Jurkat T 淋巴母細胞中四環素誘導生成抑酶干擾細胞激素的表現; The Interference of Cytokine Expressions in Inducible Antizyme Tet-On Jurkat T Cell System

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(1)!. ύ!୯!ᙴ!ᛰ!ε!Ꮲ! ୷ᘵᙴᏢࣴ‫!܌ز‬ ᅺγᏢՏፕЎ! !. Jurkat T రЃ҆ಒझύѤᕉનᇨᏤғԋ ‫׭‬䁙υᘋಒझᐟન‫߄ޑ‬౜ ! !. The Interference of Cytokine Expressions in Inducible Antizyme Tet-On Jurkat T Cell System. ! ! ࡰ!Ꮴ!௲!௤!Ǻ!ଯሎ෕!!௲!!௤! !. ӅӕࡰᏤ௲௤Ǻ!ቅӀᝬ!!ୋ௲௤! ! ࣴ!‫!ز‬ғǺ!ම‫ذ‬Ꮲ!!. !. ύ๮҇୯ΐΜΎԃΎД!.

(2) ᄔ!ा! ‫׭‬䁙཮‫ޔ‬ௗᆶಒझϣӭữғӝԋ‫ޑ‬ᜢᗖሇનചữለಥ♐䁙่ӝ ‫׎‬ԋ౦፦ΒᆫᡏǴ຾Զ٬ளചữለಥ♐䁙ӕ፦ᚈᆫӝᡏϩᚆԶѨѐࢲ ‫܄‬٠Ъ٬ചữለಥ♐䁙ӧό٩ᒘ‫ݱ‬નϐΠ೏ೈқ䁙ᡏफ़ှǶӄߏ‫ڀ‬ф ૈ‫׭ޑ܄‬䁙ϐғԋЬाࢂ‫ډڙ‬ಒझϣӭữᐚࡋ‫ޑ‬ፓ௓ǴԶЪѸሡा࿶ ၸਡᗐਡለ᠐ዸਣ+1Տ౽‫ޑ‬ၸำǶฅԶǴ‫ޔ‬ԿҞ߻ࣁЗǴ‫׭‬䁙ӧಒझ ϣ၁ಒ‫ޑ‬ғ౛фૈϝࢂ҂ܴǶ྽থ٢ᜪಒझ‫ډڙ‬Ѧٰ‫ڈ‬ᐟӵᚈި RNAs‫ࢥੰࢂ܈‬གࢉਔ཮ᇨวυᘋન‫ޑ‬ғԋǶԜѦӧεӭኧੰࢥགࢉ ΠǴTಒझӧխࣝϸᔈύ཮೏ᇨวᏤठࢲϯϷϩϯ٠Ъౢғόӕ‫ޑ‬ಒ झᐟનа‫ܢ‬ᑇੰࢥགࢉǶЎ᝘ൔ֋υᘋન‫ڀ‬Ԗ‫ࢥੰל‬ᆶ‫ל‬ಒझғߏϐ ૈΚ೽ϩࢂᙖҗ2'Æ5'ჲဏ㧿-RNase L RNAफ़ှၡ৩Ƕ߈ԃٰǴ೭చ ၡ৩ς೏᛾ჴ཮ቚуᙯ᝿ၸำၢ᠐ၸprematureಖЗஏዸϐਏ౗аϷ ӧ‫׭‬䁙ᙯ᝿ၸำύ᠐ዸਣ+1Տ౽୔ୱϐ+1Տ౽ਏ౗ቚуǶࣁΑ௖૸ӄ ߏ‫ڀ‬фૈ‫׭ޑ܄‬䁙ӧTಒझࢲϯၸำύ‫ת܌‬ᄽ‫فޑ‬ՅǴҁፕЎࣴ‫׭ز‬ 䁙‫ޑ‬ၸࡋ߄౜ჹ‫ܭ‬Jurkat Tಒझࢲϯࡰ኱੝‫ޑ܄‬ቹៜǶճҔѤᕉનᇨ Ꮴ‫س‬಍ᇨวJurkat Tಒझϣᙯ෗‫׭ޑ‬䁙୷Ӣ߄౜Ǵᢀჸว౜‫׭‬䁙‫ޑ‬ၸ ࡋ߄౜཮ቹៜ೚ӭಒझᐟન‫߄ޑ‬౜Ǵ੝ձࢂᆶխࣝࢲϯ࣬ᜢ‫ޑ‬ಒझᐟ નǴхࡴϟқન-2ǵϟқન-4ǵϟқન-5ǵϟқન-12 p40Ϸυᘋન-JǶ ‫ځ‬ύϟқન-2ӧJurkat Tಒझࢲϯၸำύ‫ת‬ᄽ๱΋ঁύЈಒझᐟન‫ޑ‬. i.

(3) ‫ف‬ՅǶࣴ‫่݀ز‬ว౜‫׭‬䁙ၸ߄౜‫܌‬Їว‫ޑ‬ϟқન-2୷Ӣ‫߄ޑ‬౜ёૈࢂ ೸ၸᙯᒵቫԛ‫ޑ‬ፓ௓Ƕ຾΋‫؁‬ϩ‫݋‬ς‫ࢲޑޕ‬ϯૻ৲໺ሀၡ৩Ǵว౜‫׭‬ 䁙ᇨวࡕϷTಒझࢲϯอਔ໔ϣǴIKKD/E‫ک‬INB-D‫ޑ‬ᕗለϯ߄౜ࢂቚ у‫ޑ‬ǴԶINB-D᏾ᡏೈқ፦߄౜ໆ߾Ԗܴᡉ‫ޑ‬෧ϿǴЪΠෞ‫ޑ‬NF-NB ӧࢲϯࡕ຾Εಒझਡ‫ޑ‬ำࡋΨࢂቚу‫ޑ‬Ƕᕴ่а΢‫ޑ‬ჴᡍ่݀Ǵҁፕ Ўගрӄߏ‫ڀ‬фૈ‫׭ޑ܄‬䁙‫ڀ‬Ԗխࣝፓ௓‫ૈޑ‬ΚǴЪ຾΋‫؁‬ቹៜϟқ ન-2‫߄ޑ‬౜຾Զѐፓ࿯TరЃಒझᅱ࿎Ϸࢲϯ฻фૈǶ. ii.

(4) Abstract Antizyme (AZ) directly binds ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis, and inactivates and degrades ODC by the 26S proteasome without ubiquitination. The production of full-length functional AZ protein depends on polyamine levels and AZ gene requires +1 translational frameshifting. However, the delicately cellular function of AZ is still unidentified. Mammalian cells in response to various stimuli such as double-stranded RNAs or viral infection can stimulate the induction of interferons (IFNs). In most viral infections, T cells can be induced to activate, differentiate and produce a number of cytokines that defend against viruses. Previous studies have shown the antiviral and antiproliferative effects of IFNs are mediated by the 2’-5’ oligoadenylate RNase L RNA decay pathway. Recently, this pathway is confirmed to increase translational readthrough efficiency at premature termination codons and increase +1 frameshift efficiency at the AZ +1 frameshift site. To elucidate the role of full-length functional AZ in T cell activation, we examined whether conditional expression of full-length AZ protein could elucidate the hallmarks of Jurkat T cell activation. Using Jurkat Tet-On System, AZ overexpression was found to affect the expression of cytokines, especially the cytokines of immuno-activation, including IL-2, IL-4, IL-5, IL-12 (p40) and IFN-J. The specific activation of IL-2 is a pivotal cytokine in Jurkat T cell activation. This study demonstrated that AZ-triggered expression of IL-2 might be through the transcriptional modulation of IL-2 gene. Further analysis on the signaling transduction revealed that phosphorylation of IKKD/E and INB-Dwere iii.

(5) increased, total INB-Dwas degraded and the nuclear translocation of NF-NB was moderately increased at early stage of activation after AZ induction. Taken together, we suggest that AZ may own the capacity of immuno-regulation and influence IL-2 expression to regulate T lymphocyte surveillance and activation.. iv.

(6) ठ!ᖴ! !!!!‫ٿ‬ԃٰ‫ޑ‬ᅺγࣴ‫ز‬ғࢲаϷՔᒿ๱ҁጇፕЎ‫ޑ‬೴΋ԋࠠǴ೭٤В ηаٰǴाགᖴ‫ޑ‬ΓϼӭǶᗨฅ೭ࢂঁᅐߏ‫ޑ‬ਓำǴԖҒౡϷधᔴ‫ޑ‬ ӣᏫǴՠനಖǴѬࠅԖ๱зΓᅈཀ‫ֽ่ޑ‬Ƕ‫׆ך‬ఈૈӛӧ೭٤Вη္ ᔅշၸ‫ޑך‬Γ߄ၲ‫ޑך‬གᖴǴхࡴ‫ޑך‬ৎΓǵԴৣǵჴᡍ࠻‫ޑ‬უՔॺ Ϸ‫܌‬΢ӕᏢॺǴᖴᖴգॺǶ! !!!!!. !!!!२Ӄाགᖴ‫ך‬ལང‫ࡰޑ‬Ꮴ௲௤ଯሎ෕Դৣ‫ޑ‬ගឫᆶྣ៝Ǵό໻ᡣ ‫ך‬ӧࣴ‫ز‬΢ϷᏢ᛽Бय़ᕇ੻‫ؼ‬ӭǴவԴৣ‫ي‬΢Ψᡣ‫ך‬Ꮲಞ‫ࣁ଺ډ‬΋ঁ Ꮲ‫ޣ‬၀Ԗ‫ޑ‬॥ጄǹќѦΨा੝ձགᖴύξᙴխࣝ‫ߏ܌܌‬ቅӀᝬԴৣ೭ ‫ٿ‬ԃٰӧჴᡍБӛ΢‫ޑ‬ൻൻ๓ᇨϷ௴วǴΨᡣ‫ך‬Αှ‫ࣽډ‬Ꮲࣴ‫ز‬ᔈ၀ Ԗ‫ࡘޑ‬ԵϷᄊࡋǶԜѦǴᗋाགᖴ‫܌‬΢ғϯࣽ‫ޑ‬ԴৣॺϷ჏ကεᏢጰ ‫݇ے‬Դৣӧჴᡍ‫ೌמ‬΢த๏ϒࡌ᝼ϷࡰᏤǶനࡕǴӧ‫ޑך‬ፕЎα၂Ϸ ፕЎቪբ΢Ǵགৱ‫ܭ‬қტ໢ԴৣǵᗛඳӀԴৣϷ‫֮׵‬ᆺԴৣ‫ޑ‬ϸᙟቩ ਡࡰᏤϷᝊ຦ࡌ᝼Ǵωૈ٬ҁፕЎϣ৒‫׳‬уֹ᏾ᝄᙣǶ! !. !!!!!ჴᡍ࠻უՔॺ‫ޑ‬ගឫ࣬ՔǴࢂ೭‫ٿ‬ԃٰЍ࡭‫ך‬ᝩុոΚ‫ޑ‬চ୏ ΚǶܴᑣᏢߏǴᖴᖴգ೭‫ٿ‬ԃٰ‫ٌޑ‬ध௲Ꮴᆶ࿶ᡍ໺‫܍‬Ǵᡣ‫ך‬Ꮲಞ‫ډ‬ ӵՖࡘԵϷှ،ୢᚒǹണЎᏢߏϷᅇ᜻ᏢߏǴᖴᖴգॺਔத๏ϒ‫ࣴך‬ ‫ز‬΢೚ӭࡌ᝼Ϸᙦ൤௴ว‫૸ޑ܄‬ፕǹа֡Ꮲ‫ۆ‬Ǵᖴᖴ‫ی‬ӧჴᡍ΢த๏. v.

(7) ϒᜢЈϷࡰᏤǹ҅એᏢߏϷ‫܃ݒ‬ǴߚதᖴᖴգॺନΑჴᡍϐѦᗋाϩ ᏼ೚ӭჴᡍ࠻ᕷख़‫ޑ‬٣୍Ǵ‫׆‬ఈգॺ‫ޑ‬ჴᡍ໩ճ຾ՉǹᗋԖ‫܃ٵ‬Ǵ‫ז‬ Γ‫ז‬ᇟ‫ޔ‬ဉη‫یޑ‬Ǵᖴᖴ‫ی‬ᔅ‫ך‬ϩᏼΑ೚ӭჴᡍ࠻ελ٣௃ǹᡉӀǴ գନΑπբϐѦᗋाᔅԆ‫ځ‬дჴᡍ࠻‫ޑ‬٣୍ǴᖴᖴգǶനࡕεᏢ೽‫ޑ‬ Ꮲ‫఼ۂ׌‬Бǵ҉ԓǵ୯ᄪǵࢀզǵۗᓄǵỜ఼ϷદഩǴᖴᖴգॺᔅշ ‫ך‬ჴᡍ΢εελλ‫ޑ‬٣௃ǴԖΑգॺჴᡍ࠻ᕴࢂкᅈ៿኷‫਻ޑ‬৲ǶԜ ѦǴΨाགᖴ‫܌‬΢ӕᏢ໡൛ǵॕ޼ǵ߷ֱǵ҅Ӻǵৎ٫ǵ໡ᄨǵ࢙‫ق‬ǾǾ ۶Ԝ࣬ϕ‫࡭ן‬ႴᓰǶനࡕǴा੝ձགᖴ‫ࢂޑ‬ύξᙴ‫ޑ‬໡‫ޱ‬Ꮲ‫ۆ‬ǴؒԖ ‫ޑی‬ᔅշϷࡰᏤǴ೭ҁፕЎࢂό཮р౜‫ޑ‬Ƕ! !. !!!!നࡕགᖴ‫ޑך‬Р҆ϷৎΓǴգॺࢂङࡕ΃‫ޔ‬Ⴔᓰ‫ך‬ӛ΢‫؃‬Ꮲ‫ޑ‬চ ୏ΚǶ೭ጇፕЎૈ୼ֹԋǴЈӸགৱ‫܌ܭ‬Ԗᔅշၸ‫ޑך‬ΓǴᙣаԜஒ ೭ࣴ‫ز‬ԋ݀᝘๏ӚՏǶ! ! ! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ම‫ذ‬Ꮲ!!ᙣᇞ! 2008.07.20 ! !. vi.

(8) Ҟ!ᒵ! ।ԛ!. ύЎᄔा!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!i! मЎᄔा!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!iii! ठᖴ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!v! Ҟᒵ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!vii! ߄Ҟᒵ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!xi! კҞᒵ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!xii! ᕭቪ߄!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!xiv! ಃ΋കǺᆣፕ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!. 1!. !!!!ಃ΋࿯ǵ‫׭‬䁙!(Antizyme). 1!. !!!!!!!!൘ǵAntizyme ‫ޑ‬ғԋᐒ‫ڋ‬. 1. ມǵAntizyme ‫ޑ‬ғ౛фૈᆶғ‫ނ‬ख़ा‫܄‬. 3!. !!!!ಃΒ࿯ǵT ಒझ‫ࢲޑ‬ϯ (T cell activation). 5!. !!!!!!!!൘ǵT ಒझࢲϯ࣬ᜢᐒᙯ!. 5. ມǵT ಒझࢲϯᆶಒझᐟન߄౜. 7. ಃΟ࿯ǵAntizyme ᆶ T ಒझϐ໔‫࣬ޑ‬ᜢ‫܄‬. 9!. !!!!!!!!൘ǵചữለಥ♐䁙!(Ornithine decarboxylase) ᆶӭữ (Polyamines) ມǵᙯᒵӢηࢲϯૻ৲ፓ௓. 9 10. !!!!ಃѤ࿯ǵࣴ‫୏ز‬ᐒϷҞ‫!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ޑ‬12! !!!!ಃϖ࿯ǵჴᡍ฼ౣ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 14!. ಃΒകǺ‫׷‬਑ᆶБ‫ݤ‬. 16!. !!!!ಃ΋࿯ǵჴᡍ‫׷‬਑!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! vii.

