHG WKH SRVVLELOLW\ WKDW 71 PD\ EH
LQIOXHQFLQJWKLVSDWKZD\'XULQJ(07ȕFDWHQLQDFFXPXODWHVLQWKHF\WRSODVPZKHUHLWIRUPVD
FRPSOH[ ZLWK F\WRVROLF WUDQVFULSWLRQ IDFWRUV DQG LV WKHQ VKXWWOHG WR WKH QXFOHXV OHDGLQJ WR
71WUHDWHGDQGXQWUHDWHGFHOOV,PPXQRVWDLQLQJUHVXOWVVKRZHGWKDWZKLOHWKHVF)YXQWUHDWHG
VWDLQLQJRIȕFDWHQLQLQWKHF\WRSODVPȕFDWHQLQH[SUHVVLRQLQWKH71WUHDWHGFHOOVZDVPDLQO\
WKH F\WRVROLF DQG PHPEUDQH IUDFWLRQV ZHUH H[DPLQHG IRU ȕFDWHQLQ E\ ZHVWHUQ EORWWLQJ XVLQJ
DQWLȕFDWHQLQDQWLERG\,QOLQHZLWKWKHLPPXQRIOXRUHVFHQFHGDWDWKHZHVWHUQEORWDOVRVKRZHG
LQFUHDVHG ȕFDWHQLQ LQ WKH PHPEUDQH IUDFWLRQ RI 71WUHDWHG FHOOV )LJ % 7KH LQFUHDVHG
PHPEUDQHORFDOL]DWLRQRIȕFDWHQLQLVH[SHFWHGWRVWUHQJWKHQWKHFDGKHULQFDWHQLQLQWHUDFWLRQV
:H WKHUHIRUH H[DPLQHG WKH H[SUHVVLRQ RI (FDGKHULQ LQ WKH FXOWXUHV DQG DV H[SHFWHG ZH
REVHUYHGFRORFDOL]DWLRQRIȕFDWHQLQDQG(FDGKHULQ )LJ$FRORFDOL]DWLRQLQ\HOORZLQPHUJHG
RULQWKHFRQWUROFHOOVJURZQLQSUHVHQFHRI2DQWLERG\.
TN64 does not act through the SMAD signaling pathway. 7*)ȕ UHFHSWRU VLJQDOLQJ FDQ DFW
GHSHQGHQW SDWKZD\V,QRUGHUWRH[DPLQHZKHWKHUWKH60$'VLJQDOLQJSDWKZD\LVLQYROYHGLQ
WKHDFWLRQRI71ZHFXOWXUHGWKHSULPDU\/(&VLQWKHSUHVHQFHRIWKHVF)YDQWLERGLHVDVZHOO
SKRVSKRU\ODWLRQ RI 60$' $V VHHQ IURP )LJXUH $ SKRVSKRU\ODWLRQ RI 60$' ZDV
15
XQDIIHFWHGLQDOOWKHFXOWXUHVUDQJLQJIURPWLPHSHULRGRIPLQWLOOKH[FHSWLQWKHSRVLWLYH
FRQWURO 6,6 ZKLFK VKRZHG LQKLELWLRQ RI SKRVSKRU\ODWLRQ PLQ SRVW7*)ȕ DGGLWLRQ 7KLV
GDWD ZDV IXUWKHU FRQILUPHG E\ LPPXQRVWDLQLQJ ZKLFK VKRZHG WKDW 71 KDG QR HIIHFW RQ
WKDW71GRHVQRWLQWHUIHUHZLWKWKHSKRVSKRU\ODWLRQRI60$'
6LQFH 7HQDVFLQ& FDQ LQGXFH (07OLNH FKDQJHV WKURXJK SKRVSKRU\ODWLRQ RI IRFDO DGKHVLRQ
FDXVHGRZQUHJXODWLRQRIS)$.$VFDQEHVHHQIURPWKHLPPXQREORWLQ)LJ&WKHOHYHORI
)$. UHPDLQHG FRQVWDQW ZKHQ FHOOV ZHUH FXOWXUHG LQ WKH SUHVHQFH RI 7*)ȕ DORQH RU LQ WKH
SUHVHQFHRI71RU2KRZHYHUFHOOVWUHDWHGZLWK71LQWKHSUHVHQFHRI7*)ȕVKRZHG
UHGXFWLRQ LQ SKRVSKRU\ODWLRQ RI )$. DW < :H DOVR H[DPLQHG ZKHWKHU WKH LQKLELWLRQ RI
(07OLNHFKDQJHVE\71ZDVPHGLDWHGE\WKH60$'LQGHSHQGHQWPLWRJHQDFWLYDWHGSURWHLQ
1WHUPLQDO NLQDVH DQG (5. H[WUDFHOOXODU VLJQDO UHJXODWHGNLQDVH ZDV DVVHVVHG LQ WKH
DOO WKHVH NLQDVHV ZDV LQKLELWHG ZKHQ FXOWXUHG LQ WKH SUHVHQFH RI 71 XQOLNH WKH LUUHOHYDQW
DQWLERG\2
'LVFXVVLRQ
7*)ȕ LV D PDMRU LQGXFHU RI HSLWKHOLDOPHVHQFK\PDO WUDQVLWLRQ (07 LQ WKH OHQV
FDWDUDFW VXUJHU\ ,Q WKLV SDSHU ZH VKRZ WKH (07LQKLELWLQJ SURSHUW\ RI DQ DQWLERG\ 71
16
GLUHFWHGDJDLQVWWKHILEURQHFWLQW\SH,,,OLNHUHSHDW LQ7HQDVFLQ7HQDVFLQKDVEHHQVKRZQ
WR SOD\ NH\ UROHV LQ FHOO DGKHVLRQ VSUHDGLQJ DQG PLJUDWLRQ RI YDULHG NLQGV RI FHOOV :H
JHQHUDWHG 71 WKURXJK SKDJH GLVSOD\ DQWLERG\ OLEUDU\ VFUHHQLQJ PHWKRGV DQG IRXQG WKDW LW
VSHFLILFDOO\UHFRJQL]HVWKH71)Q,,,±UHJLRQ0RUHDFFXUDWHO\LWELQGVLQGHSHQGHQWO\WR71
DQG 71 EXW ELQGLQJ WR 71 LV YHU\ ZHDN :H FKRVH WKLV WDUJHW VLQFH LW KDV EHHQ VKRZQ
SUHYLRXVO\ WKDW D 7HQDVFLQ& H[SUHVVLRQ LV XSUHJXODWHG XSRQ (07 RI OHQV HSLWKHOLDO FHOOV LQ
KXPDQ 3&2 E LQ WKH DEVHQFH RI 7HQDVFLQ& LQMXU\LQGXFHG (07 RI WKH PRXVH OHQV
HSLWKHOLXPLVDWWHQXDWHGF PXOWLSOHJURZWKIDFWRUVELQGZLWKKLJKDIILQLW\WRWKLVYHU\UHSHDW
SRWHQWLDOWRFURVVOLQNWKHH[WUDFHOOXODUPDWUL[E\LQWHUDFWLQJZLWKQHLJKERULQJILEURQHFWLQILEULOV
WKHH[SUHVVLRQRI003DQG003ZKLFKFDQFDXVHFOHDYDJHRIFROODJHQW\SH,9 RIWKH
EDVHPHQW PHPEUDQH RQ WKH RQH KDQG UHVXOWLQJ LQ LQFUHDVHG FHOO PLJUDWLRQ DQG RQ WKH RWKHU
