ྴಉऔapoptosis ཥԍDNA ᘇຂດพҊǫٸಉऔਘϚDNA ཥຂဵԂ 180~200 bpǫшᙍҎܓڎDNA ࡌǫҽҎ႔ݑຩጛǫࣥзࡹոԍDNA ᘇຂ gallic aicd ǫ100 Njl/well ڀ6 well plate ྴτǫσӌ໋୲ȇ24Ǭ48 Ϯ 72 hȈ; кᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBS థࢨಉऔ
ԒࡌǫҽஉಉऔЧtrypsin ಷǫܤ37ʑ୲ዺτಷ2 Ϡខࡌǫஉಉऔѱ Η٧ǫкΌ1 ml PBS Чτڠtrypsin χՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpmᙽϿ5 ϠខǫчଟΙథటǫҽкΌ1 ml PBS థࢨಉऔǫ1500 rpm ᙽϿ5 Ϡ ខǫ॰Ιథట ǫкΌ200 Njl PBS మӔ֘ϣஉಉऔԶeppendorfǫ14000 rpm ᙽϿ1 ϠខǫβϿчΙథటǫкΌ200 Njl PBS or TE bufferǫஉಉऔᝃ֘
ϣǫҽкΌ4 Njl RNase Aȇ10 mg/mlȈǫ࠲ྌᓀ5 minǫஉಉऔτRNA ྂဵǫ ҽкΌProteinase K 20 NjlǫϮBinding solution 200 Njlǫܤ70ʑДǫ10 minǫ ҽкΌ100% EtOH 200 Njlǫvotex ֘ϣȇठԓటᡆվᐑҒԼᗰటݺǫߵ
ಉऔኞၯӤǫཥॕ՜DNA ܓڎވਆܷȈǫtransfer ԶSpin columnǫ14000 rpm ᙽϿ1 ϠខȇᙽϿࡌDNA ཥbinding ڀspin column τ໋ҒԼَቢȈǫҽкΌ 300 Njl Binding solutionǫ14000 rpm ᙽϿ1 ϠខǫҽЧ700 Njl Washing solution థࢨٶԒȇ14000 rpm ᙽϿ1 ϠខȈǫടࡌԒஉcolumn τ܃ԍటᡆᙽΗ ٧ǫσкұՍsolution ދ14000rpm ᙽϿ5minǫҽܤዺ3~5 minǫஉ columnҎዺτڎзࡌǫкΌ100 Njl Elution solutionȇElution solution መҺ
ܤ70ºCДкዎǫшቑкDNA ྂဵۻǫދڀఖ٣ҋ߲ҽڎзȈǫ࠲ྌᓀ2 Ϡ ខࡌǫ14000rpmᙽϿ5minǫᙽΗటᡆղࡹ֍ԍDNA χsolutionǭஉDNA ᆭ6×
DNA loading dye మӔȇ10 Njl DNA solutionɗ2 Njl 6× DNA loading dyeȈ loading Զ2% agaroseȇ1g agaroseǬ50ml 0.5× TBE bufferǬ10 Njl Eithidium bromideȈτǭີՀ႔ݑǫടࡌܢΌUV light Ηᔗࢎٗྚࣣᓦӯǭ
ȇύȈՂЈᗱᎷݛȇWestern blottingȈ ȓᙦᄻᡦȔ
ҺஉPVDF membraneກୁӪǫҽЧmethanolฤአᔯዅࡌǫҽΌᙦӈጎጹట ȇtransfer bufferȈτǫஉກӪވᘗધҺݧӞtransfer bufferτങҋǫஉ ᙦᄻ֟ѱ້ࡌǫԼॖΗǫஉਾᆖვЙҺЧtransfer bufferዅᔯٗᎅӞ֟ ࡌڎзᙦӈጙກчӤᎠϠǫᙦӈጙࡌЧ0.05% tween 20/1X PBS థࢨ10Ϡ
blocking ᡦǫЧ࠲ྌ1βࢸҰີՀǭڎзᙦӈጙࡌܤβ౦τЧ0.05 % tween 20/1X PBS థࢨ10ϠខҼ3Ԓǭ॰థࢨటǫкΌ8 mlވતדᡆȇྂ
ܤཛྷᗩᇐχblocking solutionτǫฬក।ኞ٠σӌדᡆԍ܃σӌȈǫ4 ʑႍڰ