(9) !!!!!!!!൘ǵಒझਲ਼!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! !!!!!!!!ມǵ‫ל‬ᡏ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! !!!!!!!!ୖǵЇη!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! !!!!!!!!စǵ፦ᡏ DNA!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! !!!!!!!!Ҵǵεဉఎ๵๵ਲ਼!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!16! !!!!!!!!ഌǵϯᏢ၂Ꮚᛰࠔ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!17! !!!!!!!!ࢠǵЬाᏔ‫׷‬ሺᏔ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 20! !!!!ಃΒ࿯ǵჴᡍБ‫ !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ݤ‬21! !!!!!!!!൘ǵಒझਲ਼୻Ꭶ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!21! !!!!!!!!ມǵಒझਲ਼ғߏԔጕ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 21! !!!!!!!!ୖǵࢬԄಒझሺಒझ঒Ϋϩ‫!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!݋‬21! !!!!!!!!စǵՋБᏀᗺ‫!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ݤ‬. 22!. !!!!!!!!Ҵǵಒझ RNA ‫!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ڗܜޑ‬24! !!!!!!!!ഌǵϸᙯᒵ䁙.ᆫӝ䁙ೱᙹϸᔈ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!25! !!!!!!!!ࢠǵջਔ‫ۓ‬ໆ.ᆫӝ䁙ೱᙹϸᔈ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!26! !!!!!!!!਎ǵεဉఎ๵ϐᙯ‫ !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!׎‬27! !!!!!!!!‫ا‬ǵ፦ᡏ DNA ᇙഢ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!27! !!!!!!!!ࡵǵज़‫ڋ‬ሇનНှ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!28! !!!!!!!!ࡵ൘ǵDNA ᙯࢉ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!. 28!. !!!!!!!!ࡵມǵխࣝᑻӀࢉՅ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!29!! !!!!!!!!ࡵୖǵ಍ीϩ‫ !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!݋‬30!. ಃΟകǺ่݀!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!31! !!!!ಃ΋࿯ǵಒझਲ਼୻Ꭶ. 31!. !!!!ಃΒ࿯ǵAntizyme ‫ ܭ‬JK Tet-On ಒझਲ਼ύ‫߄ޑ‬౜. 31!. !!!!!!!!൘ǵAntizyme ၸࡋ߄౜‫܌‬Їว‫ޑ‬ಒझ঒Ϋ. 31!. viii.

(10) !!!!!!!!ມǵаՋБᏀᗺ‫ݤ‬ዴᇡ antizyme ‫ ܭ‬JK Tet-On ಒझਲ਼ύ‫߄ޑ‬౜. 32!. !!!!ಃΟ࿯ǵAntizyme ჹ‫ ܭ‬JK Tet-On ಒझਲ਼ࢲϯ‫ޑ‬ቹៜ. 32!. !!!!!!!൘ǵаϸᙯᒵ䁙.ᆫӝ䁙ೱᙹϸᔈᢀჸ antizyme ჹ‫ ܭ‬JK Tet-On ಒझਲ਼ӧόࢲϯ‫ࢲک‬ϯΠಒझᐟન୷Ӣ߄౜‫ޑ‬ቹៜ. 32. ມǵаջਔ‫ۓ‬ໆ.ᆫӝ䁙ೱᙹϸᔈϩ‫ ݋‬antizyme ჹ‫ ܭ‬IL-2 ୷Ӣ ߄౜‫ޑ‬ቹៜ. 33. ୖǵа RNAi ‫ೌמ‬υᘋ antizyme ߄౜ჹ‫ ځ‬IL-2 ୷Ӣ߄౜‫ޑ‬ቹៜ. 34. !!!!ಃѤ࿯ǵAntizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐёૈϩηᐒᙯ. 34!. !!!!!!!!൘ǵаխࣝᑻӀࢉՅᢀჸ antizyme ߦ຾ NF-NB ᙯՏ຾Εಒझਡ. 34. ມǵаխࣝᑻӀࢉՅᢀჸ antizyme ߦ຾ NF-NB Ser536 ‫ޑ‬ᕗለϯ߄౜ 35 ୖǵаՋБᏀᗺ‫ݤ‬ዴᇡ antizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐૻ৲໺Ꮴ ೈқ፦߄౜ໆ. 36. ಃѤകǺ૸ፕ. 38!. !!!!ಃ΋࿯ǵ࣬ᜢЎ᝘ຑ‫݋‬ᆶឍញ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!38! !!!!ಃΒ࿯ǵჴᡍ่݀ញፕ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!44!. ಃϖകǺ่ፕ!. 48!. ಃϤകǺୖԵЎ᝘ !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!49! ಃΎകǺკ߄. 56!. !!!!ಃ΋࿯ǵ߄. 56!. !!!!ಃΒ࿯ǵკ. 58. ! ! !. ix.

(11) ߕᒵ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!74! ߕᒵ΋ǵAntizyme ғԋᐒ‫ڋ‬. 74. ߕᒵΒǵӭữғӝԋжᖴ೼৩. 75. ߕᒵΟǵAntizymeǵODCǵAZI Ϸ polyamines ϐ໔࣬ᜢ‫܄‬. 76. ߕᒵѤǵT ಒझࢲϯૻ৲໺ሀၡ৩. 77. ߕᒵϖǵT ಒझᆶಒझᐟન߄౜. 78. ߕᒵϤǵAntizyme Gene Sequences. 79. ߕᒵΎǵAntizyme ӄߏữ୷ለ‫ׇ‬ӈ. 80. ߕᒵΖǵTet-On System. 81. ߕᒵΐǵಒझਲ਼୻ᎦБ‫ݤ‬. 82. ߕᒵΜǵDual Luciferase System. 84. ߕᒵΜ΋ǵ၂ᏊϷ጗ፂనଛᇙ. 85. ፕЎႝηᔞਢ΢ᆛ௤៾ਜ!. x.

(12) ߄Ҟᒵ! ।ԛ!. ߄΋ǵҁፕЎ‫܌‬Ҕ‫ל‬ᡏၗ਑!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!56! ߄ΒǵҁፕЎ‫܌‬ҔЇη‫ׇ‬ӈϷ‫ځ‬చҹ!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!57! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! xi.

(13) კҞᒵ! ।ԛ!. კ΋ǵჴᡍࢬำკ. 58. კΒǵJK Tet-On Cell ಒझ୻Ꭶ. 59. კ 2.1 JK Tet-On Vector ‫ ک‬JK Tet-On mAZ (1-228) ಒझਲ਼‫׎‬ᄊ. 59. კ 2.2 JK Tet-On Vector ‫ ک‬JK Tet-On mAZ (1-228) ಒझਲ਼ ғߏԔጕკ. 60. კΟǵAntizyme ᇨวಒझ঒Ϋ. 61. კ 3.1 JK Tet-On Vector ‫ ک‬JK Tet-On mAZ (1-228) ಒझ঒Ϋϐ ಒझ‫׎‬ᄊϷಒझӸࢲ౗. 61. კ 3.2 аࢬԄಒझሺϩ‫ ݋‬antizyme ၸࡋ߄౜‫܌‬Їว‫ޑ‬ಒझ঒Ϋ. 62. კѤǵAntizyme ೈқ፦߄౜ໆ. 63. კϖǵAntizyme ჹಒझᐟન୷Ӣ߄౜‫ޑ‬ቹៜ. 64. კϤǵAntizyme ჹ‫ ܭ‬IL-2 ୷Ӣ߄౜‫ޑ‬ቹៜ. 66. კΎǵAntizyme RNAi ჹ‫ ܭ‬IL-2 ୷Ӣ߄౜‫ޑ‬ቹៜ. 67. კΖǵAntizyme ቹៜ NF-NB translocation. 68. კ 8 (a) аխࣝᑻӀࢉՅᢀჸ JK Tet-On Vector ಒझਲ਼ύ antizyme ቹៜ NF-NB ᙯՏ຾Εಒझਡ. 68. კ 8 (b) аխࣝᑻӀࢉՅᢀჸ JK Tet-On mAZ (1-228) ಒझਲ਼ύ antizyme ቹៜ NF-NB ᙯՏ຾Εಒझਡ კΐǵAntizyme ቹៜ NF-NB ᕗለϯ߄౜. xii. 69 70.

(14) კ 9 (a) аխࣝᑻӀࢉՅᢀჸ JK Tet-On Vector ಒझਲ਼ύ antizyme ቹៜ NF-NB Ser536 ᕗለϯ߄౜. 70. კ 9 (b) аխࣝᑻӀࢉՅᢀჸ JK Tet-On mAZ (1-228) ಒझਲ਼ύ antizyme ቹៜ NF-NB Ser536 ᕗለϯ߄౜. 71. კΜǵAntizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐૻ৲໺ሀၡ৩ೈқ፦߄౜. 72. კΜ΋ǵAntizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐёૈϩηᐒᙯ. 73. xiii.

(15) ᕭቪ߄! AZ. Antizyme. AZI. Antizyme inhibitor. APC. Antigen presenting cell. AZIN. Ornithine decarboxylase antizyme inhibitor. AICD. Activation-induced cell death. APS. Ammonium persulfate. BSA. Bovine serum albumin. DAG. Diacylglycerol. DMSO. Dimethyl Sulfoxide. Dox. Doxycycline. FACS. Fluorescence Activated Cell Sorter. FBS. Fetal Bovine Serum. GLUL. Glutamine synthetase. IFNs. Interferons. IGJ. Immunoglobulin J chain. IKK. INB Kinase. IL. Interleukin. Ion. Ionomycin. IPA. Isopropyl alcohol. IP3. Inositol 1,4,5-trisphosphate. ITAMs. Immunoreceptor tyrosine-based activation motifs. JK cell. Jurkat T cell. OAZ1. Ornithine Decarboxylase Antizyme-1. ODC. Ornithine decarboxylase xiv.

(16) ORF. Open reading framses. PHA. Phytohemagglutinin. PI. Propidium iodide. PIP2. Phosphatidylinositol 4,5- bisphosphate. PKC. Protein kinase C. PMA/TPA. Phorbol 12-myristate 13-acetate. PVDF. Polyvinylidene fluoride. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TCR. T cell antigen receptor. TE. Trypsin-EDTA. TEMED. N,N,N',N'-Tetramethylethylenediamine. Tet-On. Tetracycline-On. ThP. T helper precursor cells. xv.

(17) ಃ΋കǵᆣፕ! ಃ΋࿯ǵ‫׭‬䁙ȐAntizymeȑ ൘ǵAntizyme ‫ޑ‬ғԋᐒ‫!ڋ‬ Antizyme (AZ)!ӧ1976ԃᙖҗOrnithine decarboxylase (ODC) ᆶ Antizyme‫܌‬ᄬԋ‫ޑޑ‬౦፦Βᆫᡏ (heterodimer) ೏᛾ܴ٠‫ۓ‬ကࣁODC ߚᝡ‫ڋ׭ݾ‬Ꮚ (Heller et al., 1976)ǶAntizyme߄౜ӭᅿࠠᄊǴନΑத ‫ޑـ‬Antizyme-1ᆶAntizyme-2ϐѦǴќѦԖಔᙃ஑΋‫ޑ܄‬Antizyme-3 ᆶԿϞߚ‫ࠠڂ‬фૈ҂‫ޑޕ‬Antizyme-4 (Pegg, 2006)Ƕӧሇ҆๵‫ک‬Γᜪ ྽ύǴantizyme‫ޑ‬ғ౛фૈЬाࢂ཮೷൩ODC‫ޑ‬फ़ှǴՠ੝ձ‫ࢂޑ‬ӧ থ٢ᜪ୏‫ނ‬ಒझύǴӄߏ‫ڀ‬фૈ‫܄‬antizyme‫ޑ‬ౢғЬाࢂ‫ډڙ‬ಒझϣ ӭữ (Polyamines) ‫ޑ‬ᐚࡋ‫܌‬ፓ௓٠Ъ໪࿶ၸᙯ᝿ਔ᠐ዸਣ+1Տ౽‫ޑ‬ ᐒ‫( ڋ‬translational +1 frameshifting)!Զғԋ!(Matsufuji et al., 1995)Ƕ!!!! Antizyme mRNAύԖ‫ঁٿ‬᠐ዸਣ (Open reading framsesǹORF)Ƕ ORF1ၨอՠࠅԖ‫ঁٿ‬ଆ‫ۈ‬ஏዸёа௴୏ᙯ᝿բҔǴՠࢂORF1࿶ᙯ᝿ р‫ޑ‬ೈқ፦‫ځ‬фૈۘ҂ܴᕕǹORF2ၨߏЪёаᙯ᝿рε೽ϩς‫ڀޕ‬ фૈ‫ޑ܄‬ೈқ፦ǴՠࢂࠅલЮଆ‫ۈ‬ஏዸǴӢԜा௴୏ORF2຾Չᙯ᝿ բҔϐ߻Ѹ໪ाӃ௴୏ORF1ǴԶ೭ύ໔߾ࢂѸ໪ा࿶ၸᙯ᝿ਔ᠐ዸ ਣՏ౽ᐒ‫܌ڋ‬ፓ௓Ƕ‫ځ‬ύǴ‫ת‬ᄽፓ௓Ԝᐒ‫ڋ‬วғ‫ޑ‬Ьा‫ف‬Յࢂಒझϣ polyaminesᐚࡋ‫ޑ‬ӭჲ (Higashi et al., 2006; Ivanov et al., 2000)Ƕ. 1.

(18) AntizymeҞ߻ς‫ࢂޕ‬ಃ΋ঁ೏ว౜ӧথ٢ᜪಒझ྽ύ཮࿶җਡ ᗐਡለ᠐ዸਣ+1Տ౽ᐒ‫ڋ‬Զౢғрٰ‫ޑ‬ೈқ፦Ƕ྽ಒझϣpolyamines Ӹӧ‫ޑ‬௃‫ݩ‬ϐΠǴORF1཮Ӄ௴୏ଆ‫ۈ‬ஏዸ຾Չᙯ᝿բҔǴ຾Չ‫ډ‬ಖ Зஏዸ (UGA) ਔǴ೸ၸԜᐒ‫ڋ‬ၢၸ΃ঁbaseǴ຾Զ໒‫ۈ‬຾ՉORF2 ‫ޑ‬ᙯ᝿բҔ٠ౢғӄߏ‫ڀ‬Ԗ҅தфૈ‫ޑ‬antizyme (1-228 a.a.)Ƕϐ߻Ў ᝘ࡰрǴԖΟঁाન཮ߦ٬polyamine-induced frameshiftingᐒ‫ޑڋ‬ౢ ғǶಃ΋೽ҽࣁӧantizyme mRNA 5’ᆄԖORF1 UGA stop codonǹಃ Β೽ҽӧORF1 UGA stop codonϐࡕmRNA཮‫׎‬ԋstem-loop่ᄬԶᆙ ௗ๱ࡕय़3’ᆄ‫΃ࢌޑ‬೽ҽ‫ׇ‬ӈ཮‫ک‬stem-loop่ᄬ‫׎‬ԋ΋ঁbase pairǴ ຾Զ‫׎‬ԋRNA pseudoknot‫ޑ‬Βભ่ᄬǹಃΟ೽ҽ߾ࢂӧ antizyme mRNA 3’ᆄ୔ୱว౜Ԗ೽ϩORF1‫ׇ‬ӈӸӧǴ‫܌‬аӃ߻Ў᝘ᇡࣁӢࣁ antizyme mRNA่ᄬ྽ύԖ೭ΟঁाનӸӧ຾Զ٬frameshifting‫ޑ‬ᐒ ‫ڋ‬ౢғ (ߕᒵ΋) (Coffino, 2001)ǶԜѦǴЎ᝘ൔ֋ࡰрਡᗐਡለ᠐ዸ ਣ‫ޑ‬Տ౽᎞๱ќ΋ঁᐒ‫܌ڋ‬ፓ࿯Ǵ྽ੰࢥགࢉਔஒ௴୏ಒझ‫ۈ߃ޑ‬ٛ ᑇϸᔈǺυᘋન(interferonsǹIFNs) ‫ࢥੰלځ‬ᆶ‫ל‬ಒझғߏૈΚ೽ϩ ࢂᙖҗ2'Æ5' oligoadenylate-RNase L (RNase L)!RNAफ़ှၡ৩ (Zhou et al., 1993)ǶΓᜪᙯ᝿ಖЗӢηeRF3཮ᆶRNase L࣬ϕբҔǴ຾Զߦ ٬premature termination codonsቚуreadthroughਏ౗аϷӧantizyme +1 frameshiftՏᗺቚу+1 frameshiftਏ౗ (Le Roy et al., 2005)Ƕ. 2.