KDQGWKLVFDQDOVRFDXVHFOHDYDJHRI7*)ȕELQGLQJSURWHLQWKHUHE\UHOHDVLQJ7*)ȕIURPLWV
(07 1HYHUWKHOHVV WKLV KLJKOLJKWV WKH LPSRUWDQFH RI WKH UHSHDW UHJLRQ RI 7HQDVFLQ& LQ
PRGXODWLQJWKHH[WUDFHOOXODUPDWUL[DVZHOODVFHOO(&0LQWHUDFWLRQVGXULQJWKHZRXQGKHDOLQJ
,W LV NQRZQ WKDW 7*)ȕ OHYHOV LQFUHDVH GUDPDWLFDOO\ DIWHU FDWDUDFW VXUJHU\ SRWHQWLDOO\
WULJHUULQJ 3&2 ZLWK 7*)ȕ NQRZQ WR EH WLPHV PRUH HIIHFWLYH WKDQ 7*)ȕ LQ FDXVLQJ
FKDQJHV DVVRFLDWHG ZLWK KXPDQ VXEFDSVXODU FDWDUDFW50 :H WHVWHG WKH HIIHFWV RI 71 RQ WZR
W\SHVRIOHQVHSLWKHOLDOFHOOVH[SRVHGWR7*)ȕWKHOHQVHSLWKHOLXPFHOOOLQH±+/(%DQGWKH
17
SULPDU\ OHQV HSLWKHOLDO FHOOV JURZQ IURP H[SODQWV REWDLQHG IURP SDWLHQWV XQGHUJRLQJ FDWDUDFW
VXUJHU\,WPD\EHQRWHGWKDWUHVXOWVVKRZLQJ71PHGLDWHGGHFUHDVHGPLJUDWLRQRI/(&V )LJ
DFWLQ VWUHVV ILEHU IRUPDWLRQ )LJ DQG ILEURQHFWLQ SRO\PHUL]DWLRQ )LJ ZHUH DOO PRUH
SURQRXQFHGZLWK SULPDU\ FXOWXUHVRI FHOOV WKDQ +/(% FHOOOLQH GHULYHG FHOOV HPSKDVL]LQJ
WKH LPSRUWDQFH RI XVLQJ VXFK FHOOV VLQFH WKH\ DUH PRUH DNLQ WR WKH SK\VLRORJLFDO SDWKRORJLF
VHWWLQJ
7*)ȕ LV NQRZQ WR LQGXFH (07 WKURXJK WKH FDQRQLFDO 60$'GHSHQGHQW DGKHVLRQ
GDWD FOHDUO\ VKRZV WKDW WKH 71PHGLDWHG LQKLELWLRQ RI (07 VWDWXV LV 60$'LQGHSHQGHQW
,PSRUWDQWO\ LW KDV EHHQ UHSRUWHG WKDW VXSSUHVVLRQ RI LQMXU\LQGXFHG (07 LQ 7HQDVFLQ& QXOO
PLFH LV DVVRFLDWHG ZLWK DWWHQXDWLRQ RI SKRVSKRU\ODWLRQ RI 60$' VLJQDOLQJ 7KLV LV QRW LQ
FRQIOLFWZLWKRXUUHVXOWVZKLOHWKHSUHYLRXVZRUNLQYROYHGPLFHODFNLQJWKHZKROHPROHFXOHRI
7HQDVFLQ& LW LV WR EH QRWHG WKDW WKH SUHVHQW ZRUN LV GLIIHUHQW RQ WZR FRXQWV L XQOLNH WKH
7*)ȕ LV ODUJHO\ SUHVHQW LQ LWV ODWHQW IRUP WKH DFWLYDWLRQ RI ZKLFK LV VXSSRUWHG E\ UHDFWLYH
R[\JHQ VSHFLHV 526 VSHFLILF LQWHJULQV WKURPERVSRQGLQ 763 RU SURWHDVHV DQG LL
XQOLNH WKH SUHYLRXV ZRUN ZKHUH 7HQDVFLQ ZDV NQRFNHG RXW FRPSOHWHO\ LQ WKH SUHVHQW ZRUN
7HQDVFLQ&LVWDUJHWHGE\DQDQWLERG\GLUHFWHGDWRQO\WKHILUVWILYHILEURQHFWLQW\SH,,,UHSHDWV
71IQ,,, ,QOLJKWRIWKHVHGLIIHUHQFHVLWPD\EHVSHFXODWHGWKDWWKHWZRVHWVRIH[SHULPHQWV
FRXOG OHDG WR GLIIHUHQW FHOOXODU HIIHFWV RI 7HQDVFLQ ,W LVWKHUHIRUH FRQFHLYDEOH WKDW DQ LQKLELWRU
VXFKDV71FDXVHVGRZQUHJXODWLRQRI(07OHDYLQJWKH60$'VLJQDOLQJSDWKZD\LQWDFW
,WKDVEHHQSUHYLRXVO\GHPRQVWUDWHGWKDW7*)ȕLQGXFHG(07FDQRFFXUWKURXJKWKHQRQ
FDQRQLFDOLQWHJULQGHSHQGHQWDXWRSKRVSKRU\ODWLRQDQGDFWLYDWLRQRI)$.RUWKURXJKWKH0$3
18
NLQDVHSDWKZD\6HYHUDORIRXUUHVXOWVDOVRLQGLFDWHWKHLQYROYHPHQWRIWKH)$.SDWKZD\LQ
71LQWKHSUHVHQFHRI7*)ȕE SKRVSKRU\ODWLRQRI)$.LVDVVRFLDWHGZLWKWKHXSUHJXODWLRQ
RI LQWHJULQ UHFHSWRUV DQG ILEURQHFWLQ FKDQJHV LQ DFWLQ VWUHVV ILEHU IRUPDWLRQ RUJDQL]DWLRQ RI
ILEURQHFWLQPDWUL[DQGFHOOPLJUDWLRQDOORIZKLFKDUHGRZQUHJXODWHGLQWKHSUHVHQFHRI71
F )$.VLJQDOLQJLVNQRZQWREHUHTXLUHGIRUGHORFDOL]DWLRQRIPHPEUDQHERXQG(FDGKHULQ
DQGRXUUHVXOWVVKRZWKDW71FDXVHVGRZQUHJXODWLRQRIWKHPHVHQFK\PDOPDUNHU1FDGKHULQ
DQGFDXVHVUHH[SUHVVLRQRIWKHFHOOFHOOMXQFWLRQDGKHVLRQSURWHLQ(FDGKHULQG )$.VLJQDOLQJ
LVDOVRLPSOLFDWHGLQILEURQHFWLQGHSHQGHQWXSUHJXODWLRQRI003VPHGLDWHGWKURXJKLQWHJULQV
RXU GDWD VKRZV WKDW 71 LV FDSDEOH RI LQKLELWLQJ WKH H[SUHVVLRQ RI ERWK 003 DV ZHOO DV
003 6LQFH )$. FDQ OLQN LQWHJULQLQLWLDWHG VLJQDOV WR WKH PLWRJHQDFWLYDWHG SURWHLQ NLQDVH
0$3. SDWKZD\ZHH[DPLQHGZKHWKHU71KDVDQ\HIIHFWRQWKHSKRVSKRU\ODWLRQRIWKHVH
NLQDVHVDQGDVH[SHFWHGSKRVSKRU\ODWLRQRIS(5.DQG-1.ZHUHLQKLELWHGE\WKHDQWLERG\
)XUWKHUVLQFH(07LQLWLDWHVLQWKHOHQVWKURXJKWKHDFWLYDWLRQRI :QWUHVSRQVLYHJHQHV
