NF-ljB ࢸಉऔਘτॐवᙦᒬәήǫՆܤಉऔ፝τཥᆭٸפڂᏁIljB ȇ Inhibitor of ljB Ȉ ฿ Ӕ ǫ ٗ σ ٷ ԍ ࢩ ۻ ވ ǫ ྴ ಉ औ ڐ proinflammatory cytokinesǬgrowth factorǬoxidative stress Ϯultraviolet light ٿᐖǫཥࢩ ϦIljB kinaseȇIKKȈcomplexǫᝠԭஉIljB ᕎᇿϦǫཥೆE3 ubiquitin ligase
ີՀubiquitinationǫٗСIljB ཥೆproteasome ॕဵǫԭកзNF-ljBǫᙽވ NF-ljB ղшtranslocate Զಉऔਘτǫተ៓ࣣ୮әᙦᒬՙҋǭಉऔτടУव ވNF-ljB ࡹҎRel-Aȇp65ȈǬNF-ljBȇp50ȈׅԂχheterodimerȇSuh and Rabson, 2004ȈǭڐNF-ljB ܃ፊொވࣣ୮әԍǫᆭפڂಉऔউԍχBcl-xLǬBcl-2 ȇChiao et al., 2002ǰLemarie et al.,2004ǰLi et al., 1999ȈǫᆭಉऔҊ߆ԍ
χIL-6ȇZerbini et al., 2004ȈϮc-mycȇSuhand Rabson, 2004ȈЧϮcyclin D1 ȇAlbanese et al., 1995Ȉา୮әࡹڐNF-ljB ܃ፊொǭ
G. p53 פᕅ୮ә 1. p53 ୮ә
p53୮әǫՆܤΊᡆ౺17ჰࢀԼᡆฤᖀI7p13.1ވՆǫࢸҎ
ϛрڀٶॸᡭ୮ჰȫղ16-20 kilobase (kb)Ȭ܃ಋԂވಉऔчਲ਼ਘᗇਘᇿ (DNA)Ϡήǫࡹፊொಉऔቑވ୮әǭΊᜡވp53୮әǫӞີϦၯสߜӯΗ٧ ވ୮әୋ࢛ǫΟࡹӞΊᜡӤᕅ੨τǫടೆพพҊࣸᠹވՆǫУवҎ11 ॸ־ҿ܃ಋԂǫٸ౺־ҿࢸߑஆዯୋǫՆܤ౺2-11־ҿ 8-10kbވՆǫ൵ۻငዝँຣၯ90%ވඛۻࣸᠹ(substitution mutations)ǫ Մܤ־ҿ5-8ǭp53ಿҒ፝ԍ5ॸଦᓅ(>90%)ߜୋ࢛ǫϠըՆܤ୮ ᇿ13-19, 117-142, 171-181,234-258ЧϮ270-286ǫٸτ୮ᇿ175Ǭ248Ǭ273 Ϯ282าՆǫΕࢸࣸᠹพҊᓅടଦވ"ћॸዎᗱ" (hotspots) ۿ୮ᇿ248Ǭ 273Ǭ175Ǭ245Ǭ249Ǭ282Ǭ176Ǭ179Ǭ220Ϯ213าՆǫࢸࣸᠹพҊᓅ
ടଦވ"Γॸዎᗱ" ( IARC, 2002 )ǭพҊܤٛ"ዎᗱ"ஆዯވࣸᠹՕ܃ԍp53ࣸ
ᠹχάҴǭӞάӤኞވငዝǫp53ވჰ୮әࣚӌพҊࣸᠹǫшિॸพ ҊᗱࣸᠹǫѓॸߵพҊիෞۻࣸᠹǭΊᜡάӤኞވp53ࣸᠹឝܤᇛ༷ࣸᠹ (missense mutation)ǫЧगౙҊॸ฿ᄣϮۻ፝צᠹΈވಿҒ፝ǫ p53ވ
Γॸዎᗱχұୋ࢛ѶพҊࣸᠹǫ p53ᗍ฿ୋղളџٸᗍ฿િΑǫີԭџч
ӤǭЅᝏോւ܃ҙǫڿp53୮әӞಉऔቑǫУवፊಉऔຳනҎG1නၯ
ڀSනǫСۊಉऔࡹոҎಉऔউ(apoptosis: programmed cell death) ԵగԭǭҊݹᏙΙވϠ݂ᡀҙǫӞፊொѼಉऔຳනވၥᙦΙǫp53୮әס ಉԿᅼᘀதџፊ੨(ataxia telangiectasia: AT)ވቲղࢸDNAॼိԍᚉǫல ޚࡇܧᑘலғጜᕅǫӌٛலޚفΙވಉऔǫڐܢোጌྚোࡌǫٸp53୮
әೆᇟพވϯᓿࡇǫGADD45୮әވౙҊԵΟൠڐڀተ៓ǭp53୮әшә
ވ฿ӔՆೆᄉ௲ǫ٣ࣸᠹp53ಿҒ፝ݛׅԂћॖᡆވ฿ᄣǭ
!!!!!!!!!!!!!!!