ີՀབྷᕆǭႍϱڎзǫӚԆતדᡆǫЧ0.05% tween 20/1X PBS థࢨᙦӈጙ 10ϠខҼ3ԒǭкΌ8mlฬក10000।ވgoat anti IgGȇHRPȈ horseradish peroxidaseconjugated antibody Ήતדᡆȇྂܤ֍2% FBSވ0.05% tween 20/1X PBSτȈǫܤ࠲ྌΗབྷᕆີՀ1βǫടࡌڎзᙦӈጙࡌЧ0.05% tween 20/1X PBS ࢨథࢨ10ϠខҼ3Ԓǭ
ȓᓯЙᡦȔȇཝ܁τີՀȈ
உᙦӈጙݧܤECL ္ᏁχమӔటȇౖӑڎ1.5 mlาБ٦మӔȈτ1Ϡខϯ ᓿǭЧٶதעተЙᗰທڧۊܤcassette ϚǫᙦӈጙٗѼॖΙܢܤᓯЙуձ ȇcassetteȈٶதעተЙτ໋ǫЧHyperfilm ೣЙܤΙቢעተЙΙǫჰྎᙦӈ ጙີՀᓯЙǫ༹ҷ໋٠ᙦӈጙΙᑲҷߔࡂۊ໋߆ฤǫँ5 ࣵԶ1 βσ าǭ༹ҷְԂࡌܢΌᡀተᏁີՀᡀተᡦȇ໋٠ძሕᡷჯۊȈǫҽЧథД
؈ࢨ30ࣵࡌܢΌۊተᏁτǫၯ30 ࣵࡌҽЧథД؈ࢨ30 ࣵǭ
౺Ζ ಄षЈݛ
ძᡄ฿ܷЧѪ֘७ከྎȇmean ± SDȈҙǫ٣ҋStudent’s t-test ٧
ۊძᡄಋᆭჰྚಋχౝǭ*ҙpɝ0.05ǫҙ಄षΙٷᡀౝǭ
౺Ζഌ ࣫ة฿ܷ
ѸძᡄЧ்૯ύ।ήᇿȇgallic acidȈჰΊᜡάಉऔޛᕅNCI-H460ಉऔχ ተ៓ǫय़Һ෪္ύ।ήᇿჰNCI-H460ಉऔχҊ߆פڂϮӯࢩχተ៓ǫϮᙍ ҎࠗᄁȇmorphologyȈΙᡷჯಉऔχᠹϦǫҽתີժҋDAPI ࢀԼϮഹಉ औᏀጛ႔ݑȇsingle cell gel electrophoresisǰ Comet assayȈᡷჯಉऔࡹոԍ ಉऔཕȇDNA damageȈׅพҊϮಉऔউȇapoptosisȈດǭΗ٧ҽ
்૯ύ।ήᇿჰNCI-H460ಉऔಉऔຳනተ៓ǫٗժҋDNA ᘇຂϠ݂ȇDNA fragmentationȈǬ౿ጌᡆጙ႔ՆȇƩƺmȈǬࢩۻਲ਼ϦݹౙҊȇROS productsȈǬ ເᙽήកܢȇcalcium releaseȈǬЧϮউಿҒcaspase-3Ǭcaspase-8Ǭcaspase-9 ࢩۻχᔗ෪ǫ٧উၘৠ்૯ǫടࡌҽЧWestern blot ٧ᔗ෪উಿҒ፝ Сᒶ໋ᗱވۦ߆ǫҤ٣பಉऔӯࢩΗॕǫԭкᛧ24ᆭ48βࡌgallic cid ჰNCI-H460ಉऔވӯࢩ50%פڂᐑࡂȇIC50ȈϠըࢸ250Ϯ300NjMǭժҋ॰
౺Ή ύ।ήᇿӞΊᜡάಉऔޛᕅಉऔਪჰಉऔຳන(Cell cycle)ވተ៓
ժҋࢣӻಉऔषኞሱຈՎσӌᐑࡂވύ।ήᇿಷ24Ϯ48βࡌǫкΌ PIǫٗᡷჯٸಉऔຳනވǭพᒶᛧݹᐑࡂވቑкǫG0/G1නχಉऔБ ٦ԍຢ٧ຢӤވᖶǫྴgallic acidᐑࡂάܤ201NjMǫG0/G1නވಉऔБ٦Ι ϩǭҎԓшތǫύ।ήᇿшЧ٣ΊᜡάಉऔޛᕅಉऔϾ૽ಉऔຳනӞG0/G1න ވଭЎ(G0/G1 arrest)ǫԭСཥϾ૽ಉऔषดۻԔՙҋǫӞაτшطsub-G1 peakǭȇӬა3.4Ǭა3.5Ȉ
౺ Ζ ᔗ ෪ ύ । ή ᇿ Ӟ Ί ᜡ ά ಉ औ ޛ ᕅ ಉ औ ਪ ࡹ ո ౙ Ҋ ࢩ ۻ ਲ਼ Ϧ ݹ )Reactive oxygen species*
ࢩۻਲ਼Ϧݹȇreactive oxygen speciesǰROSȈӞᇟᏛಉऔউ
ྴτסᄴॸॐवވظԼȇJung et al., 2001Ȉǭ౿ጌᡆࡹROSౙҊވУवՆ
ԭΕڠಉऔވԔԍȇFleury et al., 2002Ȉǭ౿ጌᡆτROSވάຽౙҊȇROS burstȈཥ೮Ԃ౿ጌᡆވлિσسȇmitochondrial dysfunctionȈǭࢸΈᔗ෪ύ
।ήᇿᇟᏛχಉऔউࡹոڠROSވౙҊԍǫӞԓժҋࢣӻಉऔሱϠ݂ಉऔ τROSވౙҊǭժҋࢣӻಉऔषኞሱຈՎᐑࡂ250 NjMވύ।ήᇿಷσӌ໋
1Ǭ3Ǭ6Ǭ12Ǭ24βǫҽкΌH2DCFDAǫٗᡷჯٸౙҊࢩۻਲ਼Ϧݹވׅǭ