IROORZLQJ WKH QXFOHDU WUDQVORFDWLRQ RI ȕFDWHQLQ LQ WKH OHQV HSLWKHOLDO FHOOV DQG )$. LV DOVR
UHTXLUHGGRZQVWUHDPRI:QWVLJQDOLQJZHH[DPLQHGZKHWKHU71DOVRRSHUDWHVWKURXJKWKH
:QW VLJQDOLQJ SDWKZD\ LQYROYLQJ ȕFDWHQLQ :H IRXQG WKDW 71 VXSSRUWV PHPEUDQH
ORFDOL]DWLRQRIȕFDWHQLQZKHUHLWORFDOL]HVZLWKWKHHSLWKHOLDO PHPEUDQHDVVRFLDWHG(FDGKHULQ
DVVRFLDWHG SKHQRW\SH
,Q WKH FRQWH[W RI ILEURVLV EORFNDGH RI 7*)ȕ ZRXOG VHHP DWWUDFWLYH KRZHYHU WKLV
F\WRNLQH SOD\V DQ LPSRUWDQW UROH LQ FRQWURO RI FHOO SUROLIHUDWLRQ DQG WXPRU VXSSUHVVLRQ
0RUHRYHU D SRSXODWLRQEDVHG FRKRUW VWXG\ KDV VKRZQ LQFUHDVHG LQFLGHQFH RI HSLWKHOLDO FHOO
PDOLJQDQFLHV LQ ILEURWLF GLVHDVHV WKHUHIRUH LW ZRXOG EH GHVLUDEOH WR GHVLJQ DQ DQWLILEURWLF
19
VWUDWHJ\ZKLFKGRHVQRWDGYHUVHO\DIIHFWDSDWKZD\LQYROYHGLQWXPRUVXSSUHVVLRQ,PSRUWDQWO\
WDUJHWHG GHOHWLRQ RI )$. LQ PDPPDU\ HSLWKHOLXP KDV EHHQ VKRZQ WR VXSSUHVV PDPPDU\
WXPRULJHQHVLV7KHLQWHJULQ)$.SDWKZD\KDVWKHUHIRUHEHHQSURSRVHGWREHDVDIHUDQGPRUH
HIIHFWLYHWKHUDSHXWLFVWUDWHJ\IRUILEURWLFGLVHDVHV
,QVXPPDU\RXUUHVXOWVVXJJHVWWKDWL WKHILEURQHFWLQW\SH,,,OLNHUHSHDWVLQ7HQDVFLQ&
PD\EHSOD\LQJDQLPSRUWDQWUROHLQ3&2LL 71LVFDSDEOHRIQHJDWLQJWKHHIIHFWVRI7*)ȕ
LQOHQVHSLWKHOLDOFHOOVLLL 71DFWVE\LQKLELWLQJWKH7*)ȕLQGXFHGSKRVSKRU\ODWLRQRI)$.
DQGQXFOHDUWUDQVORFDWLRQRIȕFDWHQLQDQGLY 71LVFDSDEOHRILQKLELWLQJWKH0$3NLQDVHV
7KHUHIRUH RXU UHVXOWV DUH LQ VXSSRUW RI WKH QHHG WR WDUJHW WKH FRGHSHQGHQW :QW VLJQDOLQJ DQG
)$. VLJQDOLQJ SDWKZD\V LQGHSHQGHQW RI WKH 60$' SDWKZD\ LQ WKDW 71 FOHDUO\ RSHUDWHV
WKURXJKWKHIRUPHUDQGQRWWKHODWWHUZKLOHLQKLELWLQJ(07DVVRFLDWHGFKDQJHV
$FNQRZOHGJHPHQWV 0/* WKDQNV WKH 'HSDUWPHQW RI 6FLHQFH DQG 7HFKQRORJ\ '67 DQG
WKDQNVWKH&6,5IRUIHOORZVKLS
5HIHUHQFHV
-DPSHO+'5RFKH16WDUN:-5REHUWV$%7UDQVIRUPLQJJURZWKIDFWRUEHWDLQ
KXPDQDTXHRXVKXPRUCurr Eye Res.
&RXVLQV 6: 0F&DEH 00 'DQLHOSRXU ' 6WUHLOHLQ -: ,GHQWLILFDWLRQ RI
WUDQVIRUPLQJ JURZWK IDFWRUEHWD DV DQ LPPXQRVXSSUHVVLYH IDFWRU LQ DTXHRXV
KXPRUInvest Ophthalmol Vis Sci.
20
$SSOH'-(VFREDU*RPH]0=DXJJ%.OHLQPDQQ*%RUNHQVWHLQ$)0RGHUQ
FDWDUDFWVXUJHU\XQILQLVKHGEXVLQHVVDQGXQDQVZHUHGTXHVWLRQV6XUY2SKWKDOPRO
6±6
:RUPVWRQH ,0 7DPL\D 6 $QGHUVRQ , 'XQFDQ * 7*)EHWDLQGXFHG PDWUL[
PRGLILFDWLRQ DQG FHOO WUDQVGLIIHUHQWLDWLRQ LQ WKH KXPDQ OHQV FDSVXODU EDJ Invest
Ophthalmol Vis Sci.
/HH (+ -RR &. 5ROH RI WUDQVIRUPLQJ JURZWK IDFWRUEHWD LQ WUDQVGLIIHUHQWLDWLRQ
DQGILEURVLVRIOHQVHSLWKHOLDOFHOOVInvest Ophthalmol Vis Sci.
0DUFDQWRQLR-09UHQVHQ*)&HOOELRORJ\RISRVWHULRUFDSVXODURSDFLILFDWLRQ
Eye (Lond) 3WE
.DSSHOKRI -3 9UHQVHQ *) GH -RQJ 37 3DPH\HU - :LOOHNHQV %/ 7KH ULQJ RI
6RHPPHUULQJ LQ PDQ DQ XOWUDVWUXFWXUDO VWXG\ Graefes Arch Clin Exp Ophthalmol.
:RUPVWRQH ,0 :DQJ / /LX &6 3RVWHULRU FDSVXOH RSDFLILFDWLRQ Exp Eye Res.
$SSOH '- 6RORPRQ .' 7HW] 05 HW DO 3RVWHULRU FDSVXOH RSDFLILFDWLRQ Surv
Ophthalmol.
:LOVRQ 0( -U 7ULYHGL 5+ 7KH RQJRLQJ EDWWOH DJDLQVW SRVWHULRU FDSVXODU
RSDFLILFDWLRQArch Ophthalmol.
%LQNKRUVW&'*RELQ0+,QMXULHVWRWKH(\HZLWK/HQV2SDFLW\LQ<RXQJ&KLOGUHQ
Ophthalmologica.
+LOHV '$ :DOODU 3+ 3KDFRHPXOVLILFDWLRQ YHUVXV DVSLUDWLRQ LQ LQIDQWLOH FDWDUDFW
VXUJHU\Ophthalmic Surg.
$SSOH'-3HQJ49LVHVVRRN1HWDO(UDGLFDWLRQRISRVWHULRUFDSVXOHRSDFLILFDWLRQ
GRFXPHQWDWLRQ RI D PDUNHG GHFUHDVH LQ 1G<$* ODVHU SRVWHULRU FDSVXORWRP\ UDWHV
QRWHG LQ DQ DQDO\VLV RI SVHXGRSKDNLF KXPDQ H\HV REWDLQHG SRVWPRUWHP
Ophthalmology.