౺Ή ࣫ةҕވ
ޛᕅވݦᕄǫӞϕЉϔࡹॸබЂވᚉǫәࢸԍԤϠχ΅ΓЧΙވல ޚǫ۪۪ຑᘇзޛᕅιࡹ੦੧ၟѺනǫԭݛ٣ҋЂೃۿܢোݦᕄาִᕄ ݛкЧਜݦǭЧ cisplatin ࢸУވϦᏙݦᕄӞၯчΉΓӺ٧Чቭݨ٣ҋӞβಉऔ ϮߑβಉऔވޛᕅலޚǫԭٸԂਆєࡹǭࢸצ๊ϦᏙݦᕄވԂਆǫӤ ཛྷވϦᏙݦᕄᛧݹ(Ӭ: gemcitabine, decetaxel)ೆ࣫พз٧ǫСᆭ cisplatin Ӕ
٪٣ҋǫพٸݦᕄޛᕅވਆܷБഹҋ cisplatine תࢸᡀǫСિۦ߆ޛᕅல ޚވҊڥǭԭǫҎܤٛϦᏙݦᕄᛧݹވ࢜ۻǬୂՙҋάǫᏁຽΙσિၯ caspase-3Ǭ8 Ϯ 9ǫടࡌ٣ވಉऔؿӒউȇapoptosisȈǫҽޚǫਲ਼Ϧኤટ ҤٷԍܫᡀפڂԿᅼཛྷҊȇangiogenesisȈχՙҋǫٸᐉᙦߵшિࡹ೯ၯᇟᏛ ಉऔވউǫӞਲ਼ϦኤટಷވાϚғಉऔȇvascular endothelial cellsȈǫ ٸшቑк FasǬp53 ᆭ intergrinǃ4 ވຽǭҎࣣ࣫ةพǫኤટടι
ೆҎॸٷԍगᕅۻވϦӔݹǫᙦԭԂࢸॸшિԍΑވדᕅᛧݹǭ
ύ।ήᇿ(gallic acid; 3,4,5-trihydroxybenzoic acid)ǫᅶҎഹშᇿτᆇڎ зވϱසݹ⇃ǫҒضาτᛧ֍ԍύ।ήᇿǫفᡆшЧफ़ҋԭᔾபύ।ή ᇿǫәԓᆭԍ࢜ۻވϦᏙݦᕄᛧݹࣣБǫᆀࡹࣣྴވӳһǭҺ߲ވ࣫ةพٸ
િ୳פڂٙᕅϮឡឿဆᕅಉऔވቑ߆Ϯٗߝ٣ᕅಉऔؿӒউǭࡹҕ߲ٸᓿ ҋܤޛᕅݦᕄވ࣫ةϔࣣྴฬ϶ǫ϶ኞ࣫ةԍพٸჰܤΊᜡβಉऔޛᕅᆭ ޛဆᕅಉऔǫٷԍ٣ಉऔؿӒউވਆܷǫԶܤٸჰܤჰܤٸѣᜡࠗވޛᕅಉ औǫӬDZάಉऔޛᕅǬᜡᕅ(carcinoid)Ϯ൵ۻ໋ғዝވተ៓ǫԶϕϔѹԍΊ࣫
ة்૯ၯǭәԓǫӞѸ࣫ةτבॱ༺व෪္ύ।ήᇿჰܤάಉऔޛᕅಉऔਪ ȇNCI-H460Ȉވדᕅࢩۻǫޛᕅಉऔәࢸ୮әވσᛐۊཥދσᘇވࣸᠹǫ
ኣވࣸᠹ܃Ꮫगדᛧۻދࡹדᕅᛧݹ܃वॖᖆވടॐፇᚉǫ܃Чǫ࣫ة້
พཛྷވՙҋЈӻᆭᐉᙦވדᕅᛧݹያძࡹټσጎވǭܤࡹǫבॱఖஉύ।ή ᇿᓿҋӞάಉऔޛᕅಉऔވדᕅ࣫ةΙǫҽޚǫύ।ήᇿχדᕅՙҋᐉᙦٗѹ
ೆְᏵވ࣫ةၯǫבॱֽΈဵύ।ήᇿࡹҎಉऔউވೳৠۿࡹಉऔᚦԔ ȇNecrosisȈވЈӻчᏛगಉऔވԔǫҤۿޚࡹ೯ၯٸЫވЈӻӬ autophage ڠ cell cycle arrest าၘৠ٧ၩڀٸדᕅࢩۻǫྴǫύ।ήᇿΟԍшિႴࡹ
Herceptin ۿࡹ STI-571 ኣࡹՙҋӞࢃॸਯވಉऔከވΙǫኣވከވ шિࡹॸगᕅ୮әǬငዝפڂәήȇtumor suppressorsȈǬҊ߆ڐᡆȇgrowth receptorsȈۿࡹᙦәήาǫ٣ЫॱσࢩϦۿࡹિୱՀѼވлિǫԭ೯ၯב ॱኣވ࣫ةǫшЧ٣בॱᇘތڀύ।ήᇿᓿҋڀᕅ੨ݦᕄވшՀۻǭ
౺Ήഌ!࣫ةЈݛ
౺ʳ ࣫ةਈ
Ǭಉऔਪ٧྄
Ѹძᡄ܃٣ҋχಉऔਪNCI-H460ࢸΊᜡάಉऔޛᕅಉऔਪȇhuman large cell carcinomaȈǫҎཛྷԥफ़ࠋηཧ࣫ة܃ȇFood Industry Research and
Development InstituteȈᖳபǭ
ΉǬಉऔਪۻ NCI-H460
Θ Tissue: human; lung; large cell carcinoma ; pleural effusion Θ Morphology: epithelial
Θ Description: derivated from the pleural fluid of a patient with large cell carcinoma prior to therapy; the cells express easily detectable p53 mRNA at levels comparable to normal lung tissue, and exhibit no gross structural DNA abnormalities; the cells stain positively for keratin and vimentin but are negative for neurofilament triplet protein; the line express some properties of neuroendocrine cell, is relatively
chemosensitive and can be clones in soft agar (with or without serum).