(19) ມǵAntizyme ‫ޑ‬ғ౛фૈᆶғ‫ނ‬ख़ा‫!܄‬ AntizymeҞ߻ς‫ୖޕ‬ᆶ‫ޑ‬ғϯжᖴନΑፓ௓ಒझϣѦpolyamines ᐚࡋϐѦǴЬाᗋхࡴΑ‫ڋ׭‬ODC䁙‫܄ࢲޑ‬ᆶፓ௓polyaminesၮଌǴ ଺ࣁpolyaminesжᖴ‫ޑ‬ፓ࿯‫( ޣ‬He et al., 1994; Mitchell et al., 1994; Sakata et al., 2000)ǶODCࢂӕ፦ᚈᆫӝᡏ (homodimer) ‫ޑ‬ሇનǴѬୖ ᆶArginine‫ޑ‬жᖴၸำǴࣁpolyamine biosynthesisᜢᗖሇન (ߕᒵΒ)Ƕ!! Antizyme‫ډڙ‬ಒझϣpolyaminesᐚࡋፓ௓࿶translational frameshifting ၸำԶғԋǴѬ཮‫ک‬ODC‫׎‬ԋ౦፦Βᆫᡏ่ᄬ (heterodimer) Їଆ ODCӕ፦ᚈᆫӝᡏ‫ޑ‬ϩᚆ٠Ъ٬ளODC೏26Sೈқ፦䁙ᡏफ़ှǴODC ‫܄ࢲޑ‬Πफ़ǶODC࿶җ26Sೈқ፦䁙ᡏफ़ှόࢂ࿶җubiquitinԶࢂ೸ ၸᆶantizyme࣬ϕ่ӝԶ຾ՉǴԜѦӧό٩ᒘubiquitinϐΠǴODC‫ޑ‬ फ़ှΨ᎞๱NAD(P)H quinine਼ϯᗋচ䁙-1‫ޑ‬ፓ࿯೏20Sೈқ፦䁙ᡏ फ़ှ (Asher et al., 2005)Ƕჹ‫ܭ‬ODC‫ޑ‬ፓ࿯ᆶфૈǴคፕantizymeࢂՏ ‫ܭ‬ಒझਡ‫܈‬ಒझ፦Ǵ೿ૈߦ٬ODC೏ೈқ፦䁙ᡏफ़ှǶନΑantizymeǵ ODC‫ک‬polyaminesϐѦǴΓᜪ‫ޑ‬antizyme inhibitor (AZI) ΨୖᆶΑ᏾ᡏ ‫ޑ‬ғϯжᖴǴѬჹ‫܌ܭ‬Ԗᅿᜪ‫ޑ‬antizymeё଺ࣁ΋૓‫ڋ׭ޑ‬ᏊǶAZI ่ӝantizymeૈΚКODCଯЪவODC:Antizyme heterodimerύញр ODCǴ຾ԶӣൺODC䁙‫܄ࢲޑ‬ǴԶӧғ‫ނ‬ᡏϣόᛙ‫ޑۓ‬AZIᆶantizyme ߾ࢂ཮࿶җubiquitinationԶࡕ೏26Sೈқ፦䁙ᡏफ़ှ (Bercovich and. 3.

(20) Kahana, 2004; Palanimurugan et al., 2004)Ƕ‫܌‬а᏾ᡏԶ‫ق‬Ǵantizymeǵ ODCǵpolyamines‫ک‬AZIѤ‫ޣ‬۶Ԝϐ໔ࢂϕ࣬‫ڀ‬ᜢᖄ‫ߕ( ܄‬ᒵΟ)Ƕ ନΑ‫ڋ׭‬ODC䁙‫܄ࢲޑ‬аϷpolyaminesжᖴ‫ޑ‬ፓ࿯‫ޣ‬฻୷ҁж ᖴфૈѦǴantizymeӧϩηቫԛᆶಒझቫԛ‫ޑ‬фૈύΨ‫ת‬ᄽख़ा‫فޑ‬ ՅǴ‫ځ‬ύхࡴΑϩϯፓ࿯ǵಒझຼයǵಒझғߏǵԝΫǵϩϯᆶဍዦ ቚғ (Feith et al., 2006; Feith et al., 2001; Fong et al., 2003; Gritli-Linde et al., 2001; Iwata et al., 1999; Mangold, 2006; Murakami et al., 1994; Tsuji et al., 2001)ǶၸѐЎ᝘ࡰрantizyme‫ڀ‬Ԗ՟ubiquitin‫ޑ‬фૈǴૈ ᔅշ‫ځ‬дೈқ፦ӧόሡ٩ᒘubiquitin௃‫ݩ‬ΠଌԿ26Sೈқ፦䁙ᡏ຾Չ फ़ှբҔ (Orlowski and Wilk, 2003)Ƕ೭٤ೈқ፦Ҟ߻ࣁЗନΑς‫ޕ‬ ‫ޑ‬ODCѦǴᗋхࡴΑᆶಒझຼයԖᜢ‫ޑ‬Cyclin D1Ϸᆶૻ৲໺ሀ‫ک‬ᙯ ᒵࢲ‫࣬܄‬ᜢ‫ޑ‬Smad1‫ک‬Snip1 (Gruendler et al., 2001; Lin et al., 2002; Murakami et al., 1992; Newman et al., 2004)ǶԜѦǴantizymeբࣁ polyaminesжᖴ‫ޑ‬ፓ࿯‫ޣ‬Ǵpolyaminesӧϩηᆶಒझቫԛ‫ޑ‬фૈύΨ ՞ख़ा‫ޑ‬ӦՏǴхࡴΑࣁಒझғߏ‫ک‬ว‫܌ػ‬ѸሡǹቚуDNAǵRNA ‫ک‬ೈқ፦‫ޑ‬ғӝԋǹೈқ፦ᐟ䁙‫ޑ‬ፓ௓ǹᙯᒵբҔ‫ޑ‬ፓ࿯Ϸፓ௓ႇᚆ η೯ၰ฻фૈ (Tabor and Tabor, 1984)Ƕᕴ่΢ॊᘜયள‫ޕ‬Ǵantizyme ғ౛фૈόᆅࢂӧ୷ҁғϯжᖴ΢‫ࢂ܈‬ϩη‫ک‬ಒझቫԛ‫ޑ‬фૈύ೿ ‫ת‬ᄽ๱ཱུख़ा‫فޑ‬ՅǶ. 4.

(21) ಃΒ࿯ǵT ಒझ‫ࢲޑ‬ϯȐT cell activationȑ! ൘ǵT ಒझࢲϯ࣬ᜢᐒᙯ Tಒझӧ᏾ঁխࣝϸᔈύǴ‫ת‬ᄽ๱ύЈ‫فޑ‬ՅǶѬ೸ၸ੝౦‫܄‬ᒣ ᛽Ѧٰ‫ל‬চǴ‫׎‬ԋբҔಒझǴᆶϩ‫ݜ‬ಒझᐟનٰፓ࿯խࣝϸᔈϐ‫܄‬፦ ᆶம১ǶTಒझ୺Չ੝‫ۓ‬фૈ‫୏௴ޑ‬Ԗᒘ‫ܭ‬ԋф‫ࢲޑ‬ϯǶTಒझ‫ࢲޑ‬ ϯёϩࣁ‫ٿ‬ᅿᜪࠠǺ΋ࢂ‫ל‬চ٩ᒘࠠǹќ΋ᅿ߾ࢂߚ‫ל‬চ٩ᒘࠠǶ‫ל‬ চ٩ᒘࠠΏࡰTಒझ߄य़‫לޑ‬চௗ‫ڙ‬Ꮤ!(T cell antigen receptorǹTCR)! ёаᒣᇡ੝‫לޑۓ‬চǴ྽TCRԏ‫לډ‬চ‫ڈ‬ᐟਔǴ཮Їଆҗಒझጢǵಒ झ፦Կಒझਡ΋ೱՍ‫ૻޑ‬৲໺ሀǴ຾Զࢲϯಒझ୺ՉфૈǹԶߚ‫ל‬চ ٩ᒘࠠ߾ࣁTಒझ‫ࢲޑ‬ϯࢂ೸ၸࢌ٤ಒझ߄य़‫ל‬চ (ӵCD2ǹCD28฻) ‫ޑ‬բҔǴ຾ԶࢲϯTಒझǶ ྽Tಒझ‫ל‬চ‫ڙ‬ᡏᆶ‫ל‬চև౜ಒझ (antigen presenting cellǹAPC) ߄य़‫ޑ‬Ьाಔᙃ࣬৒ፄӝᡏ (MHC) ΢‫לޑ‬চᴏ两 (antigen peptide) Тࢤ่ӝࡕǴᇨวTCRᆶAPCௗ᝻य़ౢғխࣝँ᝻ (immunological synapse)Ǵ൩໒‫ۈ‬໺ሀࢲϯ‫ૻޑ‬৲Ƕ྽Tಒझ࿶җTCR/CD28‫לډڙ‬চ ‫ڈ‬ᐟϐࡕ཮ᇨวSrcৎ௼ೈқ፦ᕗለᐟ䁙 (protein tyrosine kinases) Lck‫ک‬Fyn‫ࢲޑ‬ϯǶࢲϯ‫ޑ‬Lck཮຾ԶᕗለϯTCR/CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) ΢‫ޑ‬ႀữለǶZAP-70֖Ԗ‫ٿ‬ ঁSrc homology-2 (SH2) domainsǴёаᆶᕗለϯ‫ޑ‬ႀữለ่ӝǴӢԜ. 5.

(22) ᕗለϯ‫ޑ‬ITAMsᆶZAP-70่ӝǴࢲϯZAP-70ǶZAP-70೏ࢲϯࡕ཮Ծ ‫ך‬ᕗለϯ (autophosphorylation) ௗ๱ᕗለϯ೚ӭᙯௗೈқ (adaptor) ᆶೱௗೈқ (linker)Ǵхࡴ‫ঁٿ‬ჹTಒझࢲϯࡐख़ा‫ঁٿޑ‬ᙯௗೈқ LATᆶSLP-76Ƕ೭٤ᙯௗೈқ೏ࢲϯࡕᗨฅό‫ڀ‬Ԗᐟ䁙ࢲ‫܄‬Ǵՠࢂд ॺёаၟ‫ځ‬дόӕૻ৲ϩηಔԋ΋ঁૻ৲ፄӝᡏǴаߡ‫ܭ‬೏΢ෞ‫ޑ‬ᕗ ለᐟ䁙ࢲϯ (Jordan et al., 2003; Sommers et al., 2002; Wonerow and Watson, 2001)Ƕς‫ޕ‬PLC-Ȗ (phospholipase C-Ȗ)ǵVav‫ک‬Grb2೿ࢂ࿶җ ่ ӝ LAT Զ ೏ ࢲ ϯ Ƕ ࢲ ϯ ‫ ޑ‬PLC-Ȗ ཮ Н ှ phosphatidylinositol 4,5bisphosphate. (PIP2). ౢ ғ inositol. 1,4,5-trisphosphate. (IP3). ᆶ. diacylglycerol (DAG)ǶIP3ᘉණ‫ډ‬ಒझ፦ύǴᆶϣ፦ᆛ΢‫ڙޑ‬ᡏ่ӝ ᇨ ว ້ ᚆ η ញ р Ǵ ້ ᚆ η ӆ ᆶ ឫ ້ ન (calmodulin) ่ ӝ ࡕ ࢲ ϯ calcineurinǴcalcineurinࣁ΋serine/threonineѐᕗለ䁙Ǵᇨ٬NFATϷ NF-NBᙯᒵӢηவಒझ፦຾Εਡϣ୺ՉᙯᒵӢηфૈǶќѦDAGёࢲ ϯೈқ፦ᐟ䁙C (protein kinase CǹPKC)ǴPKCԖ೚ӭisoformsǴ‫ځ‬ύ PKC-TନΑ೏DAG֎ЇᙯՏԿಒझጢߕ߈ԶࢲϯǴΨ཮‫ޔ‬ௗ೏Lckᕗ ለϯǶ΋૓ᇡࣁǴPKC-T‫ࢲޑ‬ϯ཮஥୏Πෞ‫ޑ‬NF-NBǵJNKаϷAP1 ฻ૻ৲໺ሀၡ৩‫ޑ‬຾Չ (Bi et al., 2001; Siebenlist et al., 2005; Sun et al., 2000; Weil and Israel, 2006; Werlen et al., 1998)Ƕќ΋Бय़ǴVavࣁ ΋ਡ㧿ҬඤӢη (nucleotide exchange factor) ЬाࢲϯΠෞ‫ޑ‬Rhoǵ. 6.

(23) RacǴനࡕࢲϯc-jun (Billadeau et al., 2007)ǶGrb2߾ࢂ೸ၸSosࢲϯ RasǵRafϷERKǴനࡕ௴୏c-fos‫߄ޑ‬౜ǶӧಒझਡϣǴc-fosᆶc-jun ཮‫׎‬ԋ౦፦Βᆫᡏ (heterodimer) ԶᄬԋAP-1ᙯᒵӢη (ߕᒵѤ)Ƕ ࿶җTCR໺ሀ‫ࢲޑ‬ϯૻ৲Ѧӆу΢CD28‫ޑ‬Ӆӕ‫ڈ‬ᐟૻဦǴ཮ӕ ਔࢲϯኧঁख़ा‫ޑ‬ᙯᒵӢη‫܄ࢲޑ‬Ǵ຾Զ௴୏೚ӭ࣬ᜢ୷Ӣ‫߄ޑ‬౜Ǵ х֖Αϟқન-2 (interleukin-2ǹIL-2)Ϸ‫ځ‬дಒझᐟન୷Ӣ‫߄ޑ‬౜ (Okamura and Rao, 2001)Ƕ Tಒझ࡭ុ‫לڙ‬চ‫ڈ‬ᐟࢲϯࡕǴ཮ᇨวಒझ߄य़Fas‫ک‬Fas ligand ߄౜ໆቚуǴ຾Զ೷ԋᎃ߈‫ޑ‬Tಒझϐ໔೸ၸFas‫ک‬Fas ligand่ӝǴϕ ࣬໺ሀԝΫૻ৲Ꮴठಒझ঒ΫǴջ‫܌‬ᒏTಒझࢲϯठԝ‫ޑ‬ᐒᙯϐ΃ (activation-induced cell deathǹAICD) (Brunner et al., 1995; Ju et al., 1995)ǴЬाวғ‫ܭ‬TಒझࢲϯࡕයǶӧғ‫ނ‬ᡏϣǴAICD ჹᆢ࡭խࣝ ‫س‬಍‫תۓࡡޑ‬ᄽΑख़ा‫فޑ‬ՅǴаᗉխTಒझ࡭ុࢲϯቚ෗Ƕ. ມǵT ಒझࢲϯᆶಒझᐟન߄౜ T రЃ҆ಒझ࿶җࢲϯϷϩϯࡕ۳۳཮‫׎‬ԋόӕᜪࠠ‫ ޑ‬T ಒझٰ ୺Չঁձ੝‫ޑۓ‬фૈǴхࡴΑ T ᇶշಒझ (CD4+ǹTh1 Ϸ Th2 cell)ǵ T ࢥఠಒझ (CD8+ǹTc cell)ǵTh17 cell Ϸ T ፓ࿯ಒझ (Treg cell)Ƕ‫ځ‬ ύ T ಒझ‫ࢲޑ‬ϯϷϩϯၸำ۳۳౐ੋ‫ډ‬೚ӭಒझᐟન (cytokines) ‫ޑ‬ ߄౜аϷፓ௓ (Elser et al., 2002; Kanda and Watanabe, 2001; Nurieva 7.