21
%XHKO:)LQGO2(IIHFWRILQWUDRFXODUOHQVGHVLJQRQSRVWHULRUFDSVXOHRSDFLILFDWLRQ
J Cataract Refract Surg.
)LQGO 2 %XHKO : %DXHU 3 HW DO ,QWHUYHQWLRQV IRU SUHYHQWLQJ SRVWHULRU FDSVXODU
RSDFLILFDWLRQCochrane Database Syst Rev. &'
6\PRQGV -* /RYLFX )- &KDPEHUODLQ &* 'LIIHULQJ HIIHFWV RI GH[DPHWKDVRQH DQG
GLFORIHQDFRQSRVWHULRUFDSVXOHRSDFLILFDWLRQOLNHFKDQJHVLQDUDWOHQVH[SODQWPRGHO
Exp Eye Res.
:DQJ 0 =KDQJ -- -DFNVRQ 7/ 6XQ ; :X : 0DUVKDOO - 6DIHW\ DQG HIILFDF\ RI
LQWUDFDSVXODUWUDQLODVWPLFURVSKHUHVLQH[SHULPHQWDOSRVWHULRUFDSVXOHRSDFLILFDWLRQJ
Cataract Refract Surg.
.LP6<.LP-+&KRL-6-RR&.&RPSDULVRQRISRVWHULRUFDSVXOHRSDFLILFDWLRQLQ
UDEELWV UHFHLYLQJ HLWKHU PLWRP\FLQ& RU GLVWLOOHG ZDWHU IRU VHDOHGFDSVXOH LUULJDWLRQ
GXULQJFDWDUDFWVXUJHU\Clin Experiment Ophthalmol.
:DQJ*4*X+4<XDQ-46XQ+0;X<6)KHSDULQPRGLILHGLQWUDRFXODUOHQVHV
LQ5KHVXVPRQNH\VInt J Ophthalmol.
<D]LFL$7%R]NXUW(.DUD1<LOGLULP<'HPLURN$<LOPD]2)/RQJWHUPUHVXOWV
RI SKDFRHPXOVLILFDWLRQ FRPELQHG ZLWK SULPDU\ SRVWHULRU FXUYLOLQHDU FDSVXORUKH[LV LQ
DGXOWVMiddle East Afr J Ophthalmol.
7DVVLJQRQ0-*RELQ/0DWK\VHQ'9DQ/RRYHUHQ-'H*URRW9&OLQLFDORXWFRPHV
RI FDWDUDFW VXUJHU\ DIWHU EDJLQWKHOHQV LQWUDRFXODU OHQV LPSODQWDWLRQ IROORZLQJ ,62
VWDQGDUGJ Cataract Refract Surg.
6HULQL * %RFKDWRQ3LDOODW 0/ 5RSUD] 3 HW DO 7KH ILEURQHFWLQ GRPDLQ ('$ LV
FUXFLDOIRUP\RILEUREODVWLFSKHQRW\SHLQGXFWLRQE\WUDQVIRUPLQJJURZWKIDFWRUEHWD
J Cell Biol.
6HULQL * *DEELDQL * 0HFKDQLVPV RI P\RILEUREODVW DFWLYLW\ DQG SKHQRW\SLF
PRGXODWLRQExp Cell Res.
7RPDVHN -- *DEELDQL * +LQ] % &KDSRQQLHU & %URZQ 5$ 0\RILEUREODVWV DQG
PHFKDQRUHJXODWLRQ RI FRQQHFWLYH WLVVXH UHPRGHOOLQJ Nat Rev Mol Cell Biol.
22
7DQDND 6 6XPLRND 7 )XMLWD 1 HW DO 6XSSUHVVLRQ RI LQMXU\LQGXFHG HSLWKHOLDO
PHVHQFK\PDO WUDQVLWLRQ LQ D PRXVH OHQV HSLWKHOLXP ODFNLQJ WHQDVFLQ& Mol Vis.
&KXQJ &< =DUGL / (ULFNVRQ +3 %LQGLQJ RI WHQDVFLQ& WR VROXEOH ILEURQHFWLQ DQG
PDWUL[ILEULOVJ Biol Chem.
,QJKDP.&%UHZ6$(ULFNVRQ+3/RFDOL]DWLRQRIDFU\SWLFELQGLQJVLWHIRUWHQDVFLQ
RQILEURQHFWLQJ Biol Chem.
%UXPPHQGRUI 7 5DWKMHQ )* &HOO DGKHVLRQ PROHFXOHV LPPXQRJOREXOLQ
VXSHUIDPLO\Protein Profile.
%RUN 3 'RROLWWOH 5) 3URSRVHG DFTXLVLWLRQ RI DQ DQLPDO SURWHLQ GRPDLQ E\ EDFWHULD
Proc Natl Acad Sci U S A.
&KL5RVVR * *RWZDOV 3-<DQJ - HW DO )LEURQHFWLQ W\SH ,,, UHSHDWV PHGLDWH 5*'
LQGHSHQGHQW DGKHVLRQ DQG VLJQDOLQJ WKURXJK DFWLYDWHG EHWD LQWHJULQV J Biol Chem.
*XOFKHU -5 1LHV '( $OH[DNRV 0- HW DO 6WUXFWXUH RI WKH KXPDQ KH[DEUDFKLRQ
WHQDVFLQ JHQHProc Natl Acad Sci U S A.
+VLD+&6FKZDU]EDXHU-(0HHWWKHWHQDVFLQVPXOWLIXQFWLRQDODQGP\VWHULRXVJ Biol
Chem.
GH,RQJK58:HGHUHOO(/RYLFX)-0F$YR\-:7UDQVIRUPLQJJURZWKIDFWRUEHWD
LQGXFHGHSLWKHOLDOPHVHQFK\PDOWUDQVLWLRQLQWKHOHQVDPRGHOIRUFDWDUDFWIRUPDWLRQ
Cells Tissues Organs.
$QGOH\835KLP-6&K\ODFN/7-U)OHPLQJ733URSDJDWLRQDQGLPPRUWDOL]DWLRQRI
KXPDQOHQVHSLWKHOLDOFHOOVLQFXOWXUHInvest Ophthalmol Vis Sci.
6KDUPD07LZDUL$6KDUPD6HWDO)LEURWLFUHPRGHOLQJRIWKHH[WUDFHOOXODUPDWUL[
WKURXJKDQRYHO HQJLQHHUHGGXDOIXQFWLRQ DQWLERG\UHDFWLYHWRDFU\SWLFHSLWRSHRQ
WKH1WHUPLQDON'DIUDJPHQWRIILEURQHFWLQPLoS One. H
<DQJ<<H< /LQ ;:X .<X 0 ,QKLELWLRQ RI SLUIHQLGRQH RQ7*)EHWDLQGXFHG
SUROLIHUDWLRQPLJUDWLRQDQGHSLWKOLDOPHVHQFK\PDOWUDQVLWLRQRIKXPDQOHQVHSLWKHOLDO
FHOOVOLQH65$PLoS One.H
23
6KDUPD 0 /XWKUD*XSWDVDUPD 0 'HJUDGDWLRQ RI SURWHLQV XSRQ VWRUDJH DW QHDU
QHXWUDO S+ LQGLFDWLRQV RI D SURWHRO\WLFJHODWLQRO\WLF DFWLYLW\ DVVRFLDWHG ZLWK
DJJUHJDWHVBiochim Biophys Acta.
6HFKOHU-/7DNDGD<6FKZDU]EDXHU-($OWHUHGUDWHRIILEURQHFWLQPDWUL[DVVHPEO\E\
GHOHWLRQRIWKHILUVWW\SH,,,UHSHDWVJ Cell Biol.