฿ܷพᒶ໋ވۦ߆ǫԋጌࣣჰӝԍѧވᖶȇӬა3.6Ȉǫҙಉऔ ވॕ՜Ǭࣷ೯ۻވᙦᠹȇpermeability transitionȈЧϮcytochrome c Ҏ౿ጌᡆ τកзڀಉऔ፝τǫԭᇟᏛಉऔউވౙҊǫࣚࡹᆭಉऔউȇapoptosisȈԍ
ȇXia etal., 1999ȈǭѸძᡄࡹЧࢣӻಉऔሱᔗ෪ࢀԍDiOC6 χNCI-H460ಉ Fluo-31 ܢзҷȇemissionȈވࡂཥᒶಉऔϚເᙽήᐑࡂވצᠹǫԭพජ зσӌࡂވᑲҷǫࡱшҋБ٦ݛ෪பۿދ෪பވᑲҷࡂபڀເᙽήᐑࡂ
౺ϛ ժҋቴࡺ္ᡄ)Comet assay*ᡷჯύ।ήᇿჰΊᜡάಉऔޛᕅಉऔ χDNA ৄสࡂ
ഹಉऔ႔ݑϠ݂ȇsingle cell gel electrophoresis assayȈൠࡹ܃ᒆވࡺ
္ᡄȇComet assayȈǫшҋ٧Ϡ݂ϮۊຽDNA ཕȇDNA damageȈสࡂǫ
ࡹॸᘸഹǬ೩ЧϮ௬༹ࡂଦވוೃǭժҋDNA damage ࡌพҊᘇຂǫᙍҎ ႔ݑஉᘇຂވDNAܝзጙѝǫׅԂࡺݺԭڥӓχǫҎԓшᙍҎܝַވ߆ฤǫ ᡷჯDNA ވཕׅǭ
உᐑࡂϠըࢸ50Ǭ100Ǭ250Ǭ500 NjMύ।ήᇿჰNCI-H460ಉऔՙҋ48β
ࡌǫ܃೮ԂಉऔDNA ഹޟᘇຂވสࡂǭӞഄۻჰྚಋкΌ1%DMSOǫ໑ۻ ჰྚಋкΌ5 NjM H2O2ǭЧቴࡺ္ᡄଷ෪ǫᡷჯಉऔܝַވׅǫᒶᐉᒞڎ100 ᗤಉऔᡷჯܝַݠǭ฿ܷҎა3.9พDZྴύ।ήᇿᐑࡂ༾ଦǫٸDNA ܝַ
ވݠ༾ॐǫҤҙٸDNA ཕވสࡂΟ༾ଦǭ
౺΅ ժҋDAPIࢀԼᡷჯύ।ήᇿჰΊᜡάಉऔޛᕅಉऔχಉऔউ!
DAPIȇ4 -6-diamidine-2-phenyl indoleȈࡹᅶਘᇿᑲҷࢀᏁǫٸཥஈۻ ވbinding ӞDNA ᙿޟᖜ௴χβ྅ȇminor grooveȈΙǫྴಉऔউཥз
ࢀԼ፝Ꮐ฿ȇchromosomes condensation ȈǬDNA ᘇຂȇDNA fragmentationȈ
ׅพҊǫठಉऔউຢॐǫߵDNA ᘇຂຢӤԭDAPI ࢀᏁൠཥࢀΙຢӤǫ Ӟᑲҷᡀ༵ᜋΗшᡷჯڀҒԼᑲҷࡂൠຢߔǭ
ΊᜡήάಉऔޛᕅNCI-H460ಉऔЧσӌᐑࡂȇ50Ǭ100Ǭ200Ǭ300Ǭ400 NjMȈχύ।ήᇿಷࡌǫԆಉऔЧDAPI ࢀᏁஉಉऔࢀԼǫժҋՆႴᡀ༵
ᜋǫᡷჯᑲҷࡂǭבॱתີᔗಉऔԔࡹոࡹҎಉऔউၘৠǫב ॱҎDAPI staining assayǫஉDNA ᙿޟᖜ௴β྅ȇminor grooveȈΙΙDAPI ᑲҷǫӞᑲҷᡀ༵ᜋΗᡷჯǫᆭჰྚಋБၟǫάᇿಷಋᑲҷܫᡀቑкǫ฿
ܷܤ48βύ।ήᇿๆωΗǫᒶވᐑࡂቑкшพᑲҷࡂԍቑкވດǫ ҒԼᑲҷܫᡀБொڂಋߔСቑވᖶзǫшܫᡀࣥзউβᡆȇapoptotic bodyȈވಉऔзȇӬა3.10Ȉǭᡀҙύ।ήᇿཥ೮ԂάಉऔޛᕅNCI-H460 ಉऔDNA fragmentation ԭ೮Ԃউǭ
౺ ժҋࢣӻಉऔሱᔗ෪ύ।ήᇿჰάಉऔޛᕅಉऔχಉऔউࣣಿҒ
፝Caspase-3ǬCaspase-8ϮCaspase-9
Ҏ߲ुძᡄ฿ܷᡀҙǫЧύ।ήᇿಷNCI-H460ಉऔǫཥ೮Ԃಉऔຳන ވଭᅀǫٗᇟᏛಉऔؿӒಉऔউೳৠǭCaspase-3ǬCaspase-8ϮCaspase-9 ࢸ cysteine protease family χ Ԃ ঠ ǫ ࡹ ୍ ᆭ ಉ औ উ χ У व ՙ ҋ ಿ Ғ ǭ Caspase-3 ވࢩϦཥߝ٣PARPȇpolyȇADP-riboseȈpolymeraseȈϠဵǫີԭ ೮ԂDNA fagmentation χౙҊǫ೮Ԃಉऔউχດǭ