(24) et al., 2006; Sakamoto et al., 2006; Severino et al., 2006)ǶT helper ߻០ ಒझ (T helper precursor cellsǹThP) ‫לډڙ‬চև౜ಒझ APC ௗ᝻‫ڈ‬ ᐟϐࡕ཮٩όӕ‫ૻޑ‬৲ϸᔈԶϩϯԋόӕᜪࠠ‫ ޑ‬T ಒझǶ྽‫ډڙ‬ IL-12 Ϸ IFN-J‫ڈ‬ᐟਔǴThP ཮ୃӛϩϯԋ Th1ǹ࣬ჹ‫ޑ‬Ǵ྽‫ ډڙ‬IL-4 ‫ڈޑ‬ᐟਔǴThP ߾཮ୃӛϩϯԋ Th2ǴԜѦ‫ ډڙ‬TGF-E‫ ک‬IL-6 ‫ڈ‬ᐟਔǴ ThP Ψ཮ϩϯԋ Th17 cell ٰ୺Չঁձфૈ Ƕ όӕ‫׎‬Ԅ T ಒझϩձ཮ញрόӕಒझᐟનٰঁձ୺Չ‫ځ‬੝‫ޑۓ‬ фૈǶTh1 ಒझ཮ӝԋញрόӕ‫ޑ‬ಒझᐟનϩձբҔ‫ ܭ‬Macrophageǵ NK cell ‫ ک‬Tc cells ฻όӕҞ኱΢Ǵ೭٤ಒझᐟનЬाхࡴΑ IL-2ǵ IFN-JϷ TNF-DǹTh2 ಒझ߾ࢂ཮ӝԋញр IL-4ǵIL-5ǵIL-6ǵIL-10 ‫ ک‬IL-13ǴЬाբҔ‫ޑ‬Ҟ኱ࣁ B రЃಒझǶTh17 ಒझЬाӝԋញ‫ޑܫ‬ ಒझᐟનа IL-17 ࣁЬǴբҔ‫ޑ‬Ҟ኱ࣁ Stromal CellsǶќѦǴ‫ځ‬Ѭӵ Treg ಒझΨ཮ӝԋញ‫ܫ‬ಒझᐟનႽࢂ TGF-E฻ٰ୺Չ‫ځ‬੝‫ۓ‬фૈǴ Treg ೽ϩфૈࣁ‫לڋ׭‬চև౜ಒझ APC (ߕᒵϖ)Ƕ T ಒझ‫҅ޑ‬தғ౛фૈϷ߄౜‫ډڙ‬೚ӭಒझᐟન‫܌‬ፓ௓Ƕӧ೭٤ ಒझᐟન྽ύ-੝ձࢂ IL-2 ࢂ‫ࢲډڙ‬ϯ T ಒझԾᡏϩ‫܌ݜ‬ౢғ‫ޑ‬ख़ा ಒझᐟનǴΨࢂ T ಒझ‫ܭ‬ϟᏤ᏾ᡏխࣝፓ࿯ϐಒझᐟનǴЬљ T ಒ झϐғߏፓ௓ǵࢲϯǵϩϯаϷஎЬ᏾ᡏխࣝٛᑇ฻фૈ (Mukerjee et al., 2001; Wang et al., 2007; Williams et al., 2006; Zorn et al., 2006)Ƕ. 8.

(25) ಃΟ࿯ǵAntizyme ᆶ T ಒझϐ໔‫࣬ޑ‬ᜢ‫܄‬ ൘ǵചữለಥ♐䁙 (Ornithine decarboxylase) ᆶӭữ (Polyamines) ചữለಥ♐䁙 (Ornithine decarboxylaseǹODC) ࣁғ‫ނ‬ᡏϣӭữ (polyamines) ‫׎‬ԋ‫ޑ‬ᜢᗖሇન (key enzyme)ǶӧΓᜪύ‫୷ځ‬ӢՏ‫ܭ‬ಃ ΒჹࢉՅᡏp25Տ࿼΢Ǵೈқ፦߄౜ࡕЬाϩթӧಒझ፦ǴࢲϯϐODC аᚈᡏ (dimer) ‫׎‬ԄӸӧǶPolyaminesࣁ஥҅ႝữ୷‫ޑ‬λિެᜪϩη ဂǴх֖ᆭữ (putrescine)ǵ٥ᆒữ (spermidine)‫ک‬ᆒữ (spermine)Ƕ PolyaminesୖᆶಒझϣDNAǵRNAǵೈқ፦Ϸᕗለિ፦‫ޑ‬ғӝԋǴࣁ ಒझғߏ‫ک‬ว‫܌ػ‬Ѹሡ‫ޑ‬Ǵჹ‫ܭ‬ಒझғߏǵϩϯϷԝΫ‫ת‬ᄽख़ा‫ف‬ ՅǶODC‫߄ޑ‬౜ӧಒझғߏਔࢂ΋ঁѸሡ‫܄‬ሇનǴ‫ځ‬ೈқ፦߄౜Ϸࢲ ‫ܴ܄‬ᡉӦё࿶җ೚ӭғߏӢηᇨᏤԶϲଯǴғߏߦ຾Ӣηӵ಻ᅟᆾǵ ᛰ‫ނ‬ǵғߏӢη‫ک‬ဍዦߦ຾‫୼ૈނ‬٬Ԝ䁙‫ז‬ೲ߄౜Զቹៜғ౛ᡂϯǶ ODCᆶpolyaminesӧ҅த‫ޑ‬ғ౛фૈх֖खजว‫ػ‬ǵಒझຼයǵғߏ ᆶԝΫ୺Չύࣁ΋ঁѸा‫فޑ‬ՅǶၸѐЎ᝘ࡰр྽TరЃಒझࢲϯ຾ ՉԖํϩ຋߃යǴODC‫߄ޑ‬౜ໆ཮‫ډڙ‬lymphokines‫ޑ‬ቚуԶ೏ᇨᏤ рٰǴԜѦΨว౜putrescine‫ޑ‬ቚуӧTరЃಒझቚғ߃යࢂѸ໪‫ޑ‬ (Brand, 1987)ǶќѦၸѐࣴ‫ز‬Ψග‫ډ‬IL-1ԜಒझᐟનΨ཮ᙖҗᇨᏤ antizyme‫߄ޑ‬౜ᏤठODC‫߄ޑ‬౜Πफ़຾Զ‫ځڋ׭‬дಒझ‫ޑ‬ቚғǶIL-1 ЬाࢂҗѮᏘಒझ‫ࢲ܈‬ϯ‫ൂޑ‬ਡౚᇙ೷ϩ‫ݜ‬Ǵࣁ΋ᅿӭфૈ‫ޑ܄‬ಒझ. 9.

(26) ᐟનǴჹ‫ܭ‬ಒझғߏǵխࣝٛᑇǵᡏϣѳᑽࣗԿ‫ת‬ᄽխࣝᅱ࿎‫فޑ‬Յ ೿՞΋‫ۓ‬ख़ा‫( ܄‬Yang et al., 1997)ǶԿҞ߻ࣁЗǴ೚ӭЎ᝘೿ς᛾ܴ ODCᆶpolyaminesჹ‫ܭ‬ಒझ‫ޑ‬ғߏว‫ػ‬ǴࣗԿࢂDNAǵRNAϷೈқ፦ ‫ޑ‬ғӝԋ೿‫ת‬ᄽ΋‫ۓ‬ख़ा‫فޑ‬ՅǴΨว౜ODCᆶpolyaminesჹ‫ځܭ‬T ಒझ‫ޑ‬ቚғ‫ک‬ว‫ػ‬೿Ԗ‫ୖځ‬ᆶǴՠёெ‫ࢂޑ‬Ҟ߻ODCǵpolyaminesࣗ Կࢂantizymeჹ‫ܭ‬Tಒझ‫ځ‬дғ౛фૈ‫ޑ‬ፓ௓‫ٯ‬ӵࢲϯǵϩϯ‫܈‬ಒझ ঒Ϋ฻࣬ᜢࣴ‫ز‬ϝࢂό‫ޑى‬Ƕ. ມǵᙯᒵӢηࢲϯૻ৲ፓ௓ Ҟ߻ࣁЗǴantizyme ᆶ T ಒझϐ໔࣬ᜢ‫܄‬ϝࢂ҂‫ޕ‬Ƕε೽ϩЎ᝘ ௖૸೿ࢂ೸ၸ ODC ᆶ polyamines ‫ ک‬T ಒझϐ໔‫࣬ޑ‬ᜢࣴ‫ٰز‬௢ෳ antizyme ‫ ک‬T ಒझϐ໔ᜢೱ‫܄‬ǴёࢂᗲϿΓࠅ‫ޔ‬ௗ௖૸ antizyme ፓ ௓ T ಒझ‫ޑ‬ғ౛фૈǴΨӢԜၸѐ൳ЯؒԖЎ᝘Ԗග‫ ډ‬antizyme ࢂ ӵՖ‫ޔ‬ௗቹៜ T ಒझғ౛фૈǶՠ߈൳ԃٰᄌᄌΨԖ೚ӭЎ᝘໒‫ۈ‬௖ ૸ antizyme ‫ޑ‬ғ౛фૈǶ‫ځ‬ύдॺว౜ᆶૻ৲໺ሀ‫ک‬ᙯᒵࢲ‫࣬܄‬ᜢ ‫ޑ‬ೈқ፦ Smad1 ‫ ک‬Snip1 ཮࿶җ antizyme ӧ ubiquitin-independent ᐒ ‫ڋ‬Π೏ឫ஥Կ 26S proteasome ຾Չफ़ှբҔǶSnip1 Ҟ߻ς‫ࢂޕ‬ CBP/P300 ‫ڋ׭ޑ‬ೈқǴԶ CBP/P300 ߾ࣁ΋ᙯᒵӢηߦ຾ࢲϯ‫ޑ‬ೈ қ፦Ǵӧ T ಒझਡϣЬљᙯᒵӢη‫܄ࢲޑ‬Ǵ຾Զߦ٬ᙯᒵբҔ‫ޑ‬຾ ՉǴቹៜ೚ӭಒझᐟન‫ޑ‬ғԋ (Qiu et al., 1999)Ƕҗ‫ܭ‬Ҟ߻ϝคዴϪ 10.

(27) ‫ޑ‬ᐒ‫ڋ‬᛾ჴ antizyme ᆶ CBP/P300 ϐ໔‫ޑ‬ᜢ߯ǴӢԜ໻௢ෳ antizyme ёૈ೸ၸ CBP/P300 ‫߄ޑ‬౜ԶЬाቹៜ T ಒझϣᙯᒵӢη‫܄ࢲޑ‬Ǵ຾ Զቹៜ᏾ᡏ T ಒझ‫҅ޑ‬தғ౛фૈϷಒझᐟન‫߄ޑ‬౜Ƕՠჴሞ΢ antizyme ࢂӵՖፓ௓ T ಒझ‫ޑ‬᏾ᡏᐒ‫ڋ‬Ҟ߻ࣁЗϝࢂ҂‫ޑޕ‬ǶԜѦǴ ၸѐᖏ‫زࣴ׉‬ᡉҢᑡ஻࡚‫܄‬రЃዦқՈੰ (ALL) ‫ޑ‬λ‫ٽ‬஻‫ځޣ‬ൺว ำࡋёаᙖҗΟঁ୷Ӣ‫߄ޑ‬౜ٰղᘐǴхࡴΑ glutamine synthetase (GLUL) ǵ ornithine decarboxylase antizyme inhibitor (AZIN) Ϸ immunoglobulin J chain (IGJ) (Hoffmann et al., 2008)Ƕ‫ځ‬ύ AZIN ࢂ२ ࡋ೏ว౜ᇡࣁᆶరЃಒझ‫ڀ‬Ԗᜢᖄ‫܄‬ǴЪёаբࣁղᘐ ALL ൺว‫ޑ‬ ‫ځ‬ύ΋໨ࡰ኱Ƕ‫܌‬аᘜય΢ॊёள‫ޕ‬ǴODCǵantizyme ࣗԿࢂ AZI ଞჹ‫ ܭ‬T ಒझ௢ෳёૈࢂᙖҗᙯᒵቫԛ‫ޑ‬ፓ௓Զፓ࿯‫ځ‬ғ౛фૈǶӢ ԜǴҁჴᡍࣴ‫ޑز‬Ҟ‫ޑ‬ջࢂགྷा௖૸ antizyme ࢂӵՖ೸ၸᙯᒵቫԛ ‫ޑ‬ፓ௓຾Զѐቹៜ T ಒझ҅த‫ޑ‬ғ౛фૈǶ. 11.

(28) ಃѤ࿯ǵࣴ‫୏ز‬ᐒϷҞ‫!ޑ‬ җ߻य़ඔॊள‫ޕ‬Ǵӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ‫ޑ‬ғԋѸሡा࿶ၸ ਡᗐਡለ᠐ዸਣ+1 Տ౽‫ޑ‬ၸำǶਡᗐਡለ᠐ዸਣ‫ޑ‬Տ౽᎞๱΋ঁᐒ ‫܌ڋ‬ፓ࿯Ǵ྽ੰࢥགࢉਔஒ௴୏ಒझ‫ۈ߃ޑ‬ٛᑇ‫س‬಍Ǻυᘋન‫ੰלޑ‬ ࢥᆶ‫ל‬ಒझғߏૈΚ೽ϩࢂᙖҗ 2'-5' oligoadenylate-RNase L)RNase L* RNA फ़ှၡ৩‫܌‬ፓ௓Ƕੰࢥགࢉಒझਔ཮ᇨวғԋ IFNs ԶЇว ‫س‬಍‫ޑ܄‬ಒझ‫܄‬խࣝϸᔈᆶቚуӭᅿ‫ࢥੰל‬ೈқౢғǶᙖҗಒझϣ‫ޑ‬ IFNsǴRNase L ቚу antizyme +1 ਡᗐਡለ᠐ዸਣ+1 Տ౽‫ޑ‬ਏ౗ǶԜ Ѧಒझ‫܄‬ᆶੰࢥ‫܄‬ೈқ፦ᙖҗਡᗐਡለ᠐ዸਣՏ౽ᐒ‫ڋ‬ёа‫ߏۯ‬ೈ қ፦ C ᆄᙯ᝿຾ՉǴ຾Զ‫ڈ‬ᐟ‫ל‬চև౜๏ࡕϺ‫܄‬խࣝ‫س‬಍Ǵ٠υᘋ ੰࢥғӸǶӧεӭኧੰࢥགࢉΠǴT ಒझӧխࣝϸᔈ‫ת‬ᄽ๱ύЈ‫فޑ‬ ՅǴT ಒझ཮೏ᇨวᏤठࢲϯϷϩϯ٠ౢғόӕ‫ޑ‬ಒझᐟનٰ‫ܢ‬ᑇੰ ࢥགࢉǶӧҞ߻‫زࣴޑ‬྽ύǴၨϿѐ௖૸ӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ჹ‫ܭ‬ಒझϣ‫ޑ‬ғ౛фૈፓ࿯‫ޑ‬၁ಒ௃‫ݩ‬Ƕ੝ձࢂӄߏ‫ڀ‬фૈ‫ޑ܄‬ antizyme ‫ޑ‬ғԋ೽ϩࢂ‫ډڙ‬υᘋનխࣝᐒ‫܌ڋ‬ፓ௓ǴҗԜёว౜‫ډ‬ antizyme ᆶխࣝБय़‫ڀ‬Ԗ೽ϩ‫࣬ޑ‬ᜢ‫܄‬Ǵѝࢂ೭Бय़‫ޑ‬ᐒ‫ڋ‬ᗲϿΓѐ ࣴ‫ز‬ΨࡐϿԖ೭Бय़‫ޑ‬Ў᝘ѐ௖૸ǶӢԜǴҁፕЎᒧ᏷ᢀჸӧ T ಒझ ‫ࢲޑ‬ϯၸำύǴantizyme ‫ת܌‬ᄽ‫فޑ‬ՅǶҁፕЎճҔ Jurkat T Cell (Γ ᜪ࡚‫܄‬రЃ҆ಒझқՈੰ U ಒझਲ਼)!ᙯࢉӄߏ antizyme ‫ ޑ‬Tet-On. 12.

(29) System բࣁࣴ‫ޑز‬ኳࠠǶҁፕЎύ‫܌‬ᙯࢉӄߏ‫ ޑ‬antizyme ࣁಃ΋ࠠ ‫ ޑ‬antizymeǴջ antizyme-1 (ߕᒵϤ)ǶуΕ Doxycycline (Dox) ᇨว߄ ౜ӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme аϷճҔ Ionomycin calcium salt (Ion)ǵ Phorbol 12-myristate 13-acetate (PMAǹTPA) ࢲϯ Jurkat T Cell ٰᢀჸ antizyme ჹ‫ ܭ‬T ಒझࢲϯϐፓ௓Ǵ຾Զ௢ፕ᛾ܴ antizyme ዴჴ཮ቹ ៜ᏾ᡏխࣝፓ࿯ϐಒझᐟનǴ٠຾΋‫؁‬൨‫פ‬ϐ໔‫࣬ޑ‬ᜢ‫܄‬ϷᐒᙯǶ!. 13.