1DJDPRWR7(JXFKL*%HHEH'&$OSKDVPRRWKPXVFOHDFWLQH[SUHVVLRQLQFXOWXUHG
OHQVHSLWKHOLDOFHOOVInvest Ophthalmol Vis Sci.
0DKHU070R5)OR]DN$63HOHG21*RWWDUGL&-%HWDFDWHQLQSKRVSKRU\ODWHGDW
VHULQH LV VSDWLDOO\ XQFRXSOHG IURP EHWDFDWHQLQ SKRVSKRU\ODWHG LQ WKH *6.
GRPDLQLPSOLFDWLRQVIRUVLJQDOLQJPLoS One. H
0DPX\D)$:DQJ<5RRS9+6FKHLEOLQ'$=DMDF-&'XQFDQ0.7KHUROHVRI
DOSKD9 LQWHJULQV LQ OHQV (07 DQG SRVWHULRU FDSVXODU RSDFLILFDWLRQ J Cell Mol Med.
7R :6 0LGZRRG .6 ,GHQWLILFDWLRQ RI QRYHO DQG GLVWLQFW ELQGLQJ VLWHV ZLWKLQ
WHQDVFLQ&IRUVROXEOHDQGILEULOODUILEURQHFWLQJ Biol Chem.
<DS$67KHPRUSKRJHQHWLFUROHRIFDGKHULQFHOODGKHVLRQPROHFXOHVLQKXPDQFDQFHU
DWKHPDWLFUHYLHZCancer Invest.
:HQGW 0. $OOLQJWRQ 70 6FKLHPDQQ :3 0HFKDQLVPV RI WKH HSLWKHOLDO
PHVHQFK\PDOWUDQVLWLRQE\7*)EHWDFuture Oncol.
1DJDKDUX . =KDQJ ;<RVKLGD 7 HW DO 7HQDVFLQ & LQGXFHV HSLWKHOLDOPHVHQFK\PDO
WUDQVLWLRQOLNH FKDQJH DFFRPSDQLHG E\ 65& DFWLYDWLRQ DQG IRFDO DGKHVLRQ NLQDVH
SKRVSKRU\ODWLRQLQKXPDQEUHDVWFDQFHUFHOOVAm J Pathol.
3KLOOLSV *5 .UXVKHO /$ &URVVLQ ./ 'RPDLQV RI WHQDVFLQ LQYROYHG LQ JOLRPD
PLJUDWLRQJ Cell Sci. 3W
'H/DSRUWH/5LFH--7RUWHOOL)+XEEHOO-$7HQDVFLQ&SURPLVFXRXVO\ELQGVJURZWK
IDFWRUVYLDLWVILIWKILEURQHFWLQW\SH,,,OLNHGRPDLQPLoS One.H
6HQ7'XWWD$0DLW\*&KDWWHUMHH$)LEURQHFWLQLQGXFHVPDWUL[PHWDOORSURWHLQDVH
003 LQ KXPDQ ODU\QJHDO FDUFLQRPD FHOOV E\ LQYROYLQJ PXOWLSOH VLJQDOLQJ
SDWKZD\VBiochimie.
(VSDU]D-9LODUGHOO&&DOYR-HWDO)LEURQHFWLQXSUHJXODWHVJHODWLQDVH% 003
DQGLQGXFHVFRRUGLQDWHGH[SUHVVLRQRIJHODWLQDVH$ 003 DQGLWVDFWLYDWRU07
24
003 003 E\ KXPDQ 7 O\PSKRF\WH FHOO OLQHV $ SURFHVV UHSUHVVHG WKURXJK
5$60$3NLQDVHVLJQDOLQJSDWKZD\VBlood.
*RUGRQ7KRPVRQ & GH ,RQJK 58 +DOHV $0 &KDPEHUODLQ &* 0F$YR\ -:
'LIIHUHQWLDO FDWDUDFWRJHQLF SRWHQF\ RI 7*)EHWD EHWD DQG EHWD DQG WKHLU
H[SUHVVLRQLQWKHSRVWQDWDOUDWH\HInvest Ophthalmol Vis Sci.
0XUSK\8OOULFK-(3RF]DWHN0$FWLYDWLRQRIODWHQW7*)EHWDE\WKURPERVSRQGLQ
PHFKDQLVPVDQGSK\VLRORJ\Cytokine Growth Factor Rev
$QQHV-35LINLQ'%0XQJHU-67KHLQWHJULQDOSKD9EHWDELQGVDQGDFWLYDWHVODWHQW
7*)EHWDFEBS Lett.
%DUFHOORV+RII0+'L[7$5HGR[PHGLDWHGDFWLYDWLRQRIODWHQWWUDQVIRUPLQJJURZWK
IDFWRUEHWDMol Endocrinol.
<X 4 6WDPHQNRYLF , &HOO VXUIDFHORFDOL]HG PDWUL[ PHWDOORSURWHLQDVH
SURWHRO\WLFDOO\ DFWLYDWHV 7*)EHWD DQG SURPRWHV WXPRU LQYDVLRQ DQG DQJLRJHQHVLV
Genes Dev.
7KDQQLFNDO9-/HH'<:KLWH(6HWDO0\RILEUREODVWGLIIHUHQWLDWLRQE\WUDQVIRUPLQJ
JURZWK IDFWRUEHWD LV GHSHQGHQW RQ FHOO DGKHVLRQ DQG LQWHJULQ VLJQDOLQJ YLD IRFDO
DGKHVLRQNLQDVHJ Biol Chem.
Suzuki H, Uchida K, Nitta K, Nihei H. Role of mitogen-activated protein kinase in the
regulation of transforming growth factor-beta-induced fibronectin accumulation in
cultured renal interstitial fibroblasts. Clin Exp Nephrol 2004;8:188-195.
6FKDOOHU 0' %LRFKHPLFDO VLJQDOV DQG ELRORJLFDO UHVSRQVHV HOLFLWHG E\ WKH IRFDO
DGKHVLRQNLQDVHBiochim Biophys Acta.
&LFFKLQL & /DXGDGLR , &LWDUHOOD ) HW DO 7*)EHWDLQGXFHG (07 UHTXLUHV IRFDO
DGKHVLRQNLQDVH )$. VLJQDOLQJExp Cell Res.
+VLD '$ 0LWUD 6. +DXFN &5 HW DO 'LIIHUHQWLDO UHJXODWLRQ RI FHOO PRWLOLW\ DQG
LQYDVLRQE\)$.J Cell Biol.
3UDQLWKD 3 6XGKDNDUDQ 35 )LEURQHFWLQ GHSHQGHQW XSUHJXODWLRQ RI PDWUL[
PHWDOORSURWHLQDVHV LQ KHSDWLF VWHOODWH FHOOV Indian J Biochem Biophys.
25
6FKODHSIHU '' +XQWHU 7 )RFDO DGKHVLRQ NLQDVH RYHUH[SUHVVLRQ HQKDQFHV UDV
GHSHQGHQW LQWHJULQ VLJQDOLQJ WR (5.PLWRJHQDFWLYDWHG SURWHLQ NLQDVH WKURXJK
LQWHUDFWLRQVZLWKDQGDFWLYDWLRQRIF6UFJ Biol Chem.
&KRQJ && 6WXPS 5- /RYLFX )- 0F$YR\ -: 7*)EHWD SURPRWHV :QW H[SUHVVLRQ
GXULQJFDWDUDFWGHYHORSPHQWExp Eye Res.