ѸძᡄժҋPhiPhiLux-G1D1 kitȇOncoImmunin, Inc., Maryland, USAȈᔗ ෪casapse-3ǬCaspase-8ϮCaspase-9χࢩۻǭЧ250 NjM ᐑࡂχύ।ήᇿעω NCI-H460ಉऔ6~72βࡌԆಉऔǫкΌPhiPhiLux-G1D1 kit τχ୮፝ϯ ᓿǫЧ்૯ύ।ήᇿࡹոᇟᏛCaspase-3ǬCaspase-8ϮCaspase-9ࢩۻౙҊǭ ძᡄ฿ܷᡀҙǫЧύ।ήᇿಷχΊᜡάಉऔޛᕅNCI-H460ಉऔ6βࡌǫղ шܫᡀพcaspae-3ϮCaspase-8ࢩۻܫᡀቑкǫԭCaspase-9ߵܤ12βࡌพ
ࢩۻܫᡀቑкވׅǭҎԓшތǫύ।ήᇿшᇟᏛΊᜡάಉऔޛᕅNCI-H460 ಉऔχcaspase-3ǬCaspase-8ϮCaspase-9 ࢩۻቑкǫີԭߝ٣ಉऔউพҊ ȇӬა3.11Ǭ3.12Ǭ3.13Ȉǭ domain)ǰ౿ጌᡆၘৠࣣވ cytochrome CǬAIF Ϯ Apaf-1ǰϚ፝ᆒٿᐖᇷ
ၘৠࣣވ GADD153(growth arrest- and DNA damage-inducible gene 153,
औউވ caspase family ಿҒм Caspase-3ǬCaspase-8ǬCaspase-9ǫᡷჯ ٸಿҒຽᠹϦǰፊொಉऔউވ Bcl-2 family ಿҒǫмߝີಉऔউ
(pro-apoptotic)ވ Bax ڠ Badǫᆭפڂಉऔউ(anti-apoptotic)χ Bcl-2 ڠ Bcl-xl ǭ Ч GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ࢸ housekeeping gene Ϯ ǃ-actin ՙࢸ internal controlǭ
ǬಉऔጙԔڐᡆউၘৠވࣣಿҒ
Чᐑࡂ 250 NjM ύ।ήᇿಷΊᜡάಉऔޛᕅಉऔਪ NCI-H460 24 βǫժҋՂЈᎷᗱݛᡷჯพǫᒶ໋ވۦ߆ǫ FasǬFas-L Ϯ FADD ಿҒຽቑкȇӬა 3.14Ȉǭ
ΉǬ౿ጌᡆၘৠވࣣಿҒ
ྴಉऔউҎ౿ጌᡆၘৠ௫ǫཥҎ౿ጌᡆཥកз vcytochrome c ᆭ Apaf-1ǫີԭ೮ԂಉऔউǫٗࢩϦΗၘৠ caspase-9 ಿҒǭЧ ᐑࡂ 250 NjM ύ।ήᇿಷΊᜡάಉऔޛᕅಉऔਪ NCI-H460 24 βǫժ ժҋՂЈᎷᗱݛᡷჯพᒶ໋ވۦ߆ǫGADD-153 ᆭ GRP-78 ಿҒ
ຽΙϩȇӬა 3.16Ȉǭ
ћǬୱՀಉऔউވ caspase family ಿҒ
Чᐑࡂ 250 NjM ύ।ήᇿಷΊᜡάಉऔޛᕅಉऔਪ NCI-H460 24 βǫ ժҋՂЈᎷᗱݛᡷჯ caspase family ಿҒވຽǫ฿ܷพᒶ໋ވ
ۦ߆ǫcaspase-3 ಿҒຽΙϩǫcaspase-8 ಿҒຽΗॕǫcaspase-9
ಿҒຽΗॕȇӬა 3.17Ȉǭ
ύǬፊொಉऔউވ Bcl-2 family ಿҒ
Чᐑࡂ 250 NjM ύ।ήᇿಷΊᜡάಉऔޛᕅಉऔਪ NCI-H460 24 βǫ ժҋՂЈᎷᗱݛᡷჯ Bcl-2 family ಿҒވຽǫ฿ܷพᒶ໋ވۦ ߆ǫBcl-2 ڠ Bcl-xl ಿҒຽΗॕǫBad ڠ Bax ಿҒຽቑкȇӬა 3.18Ȉǭ
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౺ћഌ!૯ፌ
ύ।ήᇿࡹᅶٷӤ♆୮⇃ݹ፝ȇpolyhydroxyphenolic compoundȈǫӯ ӞӤԵසݹᆭДܷྴτǫႴᆏǬဖǬಯᆭॠᑫา[Sun J, 2002]ǭҺ
߲ވ࣫ةᛵძǫύ।ήᇿшЧథଟԵҎ୮[Inoue M,1994]ǫԭСჰҒԿ੨Ǭޛ ᕅᆭάကဆᕅಉऔވಉऔਪԍפڂቑҊވਆܷ[Kawada M, 2001; Salucci M,2002]ǫϮߜឿѼధϺಉऔ DNA ᗀդউ[Sohi KK, 2003]ǭәԓύ।ή ᇿٷԍߜឿѼಉऔǫْЎಉऔᕅϦވਆܷ[Taraphdar AK, 2001]ǭάಉऔޛ ᕅ )Large cell carcinoma* ᗟพҊՕ܃ԍປᕅಉऔވ 10%ǫәٸٷԍ ઊϚϠݓಉऔވۻǫᖆ׀ᆭޛᕅၟσӌǫٸႚࡌΟࡹ܃ԍߑβಉ