ΖǬᛧࠋ္Ꮑ
1. 4,5-dihydroxyanthraquinone-2-carboxylic acid ȇ Rhein ȈDZ ᖳԵSigma Chemical Co.
2. Dimethyl sulfoxideȇDMSOȈDZᖳԵSigma Chemical Co.
3. RPMI1640 mediumDZᖳԵGibco
5. L-GlutaminȇᎴỦᇿ , LGȈDZᖳԵGibco 6. Penicilllum StreptomycinȇPSȈDZᖳԵGibco
7. 3,3’-Dihexyloxacarbocyanine iodide ȇDioC6ȈDZᖳԵMolecular Probes
8. propidium iodideȇPIȈDZᖳԵSigma Chemical Co.
9. Trypsin-EDTADZᖳԵAmersco
10. Trypan blueDZᖳԵSigma Chemical Co.
11. Disodium hydrogen phosphateȇNa2HPO4ȈDZᖳԵMerck 12. Sodium chlorideȇNaClȈDZᖳԵMerck
13. Potassium dihydrogen phosphateȇKH2PO4ȈDZᖳԵMerck 14. Potassium chlorideȇKClȈDZᖳԵMerck
15. Propidium iodideȇPIȈDZᖳԵSigma Chemical Co.
16. PhiPhiLux®-G1D1 kitDZᖳԵOncoImmuninȇGaithersburg, MD, USAȈ
17. RNase AȇRibonuclease AȈDZᖳԵAmeresco 18. Triton X-100DZpurchase from Sigma chemical Co.
19. EthanolDZᖳԵTEDIA
20. Ammonium persulfateȇAPSȈDZᖳԵAmersco
21. Acrylamide/Bis 40ʏ solutionȇACRYL/BISTM29:1ȈDZᖳԵ Amresco 22. Bovine serum albuminȇBSAȈDZᖳԵMerck
23. GlycineDZᖳԵAmresco 24. MethanolDZᖳԵTEDIA 25. formaldehydeDZᖳԵMerck
26. ECL kitȇEnhanced chemiluminescent kitȈDZᖳԵAmersham 27. Protein assay-Dye reagent concentrateDZᖳԵBio-Rad
28. Protein markerDZᖳԵFemantas
29. 10X SDS bufferȇSodium dodecyl sulfateȈDZᖳԵAmresco
30. TEMEDȇN,N,N’,N’-Tetramethyl-ethylenediamineȈDZᖳԵAmresco 31. TrisȇTrisȇhydroxymethylȈ-aminomethaneȈDZᖳԵAmresco 32. Tween 20DZᖳԵAmresco
33. ᡀተᏁDZᖳԵKodak 34. ۊተᏁDZᖳԵKodak 35. BioMax FlimDZᖳԵKodak 36. Agarose ˞DZᖳԵAmresco
37. ਘᇿડϦ္ᏁಋȇDNA purification kitȈDZᖳԵGene Mark
38. ಿҒ፝ڎ္Ꮑȇprotein extraction solutionȈȇPRO-PREPȈDZᖳԵ iNtRON Biotechnology, INC.