(30) ಃϖ࿯ǵჴᡍ฼ౣ! ҁፕЎύࣴ‫ ޑز‬antizyme ࣣࢂࡰಃ΋ࠠ‫ ޑ‬antizymeǴջ antizyme-1Ƕ ҁፕЎ‫ޑ‬ჴᡍ฼ౣǴϩԋ൳ঁБӛǴӵკ 1 ‫܌‬ҢǶ! ಃ΃೽ϩǺ! !!!!ࡌҥᄬᑐ߄ၲΓᜪ antizyme ୷Ӣၩᡏᆶᙯࢉ੿ਡಒझǶճҔᙯࢉ БԄஒ஥Ԗ Vector Ϸӄߏ‫ ޑ‬Antizyme (‫ڮ‬Ӝࣁँᡂࠠ Antizymeǹm-AZ 1-228 a.a.) (ߕᒵΎ) ‫ ޑ‬Tet-On ‫س‬಍ pTRE2/hyg ၩᡏଌԿ Jurkat T cell ύ!(ߕᒵΖ)ǶճҔՋБᏀᗺ‫( ݤ‬Western blotting) ϷࢬԄಒझሺϩ‫݋‬ sub-G1 phase ٰ᛾ܴ Tet-On ‫س‬಍ӧԖ Doxycycline (Dox)!ӸӧϐΠӧ Jurkat T ಒझύ‫ޑ‬ዴૈ୼ᇨวӄߏ antizyme ‫ޑ‬ೈқ፦߄౜ЪᏤठಒझ ঒ΫǴ٠ЪճҔԜ Tet-On ‫س‬಍ٰ຾Չϐࡕ‫ޑ‬ჴᡍǴᢀჸӄߏ‫ڀ‬фૈ ‫ ޑ܄‬antizyme ჹ‫ ܭ‬Jurkat T ಒझࢲϯ‫ޑ‬ቹៜǶ ! ಃΒ೽ϩǺ! !!!!ଞჹTಒझ‫ࢲޑ‬ϯǴճҔ Phorbol 12-myristate 13-acetate (PMAǹ TPA)ϷIonomycin calcium salt (Ion)!ٰᇨวJurkat TಒझࢲϯǶҗ‫ܭ‬Ь ाࢂᢀჸӄߏ‫ڀ‬фૈ‫ޑ܄‬antizymeჹ‫ܭ‬Tಒझࢲϯ‫ޑ‬ቹៜǴӢԜ२Ӄ ᢀჸᆶTಒझࢲϯ࣬ᜢ‫ޑ‬ಒझᐟન‫୷ޑ‬Ӣ߄౜Ǵхࡴinterleukin-2Ǵ IFN-J Ǵ interleukin-4 Ǵ interleukin-5 Ǵ interleukin-12 p35 Ϸ p40 ‫ک‬. 14.

(31) interleukin-10฻ಒझᐟનǶӢࣁantizyme‫ޑ‬ъ૰යߚதอኩǴ۳۳ӧ อਔ໔ϣ཮फ़ှǴЪ‫ځ‬ೈқ፦߄౜և౜ࣁຼය‫ޑ܄‬ᡂϯǴ‫܌‬аᙖҗ Dox‫܌‬ᇨว‫ޑ‬antizymeѸ໪ӧ࿶җtime-dependent experimentٰᢀჸ‫ځ‬ ಒझᐟન୷Ӣ߄౜‫ޑ‬ᡂϯǶԜѦǴӆճҔRNAi‫ٰೌמޑ‬υᘋantizyme ‫߄ޑ‬౜Ǵ຾ԶᢀჸಒझᐟનࢂցӢԜΨ‫ډڙ‬ቹៜǶ‫׆‬ఈૈவ೭٤ᡂϯ ྽ύว౜٠ᑔᒧࢌ൳ᅿख़ा‫ޑ‬ಒझᐟનǴ‫୷ځ‬Ӣ߄౜ࢂ‫ډڙ‬antizyme ‫܌‬ፓ௓ǴԶ຾΋‫؁‬຾Չࣴ‫ز‬ϩ‫݋‬Ƕ ! ಃΟ೽ϩǺ! !!!!ଞჹ੝‫ޑۓ‬ಒझᐟનǴճҔኧᅿࣴ‫ز‬Б‫ٰݤ‬᛾ܴǴ‫܌‬൨ளᆶ T ಒझࢲϯ࣬ᜢ‫ޑ‬ಒझᐟનᆶӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ࢂցዴჴԖ‫ځ‬ ࣬ᜢ‫܄‬Ƕ٠຾΋‫؁‬௖૸ᆶ T ಒझࢲϯ࣬ᜢ‫ૻޑ‬৲໺ሀٰᢀჸϩ‫݋‬ᆶӄ ߏ‫ڀ‬фૈ‫ ޑ܄‬Antizyme ϐ໔‫ޑ‬ҬϕբҔᜢ߯Ǵ٠Ъᕕှ‫࣬ځ‬ᜢᐒᙯǶ! ! ! ! ! ! !. 15.

(32) ಃΒകǵ‫׷‬਑ᆶБ‫!ݤ‬ ಃ΋࿯ǵჴᡍ‫׷‬਑! ൘ǵಒझਲ਼!)Cell line*! !!!!Jurkat T cellǺΓᜪ࡚‫܄‬రЃ҆ಒझқՈੰ T ಒझਲ਼! !!!!Jurkat T (JK)-Tet-On cell (Clontech, Cat. no. 630915)Ǻ! Jurkat T (JK)-Tet-On Vector cell Jurkat T (JK)-Tet-On mAZ (1-228) cell !!!!୻Ꭶ‫ ܭ‬37ʚǴ5ʘCO2ǴRPMI-1640 ಒझ୻ᎦనǴ֖Ԗ 10ʘज! ФՈమ!(fetal bovine serumǹFBS*ǶճҔ G418 (200 Pg/ml) Ϸ Hygromycin (100 Pg/ml) ٰᆢ࡭ᙯ෗୷Ӣಒझਲ਼‫ޑ‬ᛙ‫܄ۓ‬Ƕ ມǵ‫ל‬ᡏ (Antibody) ୖྣ߄΋ ୖǵЇη!)Primer*! !!!!ୖྣ߄Β စǵ፦ᡏ DNA (Plasmid DNA) Ornithine Decarboxylase Antizyme-1 shRNA (pLKO.1-shOAZ1) Promoter of human interleukin-2 (pIL2-Luc) TK-Renilla lucferase plasmid DNA (pRL-TK) Ҵǵεဉఎ๵๵ਲ਼ (Escherichia coli) DH5DǺ୷Ӣࠠࣁ F’phi80dlacZ delta (lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 lambda-thi-1 gyrA96 16.

(33) rel-A1/F’ proAB+ lacIqZdeltaM15 Tn10 (tetr)ǴҔ‫ܭ‬፦ᡏᙯ‫!!׎‬ !!!!(transformation)ǵ፦ᡏᇙഢǶ ഌǵϯᏢ၂Ꮚᛰࠔ! 1. ΠӈౢࠔᖼԾऍ୯ Amresco ϦљǺ Tris (Cat. no. 0826-1kG)ǴAgarose (Cat. no. 0710-250G)Ǵ! TEMED (Cat. no. 0761-25ML). 2. ΠӈౢࠔᖼԾऍ୯ J.T.Baker ϦљǺ Urea (Cat. no. 4204-01)ǴGlycerol (Cat. no. 2136-01)Ǵ! Glycine (Cat. no. 4059-02)Ǵ2-Propanol (Cat. no. 9084-03)Ǵ! !!!!!!!!Ammonium persulfate (APS) (Cat. no. 0762-01)Ǵ Sodium chloride (NaCl) (Cat. no. 3624-05)ǴHEPES (Cat. no. 4018-04)Ǵ Ammonium bicarbonate (NH4HCO3) (Cat. no. 3003-01)Ǵ Dimethyl Sulfoxide (DMSO) (Cat. no. 9224-03)Ǵ Potassium Phosphate monobasic (KH2PO4) (Cat. no. 3246-01)Ǵ Ethanol (Cat. no. 8006-05)ǴChloroform (Cat. no. 9180-03)!. 3. ΠӈౢࠔᖼԾऍ୯ SIGMA ϦљǺ 2-Mercaptoethanol (2-ME) (Cat. no. M3148-100ML)Ǵ! Ampicillin sodium salt (Cat. no. A9518-25G)Ǵ Bovine serum albumin (BSA) (Cat. no. A2153-50G )Ǵ! !!!!!!!!Phosphatase Inhibitor Cocktail (Cat. no. P2850)Ǵ Protease Inhibitor Cocktail (Cat. no. P8340)Ǵ Doxycycline hyclate (Dox) (Cat. no. D9891)Ǵ Ionomycin calcium salt (Ion) (Cat. no. I0634)Ǵ Phorbol 12-myristate 13-acetate (PM˔ǹTPA) (Cat. no. P8139)Ǵ 17.

(34) Dimethyl Sulphoxide (DMSO) HYBRI MAX (Cat. no. D2650)Ǵ Propidium iodide (PI) (Cat. no. P4170). 4. ΠӈౢࠔᖼԾऍ୯ BIO-RAD ϦљǺ 30% Acrylamide/Bis (37.5:1) (Cat. no. 161-0158)Ǵ! Bradford protein assay dye (Cat. no. 500-0006)!. 5. ΠӈౢࠔᖼԾВҁ Wako પᛰπ཰ਲ਼Ԅ཮‫ޗ‬Ǻ Sodium dodecyl sulfate (SDS) (Cat. no. 196-08675)!Ǵ! Tween-20 (Cat. no. 167-1151)!. 6. ΠӈౢࠔᖼԾऍ୯ Mallinckrodt chemicals ϦљǺ Anhydrous Methyl Alcohol (Cat. no. 3041-68). 7. ΠӈౢࠔᖼԾྷγ Roche ϦљǺ Protease inhibitor cocktail tablet (Cat. no. 11836.153.001). 8. ΠӈౢࠔᖼԾՋ੤У Conda S.A.ϦљǺ Triptona (TryptonfǹTriptone) (Cat. no. 1612.00)Ǵ Extracto de levadura (Yeast extract) (Cat. no. 1702.00). 9. ΠӈౢࠔᖼԾВҁ Dojindo ϦљǺ Cell Counting Kit-8 (CCK-8) (Cat. no. CK04). 10. ΠӈౢࠔᖼԾ GIBCO ϦљǺ Geneticin (G418) (Cat. no. 11811-031)Ǵ Agar Bacteriological (Cat. no. 20001-020)Ǵ 0.25% (1X) Trypsin-EDTA (Cat. no. 25200-072)Ǵ Trypan blue (0.4%) (Cat. no. 15250-016)ǴDPBS (Cat. no. 21600-010). 18.

(35) 11. ΠӈౢࠔᖼԾ Hyclone ϦљǺ STANDARD Fetal bovine serum (FBS) (Cat. no. SH30088.03)Ǵ! RPMI-1640 Medium (1X) (Cat. no. SH30027.02). 12. ΠӈౢࠔᖼԾቺ୯ Riedel-de Haën ϦљǺ Magnesium chloride (MgCl2) (Cat. no. 31413)Ǵ Trifluoroacetic acid (TFA) (Cat. no. 61030)!Ǵ! !!!!!!! Potassium phosphate dibasic puriss (Cat. no. 63310). 13. ΠӈౢࠔᖼԾቺ୯ MERCK ϦљǺ Potassium chloride (KCl) (Cat. no. 1.04936.1000). 14. ΠӈౢࠔᖼԾྷγ Fluka ϦљǺ NP-40 (Cat. no. 74385). 15. ΠӈౢࠔᖼԾऍ୯ BIOTECX ϦљǺ Ultraspec Water (RNase Free) (Cat. no. BL-5611). 16. ΠӈౢࠔᖼԾ CalbiochemǺ Hygromycin B, Streptomyces sp (Cat. no. 400050). 17. ΠӈౢࠔᖼԾऍ୯ Invitrogen ϦљǺ TRIZOL reagent (Cat. no. 15596-026)Ǵ SYBR GreenER qPCR SuperMix (Cat. no. 11760-SAM) PureLink™ HiPure Plasmid Filter Purification Kits (Cat. no. K2100-04). 18. ΠӈౢࠔᖼԾ‫ݤ‬୯ Fermentas ϦљǺ PageRulerTM Prestained Protein Ladder Plus (Cat. no. SM1811). 19. ΠӈౢࠔᖼԾ௖઩ғ‫מ‬ϦљǺ Arrest-In Transfection reagent (Cat. no. ATR1741) 19.

(36) 20. ΠӈౢࠔᖼԾ PromegaǺ Dual-Luciferase Reporter Assay System (Cat. no. E1910). 21. ΠӈౢࠔᖼԾ GMbiolab Co., LtdǺ Oligo dT (18) (Cat. no. GM005)Ǵ2.5mM dNTP (Cat. no. GM002)Ǵ! !!!!!!!!RNase Inhibitor (Cat. no. GM2310)ǴMMLV RTase (Cat. no. GM45)Ǵ! PCR Master Mix (Cat. no. RP02-200). ࢠǵЬाᏔ‫׷‬ሺᏔ! 1. Β਼ϯᅹ୻ᎦጃǺSANYO Electric Biomedical Co, Ltd 2. ࡡྕН੎ኲǺFirStek Scientific, USA 3. ॹҥԄᡉ༾᜔ǺOLYMPUS CK2 4. ႜ৔௟ᅲӅ೫ขϩӀӀ᛼ᡉ༾᜔ǺLeica TCS SP2 5. Ծ୏ಈηीኧᏔ (Coulter Counter*ǺBeckman Coulter, Inc 6. հএᚆЈᐒǺBeckman Coulter, Inc 7. SDS-PAGE ‫ٿ‬ТԄႝ‫ݚ‬೛ഢǺBio-Rad Laboratories 8. ႝ‫ٮݚ‬ᔈᏔǺBio-Rad Laboratories 9. Odyssey Infrared Imaging System, LI-COR Biosciences 10. ࢬԄಒझϩ‫݋‬ሺǺFACSCanto, BD Biosciences! !!!!11. ᆫӝ䁙ೱᙹϸᔈᏔ (RT-PCR)ǺThermoǹApplied Biosystems 12. ਡለ‫ׇ‬ӈ‫ۓ‬ໆୀෳ‫س‬಍ (qPCR)ǺApplied Biosystems 7900HT 13. DNA ႝ‫ݚ‬೛ഢǺMupaid! !!!!14. AlphaImagerǺAlpha Innotech. !. 20.

(37) ಃΒ࿯ǵჴᡍБ‫!ݤ‬ ൘ǵಒझਲ਼୻Ꭶ!)Cell Culture*!(ߕᒵΐ)!! )ୖྣȨଯሎ෕௲௤ჴᡍ࠻ಒझ୻Ꭶ኱ྗᏹբำ‫ׇ‬ȩ Ǵጓኗ‫ޣ‬Ǻ‫ە׵‬ᑪ* ΃ǵှএಒझਲ਼ᏹբำ‫ׇ‬ Βǵᝩж୻Ꭶಒझਲ਼ᏹբำ‫ׇ‬ Οǵհএಒझਲ਼ᏹբำ‫ׇ‬ ມǵಒझਲ਼ғߏԔጕ )Cell Growth Curve* 1. ஒಒझԾಒझ୻ᎦዬѺΠǴ࿼‫ ܭ‬15 ml ᚆЈᆅύǴྗഢीᆉಒ झኧҞǶ 2. ‫ ܭ‬25T Flask ύϩձᅿΕ 1×104Ǵ3×104Ǵ1×105 cells/ml Οᅿ ಒझᐚࡋǴΟख़ፄǶ(20 ml/flask) 3. ஒ 25T Flask ࿼‫୻ྕࡡܭ‬Ꭶጃύ୻ᎦǶ 4. ಃΒϺ (day 1)ǴӃҔᡉ༾᜔ᢀჸಒझғߏ‫ݩރ‬Ǵϩձ‫ڗ‬рό ӕᐚࡋಒझӚ 0.4 ml ֡Ϭ࿼‫ ܭ‬9 ঁ༾ໆᚆЈᆅ (eppendorf)Ǵ ճҔ Trypan blue ໒‫ۈ‬ीኧಒझኧҞ 5. ಃΟϺ!(day 2)࣬ӕਔ໔ᗺǴख़ፄ day 1 ‫؁ޑ‬ᡯԿ day 8Ƕ 6. ᏾౛ day 1~day 8 ‫ޑ‬ኧᏵǴᛤᇙಒझਲ਼ғߏԔጕǶ ୖǵࢬԄಒझሺಒझ঒Ϋϩ‫)!݋‬Apoptosis analysis by FACS* 1. ‫ܭ‬6ϾዬύᅿΕ1 × 106ᗭಒझኧǴ֖1% FBS‫ޑ‬RPMI-1640. 21.

