ABSTRACT
GV RODWLRQ URZWK VVD\KU3RVWH[SRVXUHWR+'
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Reagents
530, WLVV ZHUHREWDLQHGIURP 3KDUPDFLD3LVFDWDZ 0DQQKHLP ,QGLDQD &RXOWHU &RUSRUDWLRQ ZHUHSXUFKDVHGIURP &$6 UHJLVWU\ %LRORJLFDO&HQWHU$
Lymphocyte Isola
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INTRODUCTION
dGLFKORURGLHWK\OVXOÀGH+'LVDSRWHQWYHVLFDWLQJDJHQW IEOLVWHUIRUPDWLRQLVSRRUO\XQGHUVWRRG0LFURVFRSLFH[DPL LQWLPDWHV WKH LQYROYHPHQW RI SURWHRO\VLV LQ WKH FDVFDGH RI H HSLGHUPDO²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ÁXRURJHQLF UHWKHELRPDUNHUSURWHRO\WLFDFWLYLW\LQERWK3%/VDQG+(.V RVHG WR +' 7KH PHDVXUHPHQW RI SURWHRO\WLF DFWLYLW\ ZDV VSHFWURSKRWRPHWULF DQGÁXRURPHWULFPHWKRGV6XLWDELOLW\RI VFUHHQLQJRISRWHQWLDOPHGLFDOFRXQWHUPHDVXUHHIÀFDF\ZLOO
MATERIALS AND METHODS
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tion
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DSSURYHGKXPDQXVHSURWRFRO7KHO\PSKRF\WHVZHUHLVRODWHG FHQWULIXJDWLRQ 0HLHU DQG -RKQVRQ DQG WKH DVFROOHFWHGZDVKHGDQGGLOXWHG LQ530,FRQWDLQLQJJHQWD R D ÀQDO FRQFHQWUDWLRQ RI v FHOOVPO &HOOV ZHUH WKHQ FXOWXUHSODWHVDWWKHDSSURSULDWHGHQVLW\IRUH[SHULPHQWDWLRQ
Keratinocyte Grow
1RUPDOKXPDQH IURP&ORQHWLFV7KH FXOWXUHSODWHVDWWKH
HD Exposure
+'ZDVGLOXWHGL DQGDGGHGWRWKHDSS ÀQDO+'FRQFHQWUDWL WR DOORZ K\GURO\VLV LQFXEDWRUXQGHUDKX VXUHLQFXEDWLRQSHULR
Chromozym® Ass
Keratinocytes
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Lymphocytes
6XSHUQDWHQ QO RI v YROXPHRIQO7 RIVXSHUQDWHZDVUHP ZDVSODFHGLQWKHP QPZDVGHWHUP
Intracellular Prote
$WKUSRVWH[S DGGHGWRWKHDSSURSU IRUKU DWS&)OX SODWHUHDGHUZLWKH[F
7KHFRPSRXQGV
th
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ay (24 hr Postexposure to HD)
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LFURSODWHVFDQQLQJVSHFWURSKRWRPHWHUDQGWKHDEVRUEDQFHDW LQHG
ase Activity
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RESULTS
XVHGLQWKLVLQYHVWLJDWLRQDUHIRXQGLQ)LJXUH
Chromozym® Ass
3URWHDVH DFWLYLW\ QXPEHUDQGv )LJXUH :KH UHVSRQVHZDVQREHW
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+(.WKDWKDGEH LQFUHDVHLQXURNLQDV )LJXUH7KHUH VLVWHQWZLWK SUHYLRX VWXGLHVFRQGXFWHGE
CellProbe® Assay
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ay
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LGDVHZHUHK\GURO\]HGVLJQLÀFDQWO\DERYHWKHFRQWUROYDOXHV 39 DQG ' DPLQRSHSWLGDVH VKRZHG D GHFUHDVH LQ HQ]\PH 6XEVWUDWHV$$PLQRSHSWLGDVH DQG5$PLQRSHSWLGDVH% FUHDVHLQDFWLYLW\ZKLOHVXEVWUDWH/$PLQRSHSWLGDVHVKRZHG
3URWHDVH SURÀOHV VKRZHG D VLJQLÀFDQ
SUMMARY
ZHUH GLIIHUHQW LQ WKH WZR LQ YLWURPRGHOV WHVWHG KHUH 3%/V W LQFUHDVH LQ SURWHRO\VLV XVLQJ WKH VHULQH SURWHDVH VXEVWUDWHV
&KURPR]\PŠ7+DQ LQ RXW RI WKH G JHQHUDO GHFOLQH LQ D VWUDWHV 6PLWKHWDO ZHUHDVVHVVHG1RFK WR +'ZKHQ +(.V +(.VDVVD\HGLQVLWX *DPLQRSHSWLGDVHDQ QRSHSWLGDVHZDVXQF GXH WRFXOWXULQJDQG PHWKRGWRVFUHHQFR GHWHUPLQHWKHFDXVH
G&KURPR]\PŠ75<5HSURGXFLEOHSURWHDVHDFWLYLW\ZDVIRXQG RQRUV WHVWHG:H KDYH SUHYLRXVO\ UHSRUWHG WKDW 3%/V VKRZ D OO DPLQRSHSWLGDVH DFWLYLW\ WHVWHGZKHQ XVLQJ ÁXRUHVFHQW VXE 3URWHDVHSURÀOHVZHUHPRUHFRPSOLFDWHGZKHQ+(.V DQJHVLQDPLQRSHSWLGDVHDFWLYLWLHVZHUHGHWHFWHGDIWHUH[SRVXUH ZHUH WU\SVLQL]HG DQG DQDO\]HG E\ ÁRZ F\WRPHWU\ +RZHYHU LQPLFURSODWHVVKRZHGDQLQFUHDVHLQ.DPLQRSHSWLGDVH%DQG GDGHFUHDVHLQHODVWDVHDQG'DPLQRSHSWLGDVH$EXW/DPL KDQJHG7KHUHDVRQIRUWKLVYDULDELOLW\LVXQNQRZQEXWPD\EH KDQGOLQJSURFHGXUHV%HIRUH+(.FDQEHXVHGDVDQ LQYLWUR PSRXQGVDFRQVLGHUDEOHDPRXQWRIUHVHDUFKPXVWEHGRQH WR RIWKHVHYDULDELOLWLHV
REFERENCES