39. 5× TBE bufferDZᖳԵAmresco 40. Primary antibody ȇ1°דᡆȈDZ
(a). anti-actinDZᖳԵOncogenǰcat # cp47
(b). anti-p53DZᖳԵSata cruz BiotechologyǰLot# k1102 (c). anti-p21DZᖳԵCalbiochemǰcat# op68
(d). anti-p27DZᖳԵSata Cruz Biotechnology ǰLot: G222 (e). anti-casoase-3DZᖳԵupstateǰcat# 05-654
(f). anti-caspase-8DZᖳԵCalbiochemǰcat# AM46 (g). anti-caspase-7DZᖳԵupstate
(h). anti-caspase-9DZᖳԵupstate
(i). anti-cdc25ADZᖳԵOncogenǰLot# D19466-1 (j). anti-cyclin EDZᖳԵupstate
(k). anti-cdk2DZᖳԵupstate cat#5-596
(l). anti-cyclin D3DZᖳԵupstate
(m). anti-cdk4DZᖳԵupstateǰcat# 06139 (n). anti-cdk6DZᖳԵupstate
(o). anti-FasDZᖳԵupstate
(p). anti-NF-ljb p50DZᖳԵZymedǰLot: 30979746 (q). anti-NF-ljb p65DZᖳԵZymedǰLot: 30577746 (r). anti-bcl-2DZᖳԵupstateǰcat# 05729
(s). anti-baxDZᖳԵupstate
(t). anti-cytochrome cDZCalbiochemǰcat#PC323 41. Secondary antibodyȇ2°דᡆȈDZ
(a). goat anti-mouse IgG ȇ HRP Ȉ horseradish peroxidase conjugated antibodyDZᖳԵChemiconǰAP124P
(b). gout anti-rabbit IgGȇHRPȈhorseradish peroxidase conjugated antibodyDZᖳԵChemicon
ћǬሱᏋങǬᏋ
1. ಉऔ୲ҔDZᖳԵFALCON 2. ಉऔ୲ዣDZᖳԵFALCON 3. ಉऔ୲ዺDZᖳԵNuaire
4. ಉऔषኞᏋȇHaemocytometerȈDZᖳԵBoeco
5. ॰ҜӻՆႴᡀ༵ᜋȇphase-contrast microscopeȈDZᖳԵOlympus 6. ༵ຽϱѪ (TE-200; MILLTER)
7. чᙽήДᇐ೮ᐉDZᖳԵMinipore 8. ႔྄٥ᓿᏋDZᖳԵAmersham
9. ᇿᡬ७෪ۊषȇC831ȈDZᖳԵConsort 10. PVDF membraneDZᖳԵMinipore
11. Mini-3D ShakerDZᖳԵBoeco
12. SDS-PAGE ႔ݑኩಋDZᖳԵBio-Rad 13. Transfer Cell Blot ಋDZᖳԵBio-Rad 14. кዎܿDZᖳԵLab-Line
15. ࢣӻಉऔषኞሱȇFlow cytometryȈDZᖳԵBecton Dickinson 16. ଦ೩ᙽϿᐉDZᖳԵHERMLE
17. ϠҷҷࡂषDZfrom Beckman 18. ҷᏙᡀ༵ᜋȇOlympus CH2Ȉ
19. ҷᏙᡀ༵ᜋȇNikon LABOPHOT-2Ȉ
20. ᇾટդࣔϠ݂ሱȇanthos 2020ȈDZᖳԵAnthos Labtec, Australia 21. DNA ႔ݑኩDZᖳԵ Mupid-2
౺Ή ࣫ةष
Ǭძᡄࢅᄣ
ѸძᡄЧಉऔኪӻ٧்૯ǫᡷჯύ।ήᇿgallic acid ჰΊᜡάಉऔޛᕅಉ औਪȇNCI-H460ȈҊ߆χተ៓ǫ்ٗ૯ύ।ήᇿࡹոٷԍᇟᏛH460ಉऔউ
ՙҋϮಉऔຳනވፊொՙҋ(Ӭა2.1)ǭ
ȇȈ்૯ύ।ήᇿgallic acidჰH460ಉऔҊ߆χተ៓
Ѹძᡄࢸஉσӌᐑࡂχύ।ήᇿgallic acidǫދкΌH460 ಉऔχ୲ట τǫܤ37°CǬ5% CO2 ϮбϠྊࡂΗǫܤ6Ǭ12Ǭ24Ǭ48Ǭ72 βࡌǫԆ
ಉऔǭຈՎύ।ήᇿჰH460ಉऔҊ߆ۿࡹӯࢩχተ៓ǭ
ȇΉȈ்૯ύ।ήᇿgallic acid ჰH460ಉऔຳනተ៓Ϯಉऔউχᐉڂ
Ҏӯࢩχძᡄшபχύ।ήᇿgallic acidn ཥפڂH460ಉऔਪχҊ ߆ǫٗཥᇟᏛಉऔີՀಉऔউՙҋǫࡱѸძᡄஉЧࢣӻಉऔሱᔗ෪H460ಉऔ χಉऔຳනǫ்ٗ૯ύ।ήᇿgallic acid ᇟᏛಉऔউχᐉڂǭ
୲ވ NCI-H460 ಉऔਪ
кΌσӌᐑࡂχ gallic acid
୲ 6Ǭ12Ǭ24Ǭ48Ǭ72 β
ა2.1 ύ।ήᇿgallic acid ჰΊᜡάಉऔޛᕅNCI-H460ಉऔӯࢩϮᇟᏛ ಉউڠಉऔຳනଭᅀძᡄࢅᄣ
ಉऔӯࢩ෪ۊ! Чࢣӻಉऔሱᔗ෪ᛧݹჰ ܤH460ಉऔຳනވፊொ
y ౿ဆᡆጙ႔Ն (Ʃƺm)χᔗ෪ y ࢩۻਲ਼Ϧݹ
ȇROSȈកзχ ᔗ෪
y ເᙽήកзχᔗ ෪
y DAPI staining
y ࡺ္ᡄȇComet assayȈ
DNA fragmentation assay
᧤agrose gel electrophoresis᧥
Caspase-3 ࢩۻϠ݂ϮٸЫ ಉऔউࣣಿҒ፝χᔗ෪
ΉǬ ძᡄЈݛ )* ᛧࠋᇐ
உᖳԵsigma chemical Co.χ3,4,5-trihydroxybenzoic acid monohydrate ύ।ήᇿ(gallic acid)ડϦݹǫڎ5.6846 mgǫྂܤ1ml DMSOǫᇐԂ20 mM stock solutionǫstock solution Ϡըڎσӌᐑࡂᇐ܃መᐑࡂǫᇐᐑࡂӬ ΗDZ
ϦᏙӓDZ 3,4,5-trihydroxybenzoic acid monohydrate ϠήӻDZC6H2(OH)3COOH
Ϡήຽ=188.14
188.14×103 mg/1000 mlɟ1000 mM 0.18814 mg/mlɟ1 mM
20 mM=3.7628 mg/ml
2.1!ύ।ήᇿχᇐ!