$VKWRQ*+0RUWRQ-30\DQW.HWDO)RFDODGKHVLRQNLQDVHLVUHTXLUHGIRULQWHVWLQDO
UHJHQHUDWLRQ DQG WXPRULJHQHVLV GRZQVWUHDP RI :QWF0\F VLJQDOLQJ Dev Cell.
/LNH % 0DVVDJXH - 7KH DQWLSUROLIHUDWLYH HIIHFW RI W\SH EHWD WUDQVIRUPLQJ JURZWK
IDFWRU RFFXUV DW D OHYHO GLVWDO IURP UHFHSWRUV IRU JURZWKDFWLYDWLQJ IDFWRUV J Biol
Chem.
+XEEDUG 5 9HQQ $ /HZLV 6 %ULWWRQ - /XQJ FDQFHU DQG FU\SWRJHQLF ILEURVLQJ
DOYHROLWLV$SRSXODWLRQEDVHGFRKRUWVWXG\Am J Respir Crit Care Med.
/XR 0 )DQ + 1DJ\ 7 HW DO 0DPPDU\ HSLWKHOLDOVSHFLILF DEODWLRQ RI WKH IRFDO
DGKHVLRQ NLQDVH VXSSUHVVHV PDPPDU\ WXPRULJHQHVLV E\ DIIHFWLQJ PDPPDU\ FDQFHU
VWHPSURJHQLWRUFHOOVCancer Res.
/(*(1'672),*85(6
)LJXUH 3DQHO $ (IIHFW RI 71 RQ DFWLQ VWUHVV ILEHU IRUPDWLRQ +/(% FHOOV DQG SULPDU\
FXOWXUH RI /(&V GHULYHG IURP FDSVXOHV IURP SDWLHQWV ZHUH JURZQ WR FRQIOXHQF\ RQ JHODWLQ
26
FRDWHGFRYHUVOLSVDQGLQFXEDWHGLQWKHDEVHQFH FRQWURO RULQWKHSUHVHQFHRIVF)YDQWLERGLHV
2 LUUHOHYDQW FRQWURO DQWLERG\ RU WKH WHVW DQWLERG\ 71 JPO HDFK 7KH FHOOV ZHUH
IL[HG GHWHUJHQW SHUPHDELOL]HG DQG WUHDWHG ZLWK 75,7&SKDOORLGLQ IRU DFWLQ VWUHVV ILEHUV
&RQIRFDO LPDJHV LQGLFDWH WKH IRUPDWLRQ RI H[WHQVLYH DFWLQVWUHVV ILEHUV UXQQLQJ WKURXJKRXW WKH
FHOO F\WRSODVP LQ WKH XQWUHDWHG VHW FRQWURO DQG LQ WKH SUHVHQFH RI WKH VF)Y 2 LUUHOHYDQW
VF)Y 5HDUUDQJHPHQW RI WKH DFWLQ ILEULOVZDVREVHUYHGLQWKHSUHVHQFHRIWKH71DQWLERG\
7KH VWUHVV ILEHUV ORFDOL]HG PRUH WRZDUGV WKH SHULSKHU\ LQ WKH SUHVHQFH RI WKH VF)Y DQWLERG\
71 SDUWLFXODUO\ LQ WKH FDVH RI SULPDU\ FXOWXUHV RI OHQV HSLWKHOLDO FHOOV 3DQHO % (IIHFW RI
71RQILEURQHFWLQDVVHPEO\DQGGHSRVLWLRQ&RQIRFDOLPDJHVRIILEURQHFWLQSRO\PHUL]DWLRQLQ
+/(% FHOOV DQG SULPDU\ FXOWXUH /(&6 ZLWK DQG ZLWKRXW VF)Y DQWLERGLHV )LEURQHFWLQ ZDV
YLVXDOL]HG XVLQJ UDEELW DQWLILEURQHFWLQ DQWLERG\ IROORZHG E\ ),7&FRQMXJDWHG DQWLUDEELW
DQWLERG\ 3DQHO & 4XDQWLWDWLRQ RI H[WHQW RI ILEURQHFWLQ GHSRVLWLRQ LQ WKH H[WUDFHOOXODU PDWUL[
HVWLPDWHG E\ GHR[\FKROLF DFLG '2& LQVROXELOLW\ DVVD\ OHIW SDQHO '2& LQVROXEOH IUDFWLRQ
JHQHUDWHGVWDUWLQJZLWKWKHVDPHQXPEHURIFHOOVLQHDFKFDVHZDVVHSDUDWHGRQ6'63$*(DQG
WUDQVIHUUHGRQ QLWURFHOOXORVH PHPEUDQH 7KH EORWV ZHUH GHYHORSHGXVLQJUDEELWDQWLILEURQHFWLQ
DQWLERG\ 6LJPD E\ FKHPLOXPLQHVFHQFH $PHUVKDP %LRVFLHQFHV 7KH ILJXUH LV D
UHSUHVHQWDWLYH LPDJH RI WKUHH VHSDUDWH H[SHULPHQWV 7KH ORZHU EORW LQ WKLV SDQHO VKRZV WKH
FRUUHVSRQGLQJ VLJQDOV REWDLQHG XVLQJ PRXVH DQWL*$3'+ DQWLERG\ ORDGLQJ FRQWURO 3DQHO '
'HQVLWRPHWULF TXDQWLWDWLRQ FRUUHVSRQGLQJ WR WKH ZHVWHUQ EORW LQ 3DQHO & DQDO\]HG E\ WKH
,PDJH-VRIWZDUH7KHILJXUHUHSUHVHQWVPHDQ6'FDOFXODWHGIURPWKUHHVHSDUDWHH[SHULPHQWV
)LJXUH3DWLHQWGHULYHGOHQVHSLWKHOLDOFHOOVZHUHFXOWXUHGLQFRPSOHWHPHGLDIROORZHGE\VHUXP
VWDUYDWLRQ IRU WR K &HOOV ZHUH WUHDWHG ZLWK RU ZLWKRXW VF)Y LQ WKH SUHVHQFH RI 7*)ȕ
QJPO 3DQHOV$DQG%0RXVHDQWLFROODJHQW\SH, DQGPRXVHDQWLȕLQWHJULQ
DQWLERGLHV ZHUH XVHG UHVSHFWLYHO\ IROORZHG E\ LQFXEDWLRQ ZLWK ),7& FRQMXJDWHG DQWLPRXVH
DQWLERG\ 0HDQ IOXRUHVFHQW LQWHQVLW\ RI WKH FHOOV ZDV DFTXLUHG RQ %' )$&6&DQWR
V\VWHP0),RIWKHFRQWUROZDVWDNHQDV3DQHOV&DQG'0RXVHDQWL1FDGKHULQ
DQG PRXVH DQWLDOSKD60$ DQWLERGLHV ZHUH XVHG UHVSHFWLYHO\ IROORZHG E\ LQFXEDWLRQ