(38) 2. уΕ Doxycycline 50 PM ຾Չ time-course experimentǴਔ໔ϩ ձࣁ 0ǵ12ǵ24ǵ36ǵ48 λਔǶ! 3. ‫؂‬႖΋ঁਔ໔ᗺԏ໣ಒझ pelletǴа 1X DPBS ࢱᅗ 1 ԛǶ 4. ಒझа0.2 ml DPBS ᇸ጗Ӧషӝ֡ϬಒझǴӧу΢0.8 mlӇ‫ޑ‬ 99%Όᎇషӝ֡ϬǴ‫ډܫ‬-20°C຾Չ‫ۓڰ‬Ƕ !!!!5. ႖‫ڹ‬ᚆЈ‫ڗ‬ಒझуΕ0.5 mlեᅖ೸ᓸྋన (1% Triton X-100 in DPBS and 0.5 mg/ml RNase A)ǴၸfilterǴӆуΕPIྋన (4 ȝg/ml) షӝ֡Ϭ‫ܫ‬ӧӇ΢ࢉՅ30ϩដǶ 6. ճҔFACSCantoࢬԄಒझሺ (BD Biosciences) ୀෳᑻӀ֖ໆ а຾Չϩ‫݋‬ǶᙖҗModLT software೬ᡏஒၗ਑ೀ౛ᆶϩ‫݋‬Ǵ ᢀჸapoptosisಒझຼයG1߻ය (sub-G1)Ƕ စǵՋБᏀᗺ‫ݤ‬ȐWestern blottingȑ ΃ǵኬࠔᇙഢ!)Sample preparation*! 1. ஒಒझԏԋ pelletǶ 2. уΕऊᆶ pellet ᡏᑈ฻ໆ‫ ޑ‬HEPES/NP-40 cell lysis buffer (٬Ҕ߻уΕ Protease Inhibitor Cocktail, Na3VO4, Phosphatase Inhibitor Cocktail) Ƕ 3. மਗ਼᎜ᕏ٠࿼‫ܭ‬Ӈ΢ኧϩដǴຬॣ‫ݢ‬᎜ᕏ 1 ϩដǶ 4. ᚆЈ 13,000 rpm (HERMLEǹZ216MK)Ǵ4 ºCǴ15 ϩដǶ. 22.

(39) 5. ‫ڗ‬΢మనǴBradford ೈқ፦‫ۓ‬ໆǴStandard curve r2Њ0.99 Ƕ 6. ‫ڗ‬ς‫ۓ‬ໆӳ‫ޑ‬ೈқ፦ኬࠔ, у፾ໆ HEPES/NP-40 lysis buffer Ϸ 2X sample bufferǴஒӚኬࠔ‫ޑ‬ೈқ፦֖ໆϷᡏᑈፓࣁ΋ठǶ 7. Н੎‫ݤ‬ஒ Sample ‫ݦ‬ฆ 3~5 ϩដǴࡑኬࠔԾฅհࠅࡕӆ࿼‫ܭ‬ -20 ºC ᓯᙒǶ Βǵᇙጤ )ёኩӸ‫ ܭ‬4ºC* Ꮙ໣ጤǺ5 % acrylamide gel ϩᚆጤǺ10-12 % acrylamide gel Οǵາጤ ΢ጤǺ70VǴ30 ϩដ ΠጤǺ110VǴ90 ϩដ Ѥǵೈқ፦ᙯᅄ!)transfer* 75VǴ3.5 λਔ ϖǵImmunoblotting 1. ஒᙯᅄֹԋ‫ ޑ‬PVDF membrane ‫ݰ‬Ε 1% BSA (in TBST)ύǴ ࠻ྕΠའਗ 1 ঁъλਔ (‫ ܈‬4 ºC overnight)Ƕ! 2. ‫ڗ‬р PVDF membraneǴ‫ݰ‬Ε TBSTǴའਗ 10 ϩដǴ‫ٿ‬ԛǶ 3. ‫ڗ‬р PVDF membraneǴӆ‫ݰ‬Ε֖Ԗ΋ભ‫ל‬ᡏ‫ ޑ‬TBST (‫ ܈‬1% BSA in TBST) ύǴ 4 ºC overnightǶ. 23.

(40) 4. ‫ڗ‬р PVDF membraneǴ‫ݰ‬Ε TBSTǴའਗ 10 ϩដǴѤԛǶ 5. ‫ڗ‬р PVDF membraneǴӆ‫ݰ‬Ε֖ԖΒભ‫ל‬ᡏ‫ ޑ‬TBST (‫ ܈‬1% BSA in TBST)ύǴ࠻ྕΠའਗ 1 λਔǶ 6. ‫ڗ‬р PVDF membraneǴ‫ݰ‬Ε TBSTǴའਗ 10 ϩដǴѤԛǶ ®. 7. Ҕ Odyssey Infrared Imaging System ௟ඔ‫ۭࢂ܈‬ТᓸТǴϩ‫݋‬Ƕ 8. നࡕճҔ Image-Pro PLUS ೬ᡏϩ‫݋‬ໆϯ่݀Ƕ Ҵǵಒझ RNA ‫ڗܜޑ‬ȐRNA Isolationȑ ΃ǵ֡፦ϯ!)Homogenization*! !! 1. ԏ໣ಒझ pellet а 1X DPBS ࢱᅗ 1 ԛǶ 2. уΕ 1 ml TRIZOL reagent ‫ܭ‬ಒझ pellet ύǴख़ፄ pippettingΔʳ ʳ ʳ ʳ ʳ ʳ ʳ ʳ ຾Չ cell lysisǴӆஒ Cell lysate ౽Կ eppendorf ύǴᓉ࿼‫࠻ܭ‬ ྕύ 5 minǶ Βǵ࣬ϩᚆ!)Phase separation*! !! 1. уΕ 200 Pl ‫ ޑ‬chloroformǴ᎜ᕏ 15 ࣾǴᓉ࿼࠻ྕ 2~3 minǶ! 2. ᚆЈ12,000×gΔ30ϩដǴ4°CǶ 3. ҔP200 pipetmanλЈஒНቫ!(aqueous phase) (ന΢ቫ) ౽‫!ډ‬ ཥ‫ޑ‬eppendorfύǶ! ΟǵRNA ؈ᐘ )RNA precipitation*! !!!!1. ஒ 500 Pl ‫ ޑ‬IPA уΕНቫǴషӝǴᓉ࿼ 30 ϩដǴ4°CǶ. 24.

(41) 2. ᚆЈ12,000×gΔ30ϩដǴ4°CǶ 3. ҔP200 pippetment౽ନ΢మనǶ! !!!Ѥǵమࢱ!)Wash*! !!!!1. ‫ݮ‬eppendorfᆅᏛᄌᄌуΕ1 ml -20ʚႣհ‫ޑ‬75%Όᎇ!!!!!!! (in RNase-free H2O)ǴᇸᇸའਗeppendorfǶ! 2. ᚆЈ7,500×gΔ5ϩដǴ4°CǶ! !!!!3. ҔP200 pippetment౽ନ΢మనǶ! ϖǵRNAӣྋ!)Redissolving the RNA* !!!!1. ஒpelletϷᆅᏛ΢ූ੮‫ޑ‬నᡏ॥ଳ (όाӄଳǴόӳӣྋ)Ƕ! !!!!2. Ҕ30-50 Pl of RNase-free waterஒpelletӣྋǶ! ഌǵϸᙯᒵ䁙.ᆫӝ䁙ೱᙹϸᔈȐRT-PCRȑ! 1. ஒӣྋӳ‫ ޑ‬RNA ‫ۓ‬ໆǴ‫ۓڗ‬ໆӳ‫ ޑ‬RNA у‫ډ‬༾ໆᚆЈᆅύǴ ӆуΕ RNase-free waterΔᕴᡏᑈࣁ 26 PlǶ !!!!2. ຾Չ DenatureǴ65ʚ/10 min 3. ٩‫ׇ‬уΕ RT solutionǴхࡴ 10X MMLV RT Buffer 5 PlǴ0.1 M DTT 5 PlΔ2.5 mM dNTP 10 PlǴOligo dT (18) (1 Pg/Pl) 0.5 PlǴ RNase Inhibitor 1 PlǴMMLV RT (10 U/Pl) 2.5 PlǴᕴᡏᑈനࡕ ࣁ 50 PlǶ! 4. ຾Չ Reverse TranscriptionǴ37ʚ/1 hrɡ65ʚ/10 minǴஒ RNA. 25.

(42) ϸᙯᒵࣁ cDNAǶ! 5. ӧ༾ໆᚆЈᆅύǴуΕ cDNA 1 Pl բࣁኳ‫( ހ‬template)Ǵ٩‫ׇ‬ уΕ Nuclease-free H2O (Q- H2O)!37 PlǴ10 PMʳPrimer (F* 1 PlǴ10 PM Primer (R)!1 Pl Ϸ PCR Master Mix Kit 10 PlǶ 6. షӝ֡Ϭࡕ࿼‫ܭ‬ᆫӝሇનೱᙹϸᔈᏔ (thermoǹApplied Biosystems) ‫ޑ‬ϸᔈኲύǶ 7. Ӄа 94ʚբҔ 5 ϩដǴӆа‫ຼঁ؂‬යǴ94ʚբҔ 1 ϩដ‫ ޑ‬DNA ᡂ‫܄‬ϸᔈǹբҔ΋ϩដ‫ޑ‬᜘ӝϸᔈǹ72ʚբҔ΋ϩដ‫ ޑ‬DNA ӝԋϸᔈǶ຾ՉኧঁϸᔈຼයǴϐࡕа 72ʚբҔΜϩដஒ DNA ТࢤံሸǶനࡕ೛‫ ۓ‬4ʚߥӸǶ 8. ஒ PCR DNA ౢ‫ނ‬າ 2%ᛏ✊Ꮙጤ (2% agarose gel) ຾Չႝ‫ݚ‬ ϩ‫݋‬Ƕዴ‫ ۓ‬PCR ౢ‫ނ‬Тࢤελ҅ዴǶ ࢠǵջਔ‫ۓ‬ໆ.ᆫӝ䁙ೱᙹϸᔈȐReal-Time PCRȑ 1. ӧ༾ໆᚆЈᆅύǴуΕ cDNA 4 Pl բࣁኳ‫( ހ‬template)Ǵ٩‫ׇ‬ уΕ Nuclease-free H2O (DEPC- H2O) 5.2 PlǴ10 PMʳPrimer (F) 0.4 PlǴ10 PM Primer (R) 0.4 PlǴΟख़ፄǶ! 2. షӝ֡ϬࡕуΕԿ Real-Time PCR Machine ஑Ҕ 96 ϾዬύǴ ӆӚԾуΕ SYBR Green 10 PlǴᇂ΢࠾ኳǴSpin downǴ !!!!!!!࿼‫ܭ‬ᆫӝሇનೱᙹϸᔈᏔ (Applied Biosystems 7900HT) ‫ޑ‬. 26.

(43) ϸᔈኲύǶ 3. Ӄа 50ʚբҔ 2 ϩដǴ95ʚբҔ 10 ϩដǴӆа‫ຼঁ؂‬යǴ 95ʚբҔ 15 ࣾ‫ ޑ‬DNA ᡂ‫܄‬ϸᔈǹ52ʚբҔ 1 ϩដ‫ޑ‬᜘ӝϸ ᔈǴᕴӅ຾Չ 40 cycleǴள‫ ډ‬Ct ॶǶ 4. ᙖҗABI 7900HT SDS 2.2ϩ‫݋‬೬ᡏஒၗ਑ೀ౛ᆶϩ‫݋‬Ƕ ਎ǵεဉఎ๵ϐᙯ‫)!׎‬Transformation*! 1. ஒ፦ᡏуΕ፾ᔈ‫܄‬ಒझύǴషӝࡕԿ‫ܭ‬Ӈ΢ΟΜϩដǶ 2. ӧ࿶ 42ʚϸᔈ 90 ࣾࡕǴِೲԿ‫ܭ‬Ӈ΢ 1~2 ϩដǴϐࡕуΕ 100 Pl ό֖‫ל‬ғન‫ ޑ‬LB ୻ᎦనǶ 3. ӧ 37ʚ୻Ꭶጃ୻Ꭶ΋λਔࡕǴ֡Ϭ༡ӧ֖‫ל‬ғન‫ ޑ‬LB ୻Ꭶ ୷΢Ƕ ‫ا‬ǵ፦ᡏ DNA ᇙഢ!)Preparation of!Plasmid DNA* 1. ֖Ԗ፦ᡏ‫ൂޑ‬΋๵ပǴа 100 ml TB ӧ 37°C ୻Ꭶጃ୻Ꭶ 2. ႖‫ڗڹ‬рࡕаനεೲᚆЈ 6,500ØgǴ10 ϩដࡕǴॹ௞΢మనǴ Ҕ PureLinkTM HiPure Plasmid Filter Purification kits ၂Ꮚಔ (Invitrogen) ٰપϯ፦ᡏǶ 3. २ӃѳᑽᆅࢊǺуΕ15 ml EQ1 buffer (Equilibration Buffer) 4. ຾ՉಒझLysateǺуΕ10 ml R3 buffer (Resuspension Buffer) ӆ уΕ10 ml L7 buffer (Lysis Buffer)Ǵషӝ֡ϬǴϮ᎜ᕏǶ࠻ྕ. 27.

(44) ୻Ꭶ5ϩដࡕǴӆуΕ10 ml N5 buffer (Precipitation Buffer)Ǵ షӝ֡ϬǴϮ᎜ᕏǶ 5. уΕ‫ډ‬ᆅࢊύ٠మࢱǺcell lysateᇙഢӳϐࡕǴуΕ‫ډ‬ᆅࢊύǴ ᡣlysateၸfilterࢬΕᆅࢊύǴϐࡕуΕ20 ml W8 buffer (Wash Buffer)!మࢱᆅࢊǶ! !!!!6. Eluting፦ᡏDNAǺඤཥ‫ޑ‬15 mlᚆЈᆅԿ‫ܭ‬ᆅࢊΠБǴуΕ5 ml E4 buffer (Elution Buffer)!ஒ፦ᡏDNA eluteрٰǶ !!!!7. DNA؈ᐘǺуΕ3.5 ml IsopropanolஒDNA؈ᐘǴϐࡕᚆЈѐ௞ ΢మనǴӆуΕ3 ml 70% EthanolమࢱpelletǶ 8. మࢱ pellet ϐࡕѐ௞΢మనǴ॥ଳǴനࡕа 100 Pl ‫ྐޑ‬๵ၸ‫ޑ‬ ddJH2O ӣྋ፦ᡏ DNAǶ ࡵǵज़‫ڋ‬ሇનНှȐRestrict Enzyme digestionȑ 1. ஒ plasmid DNA (1 Pg/Pl) ‫ ڗ‬1 PlǴуΕ 100X BSA 1 PlǴBuffer1 2PlǴϪՏ‫ޑ‬ज़‫ڋ‬ሇન (KpnI) 1 Pl ‫ ک‬ddH2O 15 PlǴᕴᡏᑈࣁ 20 PlǶ 2. а 37ʚբҔ 2 λਔǴஒౢ‫ނ‬າ 1%ᛏ✊ᏉጤȐ1% agarose gelȑ ຾Չႝ‫ݚ‬ϩ‫݋‬Ƕዴ‫ۓ‬Нှౢ‫ނ‬Тࢤελ҅ዴǶ ࡵ൘ǵDNA ᙯࢉȐDNA Transfectionȑ 1. ᙯࢉჴᡍ྽Ϻа serum-free ‫ ޑ‬medium ᅿΕ 1×106/ml ಒझኧ‫!ܭ‬. 28.

(45) 6cm dishǴᓉ࿼ incubator ऊ 2 λਔǴࡑಒझᛙ‫ۓ‬Ƕ! 2. ಒझ‫ޑ‬ᙯࢉࢂճҔ Arrest In (AI) Transfection Kit ၂ᏊǶஒ plasmid DNA 4 Pg/6cm dish ྋӧ serum-free medium ύǴᕴᡏ ᑈࣁ 50 PlǴvortex!ኧࣾǹӆஒ Arrest-In reagent ྋӧ serum-free medium ύǴᕴᡏᑈΨࣁ 50 PlǴvortex!ኧࣾǴஒа΢‫ޣٿ‬ష ӝ֡Ϭᓉ࿼‫ྕ࠻ܭ‬ΒΜԿΟΜϩដǴ٬ள AI/DNA complexes ‫׎‬ԋǶ٬Ҕ߻ᄇۭషӝ֡ϬǶ 3. ஒ AI/DNA complexes уΕ֖ serum-free medium ಒझύǴ‫ܭ‬ 37°CǴ5% CO2 Π୻Ꭶ 6 λਔǴӆуΕ฻ໆ 20% FBS medium ‫ܭ‬চ serum-free medium ύᝩុ୻ᎦǶ 4. ᙯࢉࡕ࿶җ 24 λਔ୻ᎦǴ໒‫ុࡕۈ‬ჴᡍǶ! ࡵມǵխࣝᑻӀࢉՅȐImmunofluorescenceȑ 1. ‫ ܭ‬12 ϾዬύᅿΕ 1 × 106/2 ml ಒझኧǴ֖ 1% FBS ‫ޑ‬ RPMI-1640ǴуΕᛰ‫ނ‬ೀ౛Ƕ 2. ԏ໣ಒझ pellet а 1X DPBS ࢱᅗǴᚆЈǴѐନ΢మన٠Ъന ࡕа 1 ml DPBS ӣྋǴషӝ֡ϬǶ 3. уΕ 100-200 Pl ಒझᝌੌన‫ܫ‬Ε channel ္ǴճҔ Cytospin ஒಒझ coating Կ coating-slide ΢Ƕచҹࣁ 800rpmǴ2 minǶ! !! 4. Ҕ 4% paraformaldehyde ‫ۓڰ‬ಒझ 20 ϩដǴϐࡕ֎ѐ 4%. 29.