ܷพǫЧσӌᐑࡂχύ।ήᇿಷ24Ϯ48βࡌǫಉऔDNAϠՠǫᒶᐑࡂ ވ ቑ к ܤ 24β ࡌ ൠ ԍ พ Ҋ ᡀ ވ G0/G1 phase arrest ԭ S phaseϮ G2/M phaseԍෞ϶ވᖶพҊǫ࣎ԶєതΗG0/G1Ϯsub-G1 peakԭιǫԓດՖЦ
ࡹಉऔݛີΌS phaseԭ٣ಉऔຳනଭᅀӞG1 phaseǭΙुٛ฿ܷᇜܫΈ ύ।ήᇿཥܫᡀ೮ԂΊᜡάಉऔޛᕅಉऔcell cycle arrestǭ
ଟԓχѝǫύ।ήᇿ܃೮ԂΊᜡάಉऔޛᕅNCI-H460ಉऔވٛՙҋΟཥ ೮ԂಉऔࠗᄁΙވצᠹǫٛצᠹࡹո౻ӔಉऔউވࠗᄁከྎǫшЧᙍҎ DAPIࢀԼ٧ያᇘǭ฿ܷพǫܤύ।ήᇿಷ48β200 NjMᐑࡂΗшЧࣥطǫ ЧDAPIࢀԼࡌχٹࠗވউβᡆȇა3.10ȈǭЅᝏࡧзǫಉऔউࠗᄁΙཥԍ ಉऔ፝ޓݧݺȇcytoplasmic blebbingȈǬಉऔਘዢᕤȇnuclear shrinkageȈǬ
ࢀԼ፝Ꮐ฿ȇchromatin condensationȈǬಉऔѝࠗσೌߵȇirregularity in shapeȈ ЧϮԆᕤވၯส[Cohen, 1993]ǭ ᙦᠹȇpermeability transitionȈЧϮcytochrome cҎ౿ጌᡆτកзڀಉऔ፝τǫ ԭᇟᏛಉऔউވౙҊǫࣚࡹᆭಉऔউȇapoptosisȈԍȇXia et al., 1999Ȉǭ Ҏࢣӻಉऔሱᔗ෪ύ।ήᇿჰΊᜡάಉऔޛᕅNCI-H460౿ጌᡆጙ႔Նχተ
៓ǫ฿ܷพmitochondria membrane
potentialȇƩƺmȈӞ24βϚԍܫᡀΗॕȇა3.7Ȉǫᡀҙύ।ήᇿཥ٣Ίᜡ
άಉऔޛᕅNCI-H460ཕԭᏛग౿ጌᡆጙ႔Նވॕ՜ǫີԭᇟᏛಉऔউވพ Ҋǭ
٧࣫ةࡧзǫᕅ੨ϦᏙႚ္ْᏁᇟᏛχಉऔউࢃϠڠࢩۻਲ਼Ϧݹ ȇreactive oxygen speciesǰROSȈވౙҊЧϮਲ਼ϦᗂϯᓿވѪᑴᚦԍ
ȇXia et al., 1999; Kluck et al., 1997Ȉǭ౿ጌᡆࡹROSౙҊވУवՆǫٗС ROSӞᇟᏛಉऔউྴτסᄴॸॐवވظԼȇJung et al., 2001; Fleury et al., 2000Ȉǭ౿ጌᡆτROSވάຽౙҊȇROS burstȈཥ೮ԂDNAވཕǫԭߝ٣
ԍЅᝏࡧзǫ Ϛ፝ᆒτເᙽήވកзᆭ౿ጌᡆጙ႔Նȇ Mitochondrial membranes potentialǰ ƩƺmȈވॕ՜ԍǫәԓבॱժҋFluo-3/AMເᙽή
்କȇcalcium probeȈЧࢣӻಉऔሱᔗ෪ύ।ήᇿჰΊᜡάಉऔޛᕅNCI-H460 ಉऔເᙽήވកзވተ៓ǭ฿ܷพເᙽήӞ౺12βԍܫᡀកзވດȇა 3.8Ȉǫχࡌ೫ᅃࡗ൱ѪᑴǫՖЦΟᆭ౿ጌᡆጙ႔ՆȇƩƺmȈӞ24βϚ Ηॕԍǫәԓבॱ෪ǫӞ້ڿ߲න܃พχDNA damageȇҎcomet assay பތȈшિࡹҎcalciumވកܢчተ៓౿ጌᡆጙ႔Նވॕ՜ࡌǫ೮Ԃ౿ጌᡆ cytochrome cވកзԭ٣பಉऔพҊউވϯᓿǭ
Caspase-3 ࢸcysteine protease family χঠǫࡹ୍ᆭಉऔউχУव