ಬԯᐑࡂȇMȈɟϠήຽ/ᡆᐿȇLȈ
20 mM of gallic acid DMSO
0.5 mM 25 Njl 975 Njl
1 mM 50 Njl 950 Njl
2.5 mM 125 Njl 875 Njl
5 mM 250 Njl 750 Njl
10 mM 500 Njl 500 Njl
உᇐӪσӌᐑࡂχύ।ήᇿҋ٧ີՀಉऔቑҊϮಉऔຳන္ᡄǫЧϮಉ औউ෪္ȇDAPI staining and Comet assayȈЧϮժҋՂЈᗱᎷݛ ȇwestern blotȈᡷჯಿҒ፝าϠ݂ǭ
ȇΉȈಉऔ୲
A. ୲Ұ
ΊᜡάಉऔޛᕅಉऔਪȇNCI-H460ȈЧRPMI1640 ୲୮ణк10%ओЛԿ థǬ1%L-glutamine Ϯ1% Penicillin Streptomycin χ୲టǫܤ5% CO2 Ϯ бϠྊࡂΗވ37 ʑވಉऔ୲ዺȇincubatorȈτ୲ǫँΉϱתඛԒ୲ 2×106×~5×106cells/mlǫమӔ֘ϣࡌܤЧከҙӪӓᅷǬኞҕϮէআЉනχէ আᅼτǫ1 ml/ᅼǭ
էআߜӯЈݛDZէআᅼܤ4 °Cǫ10 min ɿ -20 °Cǫ30 min ɿ -80 °Cǫ16~18 β ɿ!టᄁෑኩ߆නߜӯǭ
C. ᝠЭ୲
ࡈಉऔ߆ԶϠᄿǫஉ୲టڎзҞఌǫҺЧPBSȇphosphate buffer salineȈథࢨಉऔ1~2 ԒǫҽкΌ3 ml trypsin ǫܤ37ʑ୲ዺτಷ2 Ϡ
ខࡌǫкΌପ୲టЧτڠtrypsin χՙҋǫҽஉౖτ܃ԍటᡆုڀᙽϿᅼ τЧ1500 rpm ᙽϿ5 ϠខǫᙽϿࡌ॰ΙథటǫкΌ20 ml ཛྷᗩ୲టǫҽ ٣ҋ႔օຽᅼϯᙖܓօ٣֘ϣమڠǫڎ20 Njl χಉऔᝃటкΌ80 Njl trypan blue ྂటǫЧԿषኞዣȇCounting chamberȈषᆀಉऔኞҕȇԔಉऔཥೆ
trypan blue ࢀԂᙋԼǫԭࢩಉऔߵσೆࢀԼȈǭ
టǫठຣၯ48 βߵҺඛ୲టҽкΌᛧݹಷǭmicroplate well кΌ
୲టᡆᐿ2 mlǫߵк20 Njl ᛧݹǰ3 mlǫߵк30 Njlǫߵᛧݹᐑࡂೆฬក100 । PI ཥᆭDNA ᙿޟᖜ௴τχA=TǬCɥGᗍ฿ވచᗍȇhydro bondȈӞ૽ǰ ӯࢩވಉऔәٸಉऔጙְᏵPI ݛڠಉऔϚވਘᇿ฿ӔǫPI ࢀԼְԂވಉ
औшҎࢣӻಉऔሱܤ488 nm ވ႓োҷᐖพࡌԔވಉऔཥվၟࣽԼᑲ ҷǫӯࢩಉऔཥԂၟވࣽԼᑲҷЧCellQuest ೣᡆϠ݂ಉऔӯࢩǭ
ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plate τǫಉऔ٠ྚ2×105/well ᅶසܤ୲Ҕτǫၯ 24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌσӌᐑࡂވgallic acidȇ0.5 mMǬ1 mMǬ2.5 mMǬ5 mMǬ10 mMȈǫϠըࡤឲ୲12Ǭ24Ǭ48 Ϯ72 βǭк ᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBS థࢨಉऔԒࡌǫҽஉ ಉऔЧtrypsin ಷǫܤ37ʑ୲ዺτಷ2 ϠខࡌǫஉಉऔѱΗ٧ǫкΌ1 ml PBSЧτڠtrypsin χՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpm ᙽϿ5
Cell cycle Proopidium iodideȇPIȈࡹᅶᑲҷࢀᏁǫшஈވᗍ฿ވਘᇿԭቭ ݨވᓿҋܤࢣӻಉऔሱוೃτǫѼݠΗಉऔጙᆙࡤְᏵPI ݛࣷ೯ಉऔ χಉऔጙǫࡱшЧ٪ΌٸЫძᡄȇӬ:ᆭFITC ከ૭χannexinV Ҽӌଷ෪ǫЧ
ୋըӯࢩᚦԔۿউಉऔȈۿޚᆭٸЫࢀᏁҼӌ٣ҋǫຈՎಉऔӯࢩݺᄁठЧ