ZLWK ),7& FRQMXJDWHG DQWLPRXVH DQWLERG\ )OXRUHVFHQFH LQWHQVLW\ ZDV TXDQWLILHG RQ
9,&725 ;PXOWLODEHO(/,6$UHDGHU'DWDZDVSORWWHGDVSHUFHQWIOXRUHVFHQFHLQWHQVLW\7KH
ILJXUHUHSUHVHQWVPHDQ6'FDOFXODWHGIURPWKUHHVHSDUDWHH[SHULPHQWVSZDVFRQVLGHUHG
VWDWLVWLFDOO\VLJQLILFDQW3DQHO(&RQIRFDOLPDJHVFRUUHVSRQGLQJWRSDQHOV&DQG'
27
)LJXUH 3DQHO $ ,PPXQRVWDLQLQJWR VWXG\ WKH FRORFDOL]DWLRQRI(FDGKHULQDQGȕFDWHQLQLQ
WKH SUHVHQFH DQG DEVHQFH RI WKH VF)Y DQWLERGLHV XSRQ WUHDWPHQW ZLWK 7*)ȕ QJPO /HQV
HSLWKHOLDOFHOOVGHULYHGIURPWKHSDWLHQWV¶FDSVXOHVZHUHFXOWXUHGDQGWUHDWHGZLWKVF)Y71DQG
2LQWKHSUHVHQFHRI7*)ȕ QJPO IRUK&RQWUROVHWUHIHUVWRWKHFRQGLWLRQZHUHFHOOV
ZHUHWUHDWHGZLWK7*)ȕ QJPO DORQH5DEELWDQWLȕFDWHQLQDQGUDWDQWL(FDGKHULQDQWLERG\
ZHUHXVHGDVSULPDU\DQWLERGLHVIROORZHGE\),7&ODEHOHGDQWLUDEELWDQG75,7&ODEHOHGDQWL
UDWDQWLERGLHVXVHGDVVHFRQGDU\DQWLERGLHVUHVSHFWLYHO\'$3,ZDVXVHGDVDQXFOHDUG\H,QWKH
SUHVHQFH RI 71 FRORFDOL]DWLRQ RI ȕFDWHQLQ DQG (FDGKHULQ LVVHHQLQ\HOORZDORQJWKH FHOO
PHPEUDQH 3DQHO % :HVWHUQ EORW IRU H[DPLQDWLRQ RI ȕFDWHQLQ LQ F\WRVROLF DQG PHPEUDQH
IUDFWLRQV ZLWK DQWLȕFDWHQLQ DQWLERG\ ,Q WKH FRQWURO VHW ȕFDWHQLQ DSSHDUV WR EH PRUH
F\WRSODVPLF DV FRPSDUHG WR WKH FHOOV WUHDWHG ZLWK 71 ZKHUH ȕFDWHQLQ LV PRVWO\ FHOO
PHPEUDQHDVVRFLDWHG7KHFHOOVWUHDWHGZLWKWKHLUUHOHYDQWDQWLERG\2DSSHDUWREHVLPLODUWR
WKHFRQWUROFHOOV
)LJXUH(IIHFWRI71RQ7*)ȕLQGXFHGVLJQDOLQJWRPRQLWRU60$'VLJQDOLQJ 3DQHOV$
DQG% DQG)$. 3DQHO& S 3DQHO' (5. 3DQHO( DQG-1.SKRVSKRU\ODWLRQ 3DQHO)
RQWKHKXPDQOHQVHSLWKHOLDOFHOOV3ULPDU\FXOWXUHVRI/(&VZHUHJURZQWLOOFRQIOXHQF\
LQWKHSUHVHQFHRIWKHFRPSOHWHPHGLDRYHUQLJKWIROORZHGE\VHUXPVWDUYDWLRQIRUWRKDQG
WUHDWPHQW ZLWK VF)Y DQWLERGLHV RU WKH UHVSHFWLYH LQKLELWRUV ,1+ DV GHWDLOHG LQ WKH PHWKRGV
VHFWLRQ &RQWURO VHW LQGLFDWHV DEVHQFH RI VF)Y WUHDWPHQW 3DQHO $ ,PPXQREORWV IRU WLPH
GHSHQGHQW60$'VLJQDOLQJDVVHVVHGE\SURELQJZLWKSKRVSKR60$'60$'DQGEHWDDFWLQ
LQWHUQDOORDGLQJFRQWURO DQWLERGLHVUHVSHFWLYHO\6,6DVSHFLILF60$'LQKLELWRUZDVXVHGDV
DSRVLWLYHFRQWURO3DQHO%&RQIRFDOLPDJHVRI/(&VWUHDWHGDVDERYHDQGLQFXEDWHGZLWKDQWL
SKRVSKR60$' DQWLERG\ IROORZHG E\ ),7& FRQMXJDWHG VHFRQGDU\ DQWLERG\ 3DQHOV &)
,PPXQREORWVVKRZLQJUHGXFWLRQLQSKRVSKRU\ODWLRQRI)$.S(5.DQG-1.ZLWK71DV
FRPSDUHGWRWKHFRQWURO7KHOHYHORIWKHWRWDOSURWHLQVLQHDFKFDVHZDVXQDOWHUHGHLWKHULQWKH
SUHVHQFHRUWKHDEVHQFHRIWKHVF)YDQWLERG\%HWDDFWLQZDVXVHGDVORDGLQJFRQWURO
)LJXUH6FKHPDWLFGLDJUDPKLJKOLJKWLQJWKHLPSRUWDQFHRIILEURQHFWLQW\SH,,,OLNHUHSHDWV
RI7HQVFLQ&LQWKHFRQWH[WRIILEURVLV7KHUHSHDWVDUHNQRZQWRELQGWRGLIIHUHQWGRPDLQV
ZLWKLQ ILEURQHFWLQ DV ZHOO DV JURZWK IDFWRUV VXFK DV 3'*) )*) 7*)ȕ QHXURWURSKLQV
28
UHVXOWLQJ LQ ILEURQHFWLQ SRO\PHUL]DWLRQ DQG (&0 PRGXODWLRQ ,QFUHDVHG ILEURQHFWLQ
SRO\PHUL]DWLRQ UHVXOWV LQ DGKHVLRQGHSHQGHQW FHOO JURZWK DQG LQFUHDVHG 003 H[SUHVVLRQ 7KH
ODWWHU FDXVHV FOHDYDJH RI FROODJHQ ,9 LQ WKH EDVHPHQW PHPEUDQH DQG WKHUHE\ IDFLOLWDWHV FHOO
PLJUDWLRQ%HVLGHVWKHLQFUHDVHG003H[SUHVVLRQFDXVHVFOHDYDJHRI7*)ȕELQGLQJSURWHLQ
UHVXOWLQJ LQ DFWLYDWLRQ RI 7*)ȕ ZKLFK LV DYDLODEOH WR IXUWKHU ELQG WR 7HQDVFLQ& DQG DOVR
IDFLOLWDWHWKH(07SURFHVV
)LJXUH 7*)ȕ LV D NH\ PHGLDWRU RI ILEURVLV 2Q WKH RQH KDQG LW UHJXODWHV WKH LQWHJULQ)$.