ՙҋಿҒǭCaspase-3 ވࢩϦཥߝ٣PARPȓpolyȇADP-riboseȈpolymeraseȔ ϠဵǰЧϮICAD kinase ȇ Inhibitor of CAD kinase ȈࢩϦǫ ԭஉICADϡଟ ܢзCADȇcaspase-activated DnaseȈǫີԭ೮ԂDNA fagmentation χౙҊǫ ೮ Ԃ ಉ औ উ χ ດ ǭ ә ԓ ב ॱ ் ૯ ύ । ή ᇿ ჰ Ί ᜡ Ί ᜡ ά ಉ औ ޛ ᕅ (NCI-H460) ಉऔcaspase-3ࢩۻχՙҋǫพcaspase-3 ࢩۻӞ6βղԍΙ ϩވດǭLi าΊǫЧellagic acid ಷᆥવᕅT24 ಉऔǫพEllagic acid ཥ ࢩϦcaspase-3 χౙҊǫີԭᇟᏛಉऔউȇLi et al.,2005Ȉǭ
ಉ औ উ ԍ ٶ ᅶ У व ೳ ৠ DZ ౿ ጌ ᡆ ၘ ৠ ȇ Mitochondrial-initiated pathwaysȈЧϮಉऔጙԔڐᡆၘৠȇCell surface death receptor pathwayȈ ȇLi et al., 2005ȈǭҺ߲࣫ةᡀҙǫύ।ήᇿཥᏛगΊᜡάಉऔޛᕅNCI-H460 ಉऔ౿ጌᡆጙ႔ՆΗॕǫ٣ಉऔ൲ᙍ౿ጌᡆೳৠԭউǫܤάύ।ήᇿಷ
ࡌ ǫ pro-apoptoic protein—bax ಿ Ғ ຽ ԍ ቑ к ވ ດ ǰ Anti-apoptoic protein—bcl-2ಿҒຽԍॕ՜ވດǫᆭҺ߲࣫ةพp53 ވቑкǫཥ ᙦᒬࢩϦፊbax ୮әވౙҊȇMiyashita and Reed, 1995Ȉχ฿ܷࡹࣣ౻ވǫ ԭbaxཥᆭbcl-2฿Ӕԭ೮Ԃ౿ጌᡆጙ႔Նވॕ՜ߝ٣cytochrome c ࢣзԶಉ औ፝ǫcytochrome c ཥᆭApaf-1ȇapoptotic protease activing factor-1Ȉ฿Ӕǫ
ࡌࢩϦcaspase-9ǭCytochrome c ΟԍቑкވດǫԭࢩϦࠗᄁ caspase-9Ǭ caspase-3 ಿҒ೫ᅃቑкǭҙύ।ήᇿᇟᏛΊᜡάಉऔޛᕅNCI-H460 ಉऔapoptois χೳৠᆭҎ౿ጌᡆވউၘৠԍǭ
ѓѝǫѸძᡄพύ।ήᇿᇟพΊᜡάಉऔޛᕅNCI-H460ಉऔউވၯ สτCD95/Fas ϮࢩϦވcaspase-8 ຽቑкǫҙύ।ήᇿᇟᏛΊᜡήে
ᓄᕅCa Ski ಉऔވউၘৠᓿ့σєഹၘৠǫᡀҙύ।ήᇿᇟᏛCa Ski ಉ औވউၘৠۿΟڠಉऔጙΙFas ԍȇა3-14ȈǭಉऔጙΙԔڐᡆၘৠ ߵࡹࢩϦಉऔጙΙFasǬTNFR1 ϮTRAIL าԔڐᡆǫཥօϾಉऔϚވ Fas-associated death domainȇFADDȈᆭχ฿ӔǫٗСཥօϾpro-caspase-8 ׅ
ԂDISCȇdeath-inducing signaling complexȈǫີԭࢩϦcaspase-8ǫԭࡌދ
ࢩϦcaspase-3caspase-6ڠcaspase-7ǫۿޚࢩϦވcaspase-8 ཥஉಉऔ፝τBid ຂဵԂtBid ՆԶ౿ጌᡆ೮Ԃጙ႔Նॕ՜ǫԭพҊ౿ጌᡆೳৠވউϯᓿ ȇBudihardjo et al., 1999Ȉǭ
ᆌӔѸ࣫ةύ।ήᇿ܃೮ԂΊᜡάಉऔޛᕅNCI-H460ಉऔಉऔউވ
ດǫבॱፌύ।ήᇿшિ೯ၯٶᅶσӌވಉऔϯᓿᐉڂ٧פڂΊᜡάಉऔޛ ᕅNCI-H460ಉऔވቑҊǫύ।ήᇿшЧᙍҎp53 ࢩϦp21 ވၘৠԭ٣Ίᜡά ಉऔޛᕅNCI-H460ಉऔಉऔຳනଭᅀܤG0/G1 නǰѓѝǫύ।ήᇿЈॖཥᙍ Ҏເᙽήވកзǫተ៓౿ጌᡆވጙ႔Նີԭ٣cytochrome c ࢣзಉऔ፝τ೮ ԂಉऔউވพҊǫѓЈॖᙍҎಉऔጙԔڐᡆFas ވቑкǫԭࢩϦ caspase-8ǫີԭ೮ԂBid ވຂဵᙦᠹԂtBidǫΟཥተ៓౿ጌᡆጙ႔Նވॕ՜ǫ
ٛࡹύ।ήᇿ೮ԂΊᜡάಉऔޛᕅNCI-H460ಉऔ౿ጌᡆጙ႔Նॕ՜χᆌӔ
әǭࢩϦވcaspase-8 ΟшີԭࢩϦcaspase-3ǫᐖ૽caspaseᘚѩǫ٣ப laminsȇᅶਘϚಿҒȈೆॕဵǫࢀԼ፝ᐑᕤǬਘጙೆϠဵǫDNAೆϡഡԂ 180-200bpβЙ࢛ǫ೮Ԃಉऔউǭ
౺ύഌ!฿ፌᆭࡃᝳ!