(46) paraformaldehyde ٠а DPBS మࢱ 3 ԛǶ! 5. ஒ coating-slide ࿼‫ܭܫ‬Ӈ΢ǴуΕ 0.1% TritonX-100 բҔ 10 ϩដǴӆҔ DPBS మࢱ 3 ԛǶ! 6. уΕ 1% BSA/DPBSǵ࠻ྕǵ1 λਔǴ຾Չ blockingǶ! 7. ౽ѐ 1% BSA/DPBSǴуΕ΋ભ‫ל‬ᡏ (1Ǻ200Ǵ1%BSA/DPBS ีញ)Ǵ࿼‫ ܭ‬4°C ‫ܫ‬࿼႖‫ڹ‬Ƕ! 8. ಃΒϺǴӣԏ΋ભ‫ל‬ᡏǴҔ DPBS మࢱ 3 ԛǶӆуΕΒભ‫ל‬ ᡏ (1Ǻ200ǴDPBS ีញ)Ǵ࠻ྕǵ1 λਔǴख़ᗺाᗉӀǶ! 9. ౽ѐΒભ‫ל‬ᡏǴҔ DPBS మࢱ 3 ԛǶуΕ 1 ml DAPI (1Ǻ50000Ǵ DPBS ีញ) ᗉӀ࿼‫ྕ࠻ܭ‬ϸᔈ 10 ϩដǴҔ DPBS మࢱ 3 ԛǶ! 10. ᅀ‫ڗ‬΋ᅀ࠾Тన!(Mounting medium) ‫ܭ‬ၩ࣒ТύЈǴλЈ֨ ‫ڗ‬ᇂ࣒ᙟᇂ‫ܭ‬ၩ࣒Т΢Ƕ‫ܫ‬࿼࠻ྕ٬‫ځ‬Ꮙ‫ڰ‬Ƕനࡕ٬ҔӅ೫ ขႜ৔ᡉ༾᜔ᢀჸ٠ᘏ‫ڗ‬ቹႽǶ ࡵୖǵ಍ीϩ‫݋‬ȐStatistic Analysisȑ ჴᡍၗ਑ճҔSPSS 10.5‫ހ‬຾Չ಍ीϩ‫݋‬Ǵ྽಍ीኧᏵpॶλ‫!ܭ‬ !!!!!0.05*‫܈‬0.01**ਔǴж߄‫ڀ‬Ԗ಍ीᏢ΢‫ޑ‬ᡉ๱‫܄‬ཀကǶ! ! ! !. 30.

(47) ಃΟകǵ่݀! ಃ΋࿯ǵಒझਲ਼୻Ꭶ! ٩ྣȨଯሎ෕௲௤ჴᡍ࠻ಒझ୻Ꭶ኱ྗᏹբำ‫ׇ‬ȩ )ጓኗ‫ޣ‬:‫׵‬ ‫ە‬ᑪ*!຾Չಒझ୻ᎦǶJurkat T cell ࣁΓᜪ࡚‫܄‬రЃ҆ಒझқՈੰ U ಒझਲ਼Ǵ‫׎ځ‬ᄊӵკ 2.1 ‫܌‬ҢǶࣁᄇۭඓඝಒझғߏೲ౗Ϸ‫׎‬ᄊǴ຾ Չಒझਲ਼ғߏԔጕ‫ޑ‬ᛤᇙǴӵკ 2.2 ‫܌‬ҢǴхࡴΑ JK Tet-On Vector ‫ک‬ JK Tet-On mAZ (1-228) ‫ٿ‬ਲ਼ಒझǶҗғߏԔጕё௢‫ޕ‬೭‫ٿ‬ਲ਼ಒझਲ਼ ғߏԔጕ࣬՟Ǵ‫ځ‬ಒझਲ਼७ቚਔ໔ (doubling time) ऊࣁ 18-24 λਔǶ. ಃΒ࿯ǵAntizyme ‫ ܭ‬JK Tet-On ಒझਲ਼ύ‫߄ޑ‬౜ ൘ǵAntizyme ၸࡋ߄౜‫܌‬Їว‫ޑ‬ಒझ঒Ϋ ࣁΑዴᇡӄߏ‫ڀ‬фૈ‫ޑ܄‬antizymeӧJK Tet-Onಒझ‫س‬಍ύࢂԖ చҹ‫߄ޑ‬౜рٰǴಒझ๏ϒଯᏊໆ50 ȝM DoxǴϩձӧόӕਔ໔ᗺ‫ڈ‬ ᐟᢀჸಒझ঒Ϋ‫ޑ‬௃‫ݩ‬Ϸ‫ځ‬Ӹࢲ౗Ǵӵკ3.1‫܌‬ҢǶ่݀ᡉҢӧуΕ ଯᐚࡋDoxᇨวantizyme٬ளJK Tet-On mAZ (1-228) ಒझਲ਼ᒿ๱ਔ ໔‫ޑ‬ቚу‫ځ‬ಒझ‫׎‬ᄊ࿗຋‫ޑ‬ಒझኧҞቚуЪಒझӸࢲ౗ܴᡉե‫ܭ‬JK Tet-On Vectorಒझਲ਼ǶԜѦǴӧDoxӸӧᇨᏤantizyme߄౜ࡕᒿ๱ਔ ໔‫ޑ‬ቚуǴаPIࢉՅҔࢬԄಒझሺୀෳಒझຼය‫ޑ‬sub-G1යǶҗკ3.2 ่݀ᡉҢӧуΕଯᐚࡋDoxϐࡕǴᇨวӄߏ‫ڀ‬фૈ‫܄‬antizyme‫߄ޑ‬౜. 31.

(48) ٬ளJK Tet-On mAZ (1-228) ಒझਲ਼‫ޑ‬sub-G1К‫ࢂٯ‬ᒿ๱ਔ໔Զቚу Ъܴᡉଯ‫ܭ‬JK Tet-On Vectorಒझਲ਼Ƕ. ມǵаՋБᏀᗺ‫ݤ‬ዴᇡ antizyme ‫ ܭ‬JK Tet-On ಒझਲ਼ύ‫߄ޑ‬౜ !!!!җკ 4 ่݀ᡉҢǴӧ Dox ӸӧᇨวΠǴJK Tet-On mAZ (1-228) ಒ झਲ਼ύᒿ๱ᛰ‫ނ‬ᐚࡋ‫ޑ‬ቚуǴ‫ ځ‬antizyme ೈқ፦߄౜ໆΨԖቚу‫ޑ‬ ᖿ༈ǴЪ‫ ک‬JK Tet-On Vector ಒझਲ਼ٰ଺КၨǴantizyme ೈқ፦߄౜ ໆ೿Ԗᡉ๱‫ޑ܄‬ৡ౦Ƕவ Western Blotting ‫่݀ޑ‬ᡉҢ Tet-On ‫س‬಍ᄬ ᑐ߄ၲΓᜪӄߏ antizyme ୷Ӣ‫ޑ‬ਏ݀ӧԖ Dox ӸӧᇨวΠࢂёՉЪ ᡉ๱‫߄ޑ܄‬౜рٰǶ. ಃΟ࿯ǵAntizymeჹ‫ܭ‬JK Tet-Onಒझਲ਼ࢲϯ‫ޑ‬ቹៜ ൘ǵаϸᙯᒵ䁙.ᆫӝ䁙ೱᙹϸᔈᢀჸ antizyme ჹ‫ ܭ‬JK Tet-On ಒझ ਲ਼ӧόࢲϯ‫ࢲک‬ϯΠಒझᐟન୷Ӣ߄౜‫ޑ‬ቹៜ ࣁΑዴᇡӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ჹ‫ ܭ‬Jurkat T ಒझ‫ࢲޑ‬ϯࢂԖ ቹៜ‫ޑ‬Ǵӧ๏ϒಒझᏊໆ 10 ȝM Dox ᇨᏤ antizyme ߄౜ϐࡕǴᢀჸ όӕਔ໔ᗺ JK Tet-On ಒझਲ਼ӧόࢲϯ‫ࢲک‬ϯ‫ރ‬ᄊΠǴ‫࣬ځ‬ᜢಒझᐟ ન‫߄ޑ‬౜ࣁՖǶ२ӃǴӧჴᡍύճҔ PMA/Ion ೭‫ٿ‬ᅿᛰ‫ ୏௴ٰނ‬T ಒझ‫ࢲޑ‬ϯǴӵკ 5 (a) ่݀ᡉҢ PMA/Ion ‫ޑ‬ዴ཮ᇨว JK Tet-On ಒ झਲ਼‫ࢲޑ‬ϯǴ‫ࢲځ‬ϯࡰ኱ IL-2 ୷Ӣ߄౜ࢂᒿ๱ਔ໔‫ޑ‬ቚуԶቚуǶ 32.

(49) ዴ‫ࢲۓ‬ϯచҹϐࡕߡջ໒‫ۈ‬ᢀჸᇨว antizyme ߄౜ࡕǴόӕਔ໔ᗺ JK Tet-On ಒझਲ਼ӧόࢲϯ‫ࢲک‬ϯ‫ރ‬ᄊΠ࣬ᜢಒझᐟન‫߄ޑ‬౜Ƕҗკ 5 (b) ่݀ᡉҢӧ JK Tet-On ಒझਲ਼όࢲϯ‫ރ‬ᄊΠǴࢌ൳ᅿಒझᐟન‫ޑ‬ ߄౜‫ޑ‬ዴԖ‫ׯ܌‬ᡂǴ੝ձࢂᆶխࣝࢲϯ࣬ᜢ‫ޑ‬ಒझᐟનхࡴ IL-2ǵ IL-4ǵIL-5ǵIL-12 (p40) аϷ IFN-JǶ‫ځ‬ύ‫ ډڙ‬antizyme ቹៜԶ୷Ӣ ߄౜ቚу‫ޑ‬хࡴΑ IL-2 ‫ ک‬IL-4ǹ࣬ჹ‫ޑ‬Ǵ‫ ډڙ‬antizyme ቹៜԶ୷Ӣ ߄౜Πफ़‫߾ޑ‬Ԗ IL-5ǵIL-12 (p40) аϷ IFN-JǶԶӧ JK Tet-On ಒझ ਲ਼ࢲϯ‫ރ‬ᄊΠǴPMA/Ion ೀ౛ਔ໔ࣁ 1 λਔǴ่݀ӵკ 5 (c) ᡉҢǴ ‫ ډڙ‬antizyme ቹៜԶ୷Ӣ߄౜ቚу‫ޑ‬ϝࢂ IL-2 ‫ ک‬IL-4ǹ࣬ჹ‫୷ޑ‬Ӣ ߄౜Πफ़Ψࢂ IL-5ǵIL-12 (p40) аϷ IFN-JǶவ RT-PCR ‫؁߃่݀ޑ‬ ᡉҢ antizyme ၸࡋ߄౜ዴჴ཮ቹៜ JK Tet-On ಒझਲ਼ಒझᐟન‫߄ޑ‬ ౜Ǵ੝ձࢂᆶխࣝࢲϯ࣬ᜢ‫ޑ‬ಒझᐟનǴ‫ځ‬ύ IL-2 ‫ ࣁࢂ׳‬T ಒझࢲ ϯ‫ޑ‬΋ঁख़ाࡰ኱Ƕ!. ມǵаջਔ‫ۓ‬ໆ.ᆫӝ䁙ೱᙹϸᔈϩ‫ ݋‬antizyme ჹ‫ ܭ‬IL-2 ୷Ӣ߄౜ ‫ޑ‬ቹៜ வӃ߻ RT-PCR ‫؁߃่݀ޑ‬ᡉҢ antizyme ዴჴ཮ቹៜᆶխࣝࢲϯ ࣬ᜢ‫ޑ‬ಒझᐟન‫߄ޑ‬౜Ǵ‫ځ‬ύ੝ձࢂ IL-2ǴѬࣁ T ಒझࢲϯ‫ޑ‬΋ঁख़ ाࡰ኱Ǵ‫܌‬аҁჴᡍճҔ real-time PCR ٰໆϯ IL-2 ୷Ӣ߄౜‫ޑ‬ৡ౦Ƕ җკ 6 ่݀ᡉҢр྽ antizyme ၸࡋ߄౜ϩձӧ 1 λਔǵ3 λਔ‫ ک‬6 λ 33.

(50) ਔ‫ޑ‬ਔংǴӆа PMA/Ion ࢲϯ 1 λਔǴIL-2 ୷Ӣ߄౜ӧ JK Tet-On mAZ (1-228) ಒझύᡉ๱‫ޑ܄‬ଯ‫ ܭ‬JK Tet-On Vector ಒझǴԜѦΨܴᡉଯ ‫ ܭ‬0 λਔ௓‫ڋ‬ಔǶ. ୖǵа RNAi ‫ೌמ‬υᘋ antizyme ߄౜ჹ‫ ځ‬IL-2 ୷Ӣ߄౜‫ޑ‬ቹៜ ࣁΑӆԛ᛾ჴ antizyme ‫ޑ‬ዴ཮ቹៜ IL-2 ୷Ӣ‫߄ޑ‬౜ǴճҔ஥Ԗ ornithine decarboxylase antizyme1 shRNA (OAZ1 shRNA) ‫ ޑ‬፦ ᡏ DNA ᙖҗᙯࢉ‫ೌמ‬ଌԿಒझϣυᘋ antizyme ‫߄ޑ‬౜຾Զᢀჸ IL-2 ୷ Ӣ‫߄ޑ‬౜Ƕҗკ 7 ่݀ᡉҢрคፕࢂϣғ‫ ޑ܄‬antizyme (JK Tet-On Vector) ‫ࢂ܈‬Ѧྍ‫܄‬ӄߏ‫ ޑ‬antizyme (JK Tet-On mAZ 1-228)Ǵ྽ antizyme ‫୷ځ‬Ӣ߄౜‫ ډڙ‬OAZ1 shRNA ‫ޑ‬ቹៜԶ೏‫ڋ׭‬ਔǴ‫ ځ‬IL-2 ୷Ӣ‫߄ޑ‬౜ΨԖΠफ़‫ޑ‬౜ຝౢғǶӢԜவ shRNA ‫่݀ޑ‬ᡉҢрճҔ ϸ᛾‫ޑ‬БԄΨӑ᛾Α antizyme ዴჴቹៜΑ IL-2 ୷Ӣ‫߄ޑ‬౜Ǵམଛ΢ ॊ RT-PCR Ϸ real-time PCR ‫่݀ޑ‬ёܴᡉள‫ޕ‬ӄߏ‫ڀ‬фૈ‫ޑ܄‬ antizyme ӧ Jurkat T ಒझࢲϯၸำύ‫ޑ‬ዴ‫ת‬ᄽΑख़ा‫فޑ‬ՅǴѬ཮ቹ ៜᆶ T ಒझࢲϯ࣬ᜢ‫ޑ‬ಒझᐟન‫߄ޑ‬౜Ǵ‫ځ‬ύǴ੝ձࢂࢲϯࡰ኱ IL-2 ୷Ӣ‫߄ޑ‬౜‫ࢂ׳‬ᡉ๱Ƕ. ಃѤ࿯ǵAntizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐёૈϩηᐒᙯ ൘ǵаխࣝᑻӀࢉՅᢀჸ antizyme ߦ຾ NF-NB ᙯՏ຾Εಒझਡ 34.