SDWKZD\LQFUHDVHGSKRVSKRU\ODWLRQRI)$.FDXVHVLQFUHDVHGOHYHOVRIH[SUHVVLRQRILQWHJULQDQG
ILEURQHFWLQDVZHOODVGLVUXSWLRQRI(FDGKHULQDQGȕFDWHQLQRQFHOOFHOOMXQFWLRQVDORQJZLWK
LQFUHDVHGH[SUHVVLRQRI003OHYHOVUHVXOWLQJLQLQFUHDVHGFHOOPLJUDWLRQ)XUWKHU)$.FDQOLQN
LQWHJULQLQLWLDWHG VLJQDOV WR WKH PLWRJHQDFWLYDWHG SURWHLQ NLQDVH 0$3. SDWKZD\ 7KH ILJXUH
LOOXVWUDWHVWKHYDULRXVNH\VWHSVEHLQJUHJXODWHGE\WKH71DQWLERG\
/HJHQGVWRVXSSOHPHQWDU\ILJXUHV
6XSSOHPHQWDU\ )LJXUH &KDUDFWHUL]DWLRQ RI DQWL71IQ,,, SKDJHGHULYHG VF)Y DQWLERGLHV
3DQHO $ +LVWRJUDP VKRZLQJ (/,6$ YDOXHV UHFRUGHG IRU DQWLERG\FRQWDLQLQJ SHULSODVPLF
IUDFWLRQV RI FORQHV H[SUHVVHG LQ +% FHOOV ELQGLQJ WR FRDWHG 71IQ,,, 7KH \D[LV
VKRZVWKHRSWLFDOGHQVLW\DWQP7KHVHOHFWHGFORQH71LVPDUNHGZLWKDQDUURZ3DQHO%
&RRPDVVLHVWDLQHG 6'63$*( JHO VKRZLQJ SXULILHG 71 OHIW DQG LWV FRUUHVSRQGLQJ
ZHVWHUQ EORW ULJKW SUREHG ZLWK PRXVH DQWL+LV PRQRFORQDO DQWLERG\ IROORZHG E\ DQWLPRXVH
+53FRQMXJDWHGVHFRQGDU\DQWLERG\3DQHO&%LQGLQJRIYDULDQWVRI71IQ,,,ZLWKWKHVF)Y
71ODQHPROHFXODUZHLJKWPDUNHUODQH71IQ,,,ODQH71IQ,,,ODQH71IQ,,,
ODQH71IQ,,,
6XSSOHPHQWDU\ )LJXUH &KHFNLQJ WKH FURVVUHDFWLYLW\ RI 71 ZLWK RWKHU (&0UHODWHG
SURWHLQV3DQHO$+LVWRJUDPVKRZLQJ(/,6$YDOXHVUHFRUGHGIRUELQGLQJRI71DQWLERG\WR
ILEURQHFWLQ ILEULQRJHQ RVWHRSRQWLQ DQG ILEURQHFWLQOLNH W\SH ,,, UHSHDWV RI 7HQDVFLQ
71IQ,,, 7KH \D[LV VKRZV WKH RSWLFDO GHQVLW\ DW QP 3DQHO % 2VWHRSRQWLQ DQG
71IQ,,,ZHUHHOHFWURSKRUHVHGWUDQVIHUUHGRQQLWURFHOOXORVHPHPEUDQHDQGSUREHGZLWKHLWKHU
VF)Y 7Q ODQHV DQG RU VF)Y 2 ODQHV DQG 3DQHO & )LEULQRJHQ DQG ILEURQHFWLQ
ZHUH DOVR FKHFNHG IRU ELQGLQJ WR 71 /DQHV DQG ZHUH SUREHG ZLWK VF)Y 71 ZKHUHDV
29
ODQHVDQGZHUHSUREHGZLWK2 LUUHOHYDQWFRQWURO /DQHZDVSUREHGZLWKDQWLILEULQRJHQ
DQWLERG\ DQG ODQH ZLWK DQWLILEURQHFWLQ DQWLERG\ SRVLWLYH FRQWUROV /DQH 0 LQGLFDWHV WKH
PROHFXODUZHLJKWPDUNHUV
6XSSOHPHQWDU\)LJXUH ,PPXQRVWDLQLQJRIKXPDQOHQVHSLWKHOLDOFHOOVWRYLVXDOL]HWKHSRVVLEOH
FRORFDOL]DWLRQ RI WKH VF)Y DQWLERGLHV ZLWK DQWLWHQDVFLQ& DQWLERG\ 6LJPD &KHPLFDOV /HQV
HSLWKHOLDO FHOOV ZHUH SUREHG ZLWK 71 2 QHJDWLYH FRQWURO DQG DQWL7HQDVFLQ& DQWLERG\
VF)Y71 DQG 2 ZHUH YLVXDOL]HG ZLWK FP\F),7& FRQMXJDWHG DQWLERG\ ZKHUHDV DQWL
7HQDVFLQ& DQWLERG\ ZDV YLVXDOL]HG E\ DQWLPRXVH3( FRQMXJDWHG DQWLERG\ ,PDJHV ZHUH
DFTXLUHGXVLQJ2O\PSXVFRQIRFDOPLFURVFRS\
6XSSOHPHQWDU\ )LJXUH (YDOXDWLRQ RI WKH HIIHFW RI 71 RQ WKH YLDELOLW\ 3DQHO $ DQG
SUROLIHUDWLRQ 3DQHO% RIWKH+/(%FHOOV+/(%FHOOVZHUHVHHGHGDWWKHGHQVLW\RI
FHOOVSHUZHOORIZHOOSODWHLQWKHSUHVHQFHRIFRPSOHWHPHGLD 0(0)%6 DQGJURZQ
RYHUQLJKW &HOOV ZHUH VHUXP VWDUYHG DQG WUHDWHG ZLWK VF)Y DQWLERGLHV DW GLIIHUHQW GRVHV DORQJ
ZLWK 7*)ȕ QJPO IRU K LQ VHUXP IUHH FRQGLWLRQ )RU FHOO YLDELOLW\ 077 DVVD\ ZDV
SHUIRUPHG VKRZLQJ QR VLJQLILFDQW GHFUHDVH LQ WKH YLDELOLW\ WLOO D FRQFHQWUDWLRQ RI XJPO RI
71 2 VKRZHG QR HIIHFW RQ YLDELOLW\ DW DQ\ FRQFHQWUDWLRQ )RU SUROLIHUDWLRQ %UG8
LQFRUSRUDWLRQDVVD\ZDVGRQH%UG8ZDVDGGHGDIWHUKRIDGGLWLRQRIVF)YDQWLERGLHVDQGFHOOV
ZHUH IXUWKHU LQFXEDWHG IRU K 71 VKRZHG VLJQLILFDQW GHFUHDVH LQ SUROLIHUDWLRQ VWDUWLQJ
IURP D FRQFHQWUDWLRQ RI XJPO VF)Y 2 KDG QR HIIHFW RQ SUROLIHUDWLRQ S ZDV
FRQVLGHUHGVWDWLVWLFDOO\VLJQLILFDQW
6XSSOHPHQWDU\ )LJXUH (YDOXDWLRQ RI H[SUHVVLRQ OHYHOV FROODJHQ W\SH, 3DQHO $ DQG ȕ
LQWHJULQ 3DQHO % E\ IORZ F\WRPHWU\ 3DWLHQW GHULYHG OHQV HSLWKHOLDO FHOOV ZHUH FXOWXUHG LQ
FRPSOHWHPHGLDIROORZHGE\VHUXPVWDUYDWLRQIRUWRK&HOOVZHUHWUHDWHGZLWKDQGZLWKRXW
VF)Y DQWLERGLHV DORQJ ZLWK 7*)ȕ QJPO )RU FROODJHQ W\SH, FHOOV ZHUH IL[HG
SHUPHDELOL]HG EORFNHG DQG LQFXEDWHG ZLWK PRXVH DQWLFROODJHQ W\SH, DQWLERG\ 6LJPD
&KHPLFDOV )RUȕLQWHJULQFHOOVZHUHKDUYHVWHGDQGLQFXEDWHGLQEORFNLQJEXIIHUIROORZHGE\
LQFXEDWLRQ ZLWK SULPDU\ PRXVH DQWLȕLQWHJULQ DQWLERG\ 6LJPD &KHPLFDOV ),7& FRQMXJDWHG
DQWLPRXVHDQWLERG\ZDVXVHGDVVHFRQGDU\DQWLERG\
30