!
Ѹ࣫ةτЧύ।ήᇿಷΊᜡάಉऔޛᕅNCI-H460ಉऔǫ்૯ύ।ήᇿވ דငዝࢩۻϮፊொχϠήᐉڂǭ฿ܷபތǫύ।ήᇿٷԍܫᡀפڂΊᜡάಉऔ ޛᕅNCI-H460ಉऔҊ߆ՙҋЧϮᇟᏛಉऔຳනଭᅀӞG0/G1 නڠಉऔউՙ ҋǭసΌ்૯Ϡήᐉڂǫพύ।ήᇿተ៓ϮፊொވϠήቢॖࣣྴቭǫм
ٛເᙽήވѪᑴǬ౿ጌᡆጙ႔Նވॕ՜ǬಉऔጙԔڐᡆFas تҿވԔ
ЧϮᙦᒬәήาǭЩಉϠ݂ٛڐተ៓ވϠή໋ࣣόވᕻۻǫ Ҏძᡄ฿
ܷшபތǫύ।ήᇿๆωΊᜡάಉऔޛᕅNCI-H460ಉऔ12βϚൠพເᙽ ήވកзǫԭተ៓౿ጌᡆވጙ႔Նີԭ٣cytochrome c កз೮Ԃಉऔউވ พҊǫٗС೮ԂDNAཕǫߝ٣p53ಿҒވࢩϦີԭ٣ಉऔଭᅀܤG0/G1
නǰѓЈॖ٣ப౿ጌᡆউၘৠވີՀǰСבॱΟพಉऔጙԔڐᡆFas ވቑкǫߝ٣pro-caspase-8 ࢩϦǫԭދࢩϦcaspase-3ԭ೮Ԃಉऔউ
)Ӭა3.19*ǭ
Ѹ࣫ةτǫבॱያძᛵܫΈύ।ήᇿވדΊᜡάಉऔޛᕅNCI-H460ಉऔ ਪငዝࢩۻǫࡹҎᇟᏛಉऔউވพҊǫϮಉऔຳනވଭᅀǭәԓǫύ।ή ᇿᗟۏѹિҜղᓿҋܤᖆ׀ᕅ੨ݦᕄΙǫϔٷԍพ্Ԃཛྷᜡᕅ੨ϦᏙႚْ
္ᏁވΑǭ
୍ԬЅᝏ
!
ጸҊһ୦ጸҊ಄ष၎ᆒ(Health and National Health Insurance Annual Statistics Information service)(2005-2006)DZѾ୦94-95Ӻ୦ΊУवԔә಄ष၎
ਈ (http://www.doh.gov.tw/statistic/index.htm)
ᐡǫംܫǫѼᐒ)2000*ǫᆬᡳވසݹ㓆ǫՀࡰଚၤཧڸঠཥǬτѾ ୦ᕀდᆏϦڇཥጊӈǭ(http://subject.forest.gov.tw/species/gall/)
!
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Concentrations of gallic acid (PM)
0 50 100 150 200 300 400 500
Relative of cell viability (%)
0
Concentrations of gallic acid (PM)
0 50 100 150 200 300 400 500
Relative of cell viability (%)
0
ა3.1 σӌᐑࡂχύ।ήᇿgallic acidಷNCI-H460ಉऔ24βӯࢩǭ٣ҋ σӌᐑࡂވύ।ήᇿϠըಷ24βࡌǫӞΊᜡάಉऔޛᕅಉऔਪ܃
ᔗ෪ڀވӯࢩׅǭኞᏬ฿ܷЧmean±SD㨤ҙǫn=3 ( * p<
0.05ǫ** p< 0.01) ǭ
ა3.2 σӌᐑࡂχύ।ήᇿgallic acidಷNCI-H460ಉऔ48βӯࢩǭኞᏬ
฿ܷЧmean±SD 㨤ҙǫn=3 ( * p< 0.05ǫ** p< 0.01) ǭ
IC50: 250 ȝM
IC50: 300ȝM
ა3.3 ժҋ॰ҜӻࣣՆᡀ༵ᜋᡷჯಉऔވࠗᄁǭкΌσӌᐑࡂވύ।ήᇿಷ
24 βࡌǫพᒶᛧݹᐑࡂވΙϩǫಉऔኞҕԍܫᡀΗॕވᖶǫ СԍಉऔࠗᄁσְᏵǬಉऔጙዢᕤᆭಉऔޓݧϦވດǫܢά।200 Xǭʗ
100 NjM
Control 200 NjM
300 NjM 400 NjM 500 NjM
A
B.
Control 50 NjM 100 NjM
200 NjM
150 NjM 300 NjM
C.