ᆉஉಉऔጙѱࢧڧۊࡌǫີΌಉऔϚχPI шᆭਘᇿີՀᗍ฿ǫժҋࢣӻಉ औሱȇFlow cytometry; FACSȈଷ෪PI ܃ૼᝏχᑲҷǫߵшЧϯᓿಉऔϚχDNA ݺᄁۿີՀಉऔຳනϠ݂ȇDarzynkiewicz et al., 1992Ȉǭ
ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plate τǫಉऔ٠ྚ2×105/well ᅶසܤ୲Ҕτǫၯ 24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌσӌᐑࡂވgallic acidȇ0.5 mM, 1 mM,
ಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBS థࢨಉऔԒࡌǫҽஉಉऔЧtrypsin ಷǫܤ37 ºC ୲ዺτಷ2 ϠខࡌǫஉಉऔѱΗ٧ǫкΌ1 ml PBS Ч τڠtrypsinχՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ 1500 rpm 5 ϠខވᙽϿȇॐ ጻԓᡦԒȈǫчଟΙథటǫܤ᎓ᕆᏋᇯᇯ᎓ᕆҽкΌ70% EtOH/PBSǫ
ᄷᄷᄷΌᙽϿᅼτЧڧۊಉऔǫ٣ಉऔְһ֘ϣϠජܤ70% EtOH/PBS τǫᓀ-20 ºCԶ϶βǫڎзಉऔࡌᙽϿȇ1500 rpm 5 ϠខȈǫ॰Ι థటкΌPBS 3ml
థࢨಉऔǫᙽϿчଟΙథటǫкΌcycle PI ࢀᏁ350 Njlǫஉಉऔѱජǫᗀҷ 30 ϠខǫЧࢣӻಉऔሱՀኣࠋϠ݂ǫ܃பχ฿ܷЧModfit LT ೣᡆϠ݂ǭ C. ౿ဆᡆጙ႔ՆȇMitochondria membrane potential; ƩƺmȈχᔗ෪
ಉऔጙ႔Ն்କǫDioC6ȇ3,3’-Dihexyloxacarbocyanine iodideȈࡹᅶш ࡧከȇPetit et al., 1990Ȉǭ
ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plateτǫಉऔ٠ྚ2×105/wellᅶසܤ୲Ҕτǫၯ24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌ5 mM gallic aicdǫ20 Njl/wellڀ12 well plateྴτȇടᐑࡂࢸ50 NjMȈǫσӌ໋୲ȇ1Ǭ3Ǭ6Ǭ12Ϯ24 hȈǫ кᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBSథࢨಉऔԒࡌǫҽ உಉऔЧtrypsinಷǫܤ37ʑ୲ዺτಷ2ϠខࡌǫஉಉऔѱΗ٧ǫкΌ1 ml PBSЧτڠtrypsinχՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpmᙽϿ5Ϡ
ខǫчଟΙథటǫҽкΌ1 ml PBSథࢨಉऔǫ1500 rpmᙽϿ5ϠខǫчଟΙథ ట ǫ ڎ MMP ȇ Ʃƺm Ȉ ࢀ Ꮑ ȇ 10 Njl DioC6/500Njl PBS Ȉ DioC6 ȇ3,3’-Dihexyloxacarbocyanine iodideȈࢀᏁᅼкΌ500 Njlǫመԍᅼblank σкᛧΟσкࢀᏁǫܤ37 ºC୲ዺᗀҷ୲30 minࡌǫtransferԶFACSᅼτǫ ЧࢣӻಉऔሱີՀኣࠋϠ݂ǫኣࠋԆ10000ᗤಉऔЧCellQuestೣᡆϠ݂ǭ உblankȇਨጌވpeakȈፊӞ100~101χ໋ǫcontrolȇ௰ጌވpeakȈፊӞ101~102 χ໋ǫM1 gatedँ75ʔpeakǫЧΙsampleΙᐉࡌǫϠ݂MMPȇpeak۪ъσౙ Ҋapoptosisǫ۪ѧౙҊapoptosisȈǭ
ა2.2 MMP ೣᡆވϠ݂ა D. ࢩۻਲ਼ϦݹౙҊχᔗ෪