(51) ၸѐЎ᝘ࡰрճҔ PMA/Ion ٰᇨᏤ T ಒझ‫ࢲޑ‬ϯЬाૻ৲໺ሀ ၡ৩ࣁ೸ၸ PKCǵIKKǵINB Կ NF-NB ࢲϯᙯՏ຾ΕಒझਡϣǶҁჴ ᡍ߾ճҔխࣝᑻӀࢉՅᙖҗӅ೫ขႜ৔ᡉ༾᜔ᢀჸ antizyme ቹៜ NF-NB Εਡ‫ݩރޑ‬Ƕҗკ 8 (a) ่݀ᡉҢрӧ JK Tet-On Vector ಒझ ύǴ௓‫ڋ‬ಔϷൂᐱуΕ Dox ᛰ‫ނ‬ೀ౛ಔձ‫ ځ‬NF-NB Εਡ٠คቚу‫ޑ‬ ௃‫ݩ‬р౜ǹՠ࣬ჹ‫ൂޑ‬ᐱуΕ PMA/Ion ‫ࢂ܈‬ӝ‫ٳ‬ᛰ‫ނ‬ೀ౛‫ޑ‬ಔձǴ NF-NB ԖΕਡቚу‫ޑ‬౜ຝр౜Ƕკ 8 (b) ࣁ JK Tet-On mAZ (1-228) ಒझਲ਼Ƕ่݀ᡉҢр௓‫ڋ‬ಔϝค NF-NB Εਡ‫ޑ‬௃‫ݩ‬Ǵՠ੝ձ‫ࢂޑ‬ӧ Ԗ Dox Ӹӧᇨว antizyme ಔձᆶ௓‫ڋ‬ಔ࣬КၨǴNF-NB Εਡ߾Ԗ٤ ༾ቚу‫ޑ‬ᖿ༈р౜ǶԜѦൂᐱуΕ PMA/Ion ‫ࢂ܈‬ӝ‫ٳ‬ᛰ‫ނ‬ೀ౛‫ޑ‬ಔ ձǴNF-NB ΨԖΕਡቚу‫ޑ‬౜ຝр౜ǶவխࣝᑻӀࢉՅ่݀߃‫؁‬ᡉҢ р JK Tet-On mAZ (1-228) ಒझӧԖ Dox Ӹӧᇨว antizyme ߄౜ΠǴ όᆅࢂᆶ௓‫ڋ‬ಔ‫ کࢂ܈‬JK Tet-On Vector ಒझ࣬КၨǴantizyme ዴჴ ཮ߦ຾ NF-NB ᙯՏ຾ΕಒझਡϣǴ຾Զቹៜ IL-2 ୷Ӣ‫߄ޑ‬౜Ƕ. ມǵаխࣝᑻӀࢉՅᢀჸ antizyme ߦ຾ NF-NB Ser536 ‫ޑ‬ᕗለϯ߄౜ ࣁΑዴᇡ antizyme ‫ޑ‬ዴ཮ቹៜ NF-NB ‫܄ࢲޑ‬຾ԶᏤठ NF-NB ຾ Εಒझਡϣቹៜಒझᐟન IL-2 ୷Ӣ‫߄ޑ‬౜ǴҁჴᡍᝩុճҔխࣝᑻ ӀࢉՅᙖҗӅ೫ขႜ৔ᡉ༾᜔ᢀჸ antizyme ቹៜ NF-NB ᕗለϯ‫߄ޑ‬ ౜Ǵ੝ձࢂӧՏ࿼ Ser536 ‫ޑ‬ᕗለϯǴѬЬाቹៜ NF-NB ᙯՏ຾Εಒ 35.

(52) झਡ‫߄ޑ‬౜Ƕკ 9 (a) ่݀ᡉҢӧ JK Tet-On Vector ಒझύǴ௓‫ڋ‬ಔ ϷൂᐱуΕ Dox ᛰ‫ނ‬ೀ౛ಔձ‫ ځ‬NF-NB Ser536 ᕗለϯ‫߄ޑ‬౜٠คܴ ᡉ‫ޑ‬ᡂϯǴԶൂᐱуΕ PMA/Ion ‫ࢂ܈‬ӝ‫ٳ‬ᛰ‫ނ‬ೀ౛‫ޑ‬ಔձǴNF-NB Ser536 ᕗለϯ‫߄ޑ‬౜ӧಒझਡϣ߾Ԗቚу‫ޑ‬౜ຝǶ࣬ჹ‫ޑ‬Ǵკ 9 (b) ࣁ JK Tet-On mAZ (1-228) ಒझਲ਼Ǵ‫่݀ځ‬ᡉҢр௓‫ڋ‬ಔ NF-NB Ser536 ᕗለϯ߄౜ؒԖ੝ձᡂϯǴՠ੝ձ‫ࢂޑ‬ӧԖ Dox Ӹӧᇨว antizyme ಔձᆶ௓‫ڋ‬ಔ࣬КၨǴNF-NB Ser536 ᕗለϯ‫߄ޑ‬౜ӧಒझਡϣ߾Ԗቚ у‫ޑ‬౜ຝǴԜѦൂᐱуΕ PMA/Ion ‫ࢂ܈‬ӝ‫ٳ‬ᛰ‫ނ‬ೀ౛‫ޑ‬ಔձǴNF-NB Ser536 ᕗለϯ‫߄ޑ‬౜Ψࢂቚу‫ޑ‬౜ຝǶவკ 8 Ϸკ 9 խࣝᑻӀࢉՅ่ ݀ᡉҢр JK Tet-On mAZ (1-228) ಒझӧԖ Dox Ӹӧᇨว antizyme ߄ ౜ΠǴዴჴ཮ߦ٬ NF-NB Ser536 ᕗለϯ߄౜ໆቚуЪ NF-NB ᙯՏ຾ ΕಒझਡϣໆΨቚуǴ຾ԶᏤठቹៜ IL-2 ୷Ӣ‫߄ޑ‬౜Ƕ. ୖǵаՋБᏀᗺ‫ݤ‬ዴᇡ antizyme ߦ຾ IL-2 ୷Ӣ߄౜ϐૻ৲໺Ꮴೈқ ፦߄౜ໆ җ‫ܭ‬Ӄ߻ග‫ ډ‬antizyme ቹៜߦ຾ IL-2 ୷Ӣ߄౜ёૈࢂ೸ၸ PKCǵIKKǵINB Կ NF-NB ೭చૻ৲໺ሀၡ৩‫܌‬ፓ௓ǴӢԜҁჴᡍճ Ҕ Western Blotting ٰᢀჸ೭٤ೈқ፦‫ޑ‬ᡂϯǶ२ӃճҔ JK Tet-On Vector ‫ ک‬JK Tet-On mAZ (1-228) ‫ٿ‬ਲ਼ಒझӃуΕ Dox ௴୏Ѥᕉનᇨ Ꮴ‫س‬಍ 3 λਔࡕǴӆуΕ PMA/Ion ຾Չਔ໔ᗺ‫ޑ‬ჴᡍࢲϯ T ಒझǴ 36.

(53) ϩձࣁ 0ǵ15ǵ30ǵ45ǵ60 ϩដ฻όӕਔ໔ᗺǴᢀჸ IKKǵINB Ϸ NF-NB ᕗለϯ‫ࢂ܈‬ҁ‫ي‬ೈқ፦‫߄ޑ‬౜ໆࢂցԖ‫܌‬ৡ౦Ƕҗკ 10 ่݀ ᡉҢᒿ๱ਔ໔ᗺ‫ޑ‬ቚуǴӧ JK Tet-On mAZ (1-228) ಒझਲ਼ύǴ IKKD/EϷ INB-Dᕗለϯ‫߄ޑ‬౜ໆ೿ࢂ΢ϲ‫ޑ‬ǴIKKD/E᏾ᡏೈқ፦߄ ౜ໆؒԖܴᡉᡂϯǴՠࢂ INB-D᏾ᡏೈқ፦߄౜ໆࠅࢂܴᡉ‫ޑ‬Πफ़ϐ ࡕΞԖӣൺ‫ޑ‬౜ຝౢғǶԜѦΨว౜ӧ JK Tet-On mAZ (1-228) ಒझ ਲ਼ύ NF-NB Ser536 ᕗለϯ‫ޑ‬ೈқ፦߄౜ໆ‫ ک‬JK Tet-On Vector ࣬Кၨ Ԗܴᡉ‫ޑ‬ቚуǴԶ NF-NB ᏾ᡏೈқ፦߄౜ໆคܴᡉᡂϯǶ‫܌‬аவխ ࣝᑻӀࢉՅϷ Western Blotting ่݀ᡉҢр antizyme ዴჴቹៜ NF-NB ૻ৲໺ሀၡ৩຾Զߦ຾ IL-2 ୷Ӣ‫߄ޑ‬౜Ƕ. 37.

(54) ಃѤകǵ૸ፕ! ಃ΋࿯ǵ࣬ᜢЎ᝘ຑ‫݋‬ᆶឍញ!!!! AntizymeǴύЎӜᆀΞᆀࣁ‫׭‬䁙ǴЬљፓ௓ಒझϣODC‫܄ࢲޑ‬ᆶ polyamines‫ޑ‬ၮଌϷжᖴ࣬ᜢ‫ޑ‬ೈқ፦Ƕ੝ձ‫ࢂޑ‬Ǵantizyme‫ޑ‬ғԋ ЬाΨࢂ‫ډڙ‬ಒझϣpolyaminesᐚࡋ‫܌‬ፓ௓ǴԶЪѸሡा࿶ၸਡᗐਡ ለ᠐ዸਣ+1Տ౽‫ޑ‬ᐒ‫ڋ‬ԶғԋǶԿҞ߻ࣁЗǴӧথ٢ᜪ୏‫ނ‬྽ύѝԖ antizymeࢂ୤΋ᙖ๱ਡᗐਡለ᠐ዸਣྖ୏‫ޑ‬ᐒ‫ڋ‬ౢғӄߏ٠‫ڀ‬Ԗਏ ૈ‫ޑ‬ೈқ፦ǶAntizymeૈ߄౜ӭᅿࠠᄊǴόᆅࢂӧΓᜪ‫ځࢂ܈‬дথ٢ ᜪ୏‫ނ‬྽ύǴAntizyme-1‫ک‬Antizyme-2ࢂӧᡏϣӚঁಔᙃ‫܈‬ಒझ྽ύ നத߄౜‫ࠠޑ‬ᄊǴќѦᗋхࡴΑಔᙃ஑΋‫ޑ܄‬Antizyme-3аϷߚ‫ࠠڂ‬ фૈ҂‫ޑޕ‬Antizyme-4Ƕ‫ځ‬ύǴ Antizyme-3നЬा߄౜‫ޑ‬೽ՏࢂӧΓ ᜪ‫ޑ‬࿐ΤаϷတ೽Ǵ‫ځ‬ғ౛բҔࣁፓ௓ᆒη‫ޑ‬ғԋ (spermatogenesis) (Lopez-Contreras et al., 2006)Ƕ!!! !!!!Antizyme-1ࢂಃ΋ঁ೏ว౜཮‫ൂک‬ᡏODC่ӝ‫׎‬ԋ౦፦Βᆫ ᡏǴ٬ளODCѨѐࢲ‫܄‬٠Ъ٬ODCӧubiquitin-independent௃‫ݩ‬Π೏ 26S proteasomeफ़ှ‫ޑ‬ೈқ፦ (Murakami et al., 1996)ǶၸѐЎ᝘ς௖ ૸೚ӭԖᜢantizyme‫ޑ‬ғ౛фૈǴନΑхࡴ‫ڋ׭‬ODC䁙‫܄ࢲޑ‬аϷ polyaminesжᖴ‫ޑ‬ፓ࿯‫ޣ‬฻୷ҁжᖴфૈѦǴantizymeӧϩηቫԛᆶ ಒझቫԛ‫ޑ‬фૈύΨ‫ת‬ᄽख़ा‫فޑ‬ՅǴ‫ځ‬ύхࡴΑϩϯፓ࿯ǵಒझຼ. 38.

(55) යǵಒझғߏǵԝΫǵϩϯᆶဍዦቚғǶ! җӃ߻ඔॊள‫ޕ‬Ǵӄߏ‫ڀ‬Ԗਏૈ‫ ޑ‬antizyme ‫ޑ‬ғԋѸሡा࿶ၸ +1 translational frameshifting ‫ޑ‬ၸำǴԶ Frameshifting ࢂ᎞๱΋ঁᐒ ‫܌ڋ‬ፓ࿯Ƕ྽ੰࢥགࢉਔυᘋનஒ೏ᇨᏤрٰǴ‫ࢥੰלځ‬ᆶ‫ל‬ಒझғ ߏૈΚ೽ϩࢂᙖҗ 2'-5' RNase L RNA decay pathway ‫܌‬ፓ௓Ƕੰࢥག ࢉಒझਔ཮ᇨวғԋ IFNs ԶЇว‫س‬಍‫ޑ܄‬ಒझ‫܄‬խࣝϸᔈᆶቚуӭ ᅿ‫ࢥੰל‬ೈқౢғǶᙖҗಒझϣ‫ ޑ‬IFNs ቚуǴRNase L ቚу antizyme +1 frameshifting ‫ޑ‬ਏ౗ǶԜѦಒझ‫܄‬ᆶੰࢥ‫܄‬ೈқ፦Ψёаᙖҗਡᗐ ਡለ᠐ዸਣ+1 Տ౽ᐒ‫ߏۯڋ‬ೈқ፦ C ᆄᙯ᝿຾ՉǴ຾Զ‫ڈ‬ᐟ‫ל‬চև ౜๏ࡕϺ‫܄‬խࣝ‫س‬಍Ǵ٠υᘋੰࢥғӸǶ ၸѐխࣝᏢਜύςග‫ډ‬խࣝϸᔈхࡴΑӃϺ‫܄‬ϷࡕϺ‫܄‬խࣝϸ ᔈЪӃϺ‫܄‬խࣝϸᔈ཮஥୏ᇨวࡕϺ‫܄‬խࣝϸᔈԶ୺Չ‫׳‬மεЪԖ ਏ౗‫ޑ‬ٛᑇᐒ‫ڋ‬ǶԶӧӭኧੰࢥགࢉΠǴε೽ϩ୺Չխࣝٛᑇٰ‫לܢ‬ ੰࢥགࢉ‫ޑ‬ЬाࢂࡕϺ‫܄‬խࣝϸᔈǴ‫ځ‬ϸᔈ‫ޑ‬ಒझа T రЃಒझ‫ ک‬B రЃಒझࣁЬǴ‫ځ‬ύ T రЃಒझӧ᏾ᡏխࣝϸᔈ‫ת‬ᄽ๱ύЈ‫فޑ‬ՅǶ T ಒझ࿶ᇨวᏤठࢲϯϷϩϯԋόӕࠠᄊ‫ ޑ‬T ಒझٰ୺ՉঁձфૈǴ ԜѦΨౢғόӕ‫ޑ‬ಒझᐟન٠ᙖҗಒझᐟનϩձբҔ‫ܭ‬όӕҞ኱х ࡴΑѮᏘಒझǵB రЃಒझ‫ ࢂ܈‬NK ಒझ฻ٰၲ‫ܢډ‬ᑇੰࢥགࢉ‫ޑ‬Ҟ ‫ ޑ‬Ƕ ࡽ ฅ antizyme ‫ ޑ‬ғ ԋ ೽ ϩ ཮ ‫ ډ ڙ‬խ ࣝ ᐒ ‫ ܌ ڋ‬ፓ ௓ Զ ቚ у. 39.

(56) translational frameshifting ‫ޑ‬ਏ౗ǴҁፕЎགྷा૸௖‫ ځ‬antizyme ᆶխࣝ ᐒ‫ڋ‬ϐ໔‫࣬ޑ‬ᜢ‫܄‬Ǵ‫ځ‬ύǴҁፕЎ๱ख़‫ޑ‬Ҟ኱ࢂ antizyme ᆶ T రЃ ಒझϐ໔‫ޑ‬ᜢೱ‫܄‬Ǵ຾΋‫؁‬ᢀჸ antizyme ӧ T రЃಒझࢲϯၸำύ ‫ת܌‬ᄽ‫فޑ‬ՅǶёெ‫ࢂޑ‬Ǵӧ౜Ϟࣴ‫ز‬྽ύǴӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ჹ‫ܭ‬ಒझϣғ౛фૈፓ࿯‫ޑ‬၁ಒ௃‫ݩ‬ԿϞϝคዴϪ‫ۓޑ‬ፕǴᆶ T రЃ ಒझϐ໔‫ޑ‬ᜢೱ‫ޑ܄‬௖૸‫ࢂ׳‬ϿϐΞϿǶ Ҟ߻ᜢ‫ܭ‬antizymeᆶTరЃಒझϐ໔‫࣬