ა3.4 σӌᐑࡂχύ।ήᇿgallicaicdჰNCI-H460ಉऔಉऔຳනϠՠተ៓-12β
ǭኞᏬ฿ܷЧmean±SD 㨤ҙǫn=3 ( * p< 0.05ǫ** p< 0.01) ǭ
A.
B.
Control 50 NjM
100 NjM 150 NjM 200 NjM
C.
ა3.5 σӌᐑࡂχύ।ήᇿgallicaicdჰNCI-H460ಉऔಉऔຳනϠՠተ៓-48β
ǭኞᏬ฿ܷЧmean±SD 㨤ҙǫn=3 ( * p< 0.05) ǭ
A.
B.
C.
ა3.6 ժҋࢣӻಉऔषኞሱຈՎΊᜡάಉऔޛᕅಉऔҎๆω250NjMχύ।ή ᇿӞσӌ໋܃೮ԂROS ౙҊވተ៓ǭ฿ܷพԋጌԍѧވᖶ(ა AǬ B)ǫҙٗ؉ԍࢩۻਲ਼ϦݹROSౙҊቑкވດ(აC)ǭ
$
%
C.
ა3.7 ΊᜡάಉऔޛᕅಉऔҎๆωᐑࡂ250NjMχύ।ήᇿ܃೮Ԃ౿ဆᡆጙ႔ ՆȇƩƺmȈᠹχຽϦაǭ฿ܷพᒶ໋ᗱވۦ߆ǫMMP ኞ७Ο ᒶχΗॕǫӞ24βǫಉऔ౿ጌᡆጙ႔Նވॕ՜ടܫᡀǭኞᏬ฿ܷЧ mean±SD 㨤ҙǫn=3 ( * p< 0.05) ǭ
$
%
ა3.8 ΊᜡάಉऔޛᕅಉऔҎๆωᐑࡂ250NjMχύ।ήᇿ܃೮Ԃເᙽήᐑࡂ ΙϩވຽϦაǭ฿ܷพᒶ໋ᗱވۦ߆ǫCa2+ኞ७ΟᒶχΙϩǫ
ҙເᙽήᐑࡂቑкǭኞᏬ฿ܷЧmean±SD 㨤ҙǫn=3ǭ( * p< 0.05ǫ
** p< 0.01)ǭ
ა3.9 Ҏσӌᐑࡂވύ।ήᇿಷΊᜡάಉऔޛᕅಉऔ48βࡌǫЧቴࡺ္
ᡄϠ݂ٸჰಉऔDNA ৄވสࡂǭഄۻჰྚಋкΌ1%DMSOǫ໑ۻჰྚಋкΌ 5 NjM H2O2ǫՙҋ24 βǭ
Positive Control (5 NjM H2O2)
Negative Control (1% DMSO)
50 NjM
100 NjM 250 NjM 500 NjM
ა3.10 σӌᐑࡂވύ।ήᇿಷΊᜡάಉऔޛᕅNCI-H460ಉऔ 24βࡌDAPIࢀԼᡷჯ
A.
Control 50 NjM 100 NjM
200 NjM 300 NjM 400 NjM
B.
C.
ა3.11 ύ।ήᇿჰჰΊᜡάಉऔޛᕅಉऔcaspase-3ࢩۻχተ៓
A.
B.
C.
ა3.12 ύ।ήᇿჰჰΊᜡάಉऔޛᕅಉऔcaspase-8ࢩۻχተ៓
A.
B.
C.
ა3.13 ύ।ήᇿჰჰΊᜡάಉऔޛᕅಉऔcaspase-9ࢩۻχተ៓
ა3.14 ժҋՂЈᎷᗱݛᡷჯᆭapoptosisԍވFasǬFas-LǬFADDވಿ
ҒຽᠹϦǫЧGAPDHྴՙinternal controlǭ
ა3.15 ժҋՂЈᎷᗱݛᡷჯᆭapoptosis ԍވcytochrome cǬAIFǬApaf-1 Ϯcaspase-9ಿҒຽᠹϦǫЧGAPDHྴՙinternal controlǭ
Fas
Fas-L
FADD
GAPDH
GAPDH Caspase-9 Apaf-1
AIF Cytochrome c
ა3.16 ժҋՂЈᎷᗱݛᡷჯᆭapoptosis ԍވGADD153ǬGRP78ಿ
ҒຽᠹϦǫЧ GAPDH ྴՙ internal controlǭ
ა3.17 ժҋՂЈᎷᗱݛᡷჯᆭapoptosis ԍވcaspase familyಿ
ҒຽᠹϦǫЧ ǃ-actin ྴՙ internal controlǭ
Caspase-8
Caspase-9
Procaspase-3 Cleaved caspase-3
ǃ-actin
ა3.18 ժҋՂЈᎷᗱݛᡷჯᆭapoptosis ԍވBcl-2 familyಿ
ҒຽᠹϦǫЧ ǃ-actin ྴՙ internal controlǭ
ǃ-actin Bcl-xl Bcl-2
Bax Bad
!
!
ა 3.19 ύ।ήᇿᇟᏛΊᜡάಉऔޛᕅ NCI-H460 ಉऔਪಉऔຳ නଭᅀϮಉऔউχၘৠა (ጌҴۏѹძᡄᛵძ)