դࣔಉऔີՀመਲ਼ۻ྇ၯสτǫཥӞಉऔϚ௫೨Մވਲ਼Ϧᗂϯ ᓿǫәԭౙҊٛOxidative MetabolitesǫӬH2O2 ǫO2- free radicalǭӬఖЧ ࢣ ӻ ಉ औ ሱ ෪ ຽ ٛ Э ᖫ ݹ ވ ౙ ຽ ǫ ш ҋ Dihydrorodamine 123 Ǭ 2’7’-dichlorofluoresceinȇfor H2O2Ȉۿhydroethidineȇfor O2- free radicalȈา ᑲ ҷ ࢀ Ꮑ ǫ ჰ ಉ औ ີ Հ ࢀ Լ ǫ ᙍ Ҏ 2’,7’-dichlorofluorescein diacetate ȇH2DCF-DAȈౙҊᑲҷ٧෪ຽROS ވౙҊǫH2DCF-DA ࡹᅶٷԍᑲҷۻ፝ǫ шᅍ೯ಉऔጙౝۻވଊᙢຈՎROS ވౙҊǭH2DCF-DA ཥೆಉऔϚވㄴ
✁䁐ȇesterasesȈчㄴϦȇdeacetylatedȈԂߑᑲҷۻވDCFHǫDCFH ཥӞ
ಉऔϚೆH2O2ਲ਼ϦԂᑲҷۻ፝ވDCFǫٗᆢӞ౿ဆᡆτǫ܃พජᑲҷߵшϯ
ࡷзಉऔϚH2O2ވᐑࡂȇRoyall et al., 1993Ȉǭ ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plateτǫಉऔ٠ྚ2×105 /wellᅶසܤ୲Ҕτǫၯ24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌ5 mM gallic acidǫ20 Njl/wellڀ12 well plateྴτȇടᐑࡂࢸ50 NjMȈǫσӌ໋୲ȇ0.5Ǭ1Ǭ2Ǭ4Ϯ6 hȈǫк ᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBSథࢨಉऔԒࡌǫҽஉ ಉऔЧtrypsinಷǫܤ37ʑ୲ዺτಷ2ϠខࡌǫஉಉऔѱΗ٧ǫкΌ1 ml PBSЧτڠtrypsinχՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpmᙽϿ5Ϡខǫ чଟΙథటǫҽкΌ1 ml PBSథࢨಉऔǫ1500 rpmᙽϿ5ϠខǫчଟΙథటǫ ڎROSࢀᏁH2DCF-DAࢀᏁȇ1 Njl
H2DCF-DA /500 Njl PBSȈᅼкΌ500 NjlǫመԍᅼblankσкᛧΟσкࢀᏁǫ єкΌ500 Njl PBSǫӞܤ37 ºC୲ዺᗀҷ୲30 minࡌǫtransferԶFACSᅼ τǫЧࢣӻಉऔሱີՀኣࠋϠ݂ǫኣࠋԆ10000ᗤಉऔЧCellQuestೣᡆϠ
݂ǭஉblankȇਨጌވpeakȈፊӞ100~101χ໋ǫcontrolȇ௰ጌވpeakȈፊӞ101~102 χ໋ǫM1 gatedँ75ʔЧΙǫsampleΙᐉࡌǫϠ݂ROSȇpeak۪ъࡹౙҊԵҎ ୮ǫ۪ѧࡹדਲ਼ϦȈǭ
ა2.3 ROS ೣᡆϠ݂ა E. ເᙽήកзχᔗ෪
औϚເᙽήՙࢸಉऔߕဝᇷވߕǫࡹಉऔᐖࢩၯสτॐवވлિ୍ኞǭᑲ ҷࢀᏁȇӬFura-RedǬFluo-3ڠIndo-1าȈ೦ၯㄴҏ✁ȇAcetatoxymethyl EsterǰAEȈᏛΌಉऔࡌǫIndo-1ཥᆭເᙽήౝۻ฿ӔǭٛᑲҷࢀᏁވ฿ᄣ ӻǫᆭEDTAࣣՖǫш⽳Ӕເᙽήǫ⽳ӔເᙽήχᑲҷࢀᏁཥԍҷᏙۻ ΙވצᠹǫӞȇโѝҷȈUV ވᐖพΗǫIndo-1 ܢзҷȇemissionȈވࡂཥ ᒶಉऔϚເᙽήᐑࡂވצᠹǫԭพජзσӌࡂވᑲҷǫࡱшҋБ٦ݛ෪ப ۿދ෪பވᑲҷࡂபڀເᙽήᐑࡂވࣣჰ७ǫძሕᐑࡂመਗჰࡌᔾபǭ ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plateτǫಉऔ٠ྚ2×105 /wellᅶසܤ୲Ҕτǫၯ24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌ5 mM gallic acidǫ20 Njl/wellڀ12 well plateྴτȇടᐑࡂࢸ50 NjMȈǫσӌ໋୲ȇ0.5Ǭ1Ǭ2Ǭ4Ϯ6 hȈǫк ᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBSథࢨಉऔԒࡌǫҽஉ ಉऔЧtrypsinಷǫܤ37ʑ୲ዺτಷ2ϠខࡌǫஉಉऔѱΗ٧ǫкΌ1 ml PBSЧτڠtrypsinχՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpmᙽϿ5Ϡខǫ чଟΙథటǫҽкΌ1 ml PBSథࢨಉऔǫ1500 rpmᙽϿ5ϠខǫчଟΙథటǫ
1000 Njl PBSǫӞܤ37ºC୲ዺᗀҷ୲1hǫ10 minΙΗమӔԒǫ1hࡌ кΌPBSࢨ2Ԓǫ1500 rpmᙽϿ5 minǫ॰ΙథటǫᅼкΌ400 Njl PBSǫҽ transferԶFACSᅼτǫЧࢣӻಉऔሱີՀኣࠋϠ݂ǫኣࠋԆ10000ᗤಉऔ ЧCellQuestೣᡆϠ݂ǭஉblank(ਨጌވpeak)ፊӞ100~101χ໋ǫcontrolȇ௰ጌ ވpeakȈፊӞ100~101χ໋ǫM1 gatedँ0ʏǫЧΙsampleΙᐉࡌǫϠ݂calcium release(peak۪ъࢸເᙽήកз*ǭ!
ა2.4 ເᙽήೣᡆϠ݂ა
F. Caspase-3ǬCaspase-8ǬCaspase-9 ࢩۻϠ݂
ժҋPhiPhiLux-G1D1 kit ٧ᔗ෪উಉऔcaspase-3Ǭcaspase-8Ǭcaspase-9 χౙҊǫPhiPhiLux- G1D1kit ୮፝ࡹᅶ֍ԍᑲҷݹ፝χỦ୮ᇿ־ҿȇamino acid sequenceȈǫԭࢩϦχcaspase-3Ǭcaspase-8Ǭcaspase-9 шЧຂဵỦ୮ᇿ־
ҿχۊՆǫԭࡹᑲҷݹ፝កܢз٧ǫҽҎࢣӻಉऔሱϠ݂ǫшபތठᑲ ҷౙຽຢӤߵౙҊࢩۻχcaspase-3Ǭcaspase-8Ǭcaspase-9 ຢӤȇKomoriya et al., 2000Ȉǭ
ȓძᡄᡦȔ
உಉऔᅶසܤ12 well plate τǫಉऔ٠ྚ2×105 /well ᅶසܤ୲Ҕτǫၯ 24 βᓀ୲ࡌǫࡈಉऔທߌࡌкΌ5 mM gallic acid 20 Njl/well ڀ12 well plate ྴτȇടᐑࡂࢸ50 NjMȈǫσӌ໋୲ȇ6Ǭ12Ǭ24Ǭ48 Ϯ72 hȈǰ кᛧ໋ࡌǫԆಉऔǫஉΙቢటԶᙽϿᅼτǫкΌPBS థࢨಉऔԒࡌǫҽ உಉऔЧtrypsin ಷǫܤ37ʑ୲ዺτಷ2 ϠខࡌǫஉಉऔѱΗ٧ǫк Ό1 ml PBS Чτڠtrypsin χՙҋǫҽஉ܃ԍటᡆုڀᙽϿᅼτǫ1500 rpm ᙽ Ͽ5 ϠខǫчଟΙథటǫҽкΌ1 ml PBS థࢨಉऔǫ1500 rpm ᙽϿ5 Ϡខǫ
чଟΙథటǫڎ10 NjM substrateȇPhiphilux green for caspase-3ȈȇPhiphilux red for mitochondriaȈᅼкΌ25 NjlǫӞܤ37 ºC ୲ዺᗀҷ୲1 hǫ1 h ࡌкΌ1 ml PBS ࢨ1 Ԓǫ1500 rpm ᙽϿ5minǫ॰ΙథటǫᅼкΌ500 Njl PBSǫҽtransfer ԶFACS ᅼτǫЧࢣӻಉऔሱີՀኣࠋϠ݂ǫኣࠋԆ10000 ᗤಉऔЧCellQuest ೣᡆϠ݂ǭஉblankȇਨጌވpeakȈፊӞ100~101χ໋control ȇ௰ጌވpeakȈፊӞ101~102 χ໋ǫM1 gated ँ75%ǫЧΙsampleΙᐉࡌǫ Ϡ݂caspase-3 ࢩۻȇpeak ۪ъࢸcaspase-3 ౙҊȈǭ
ა2.5 Caspase-3 ೣᡆϠ݂ა
ȇћȈDNA ཕᔗ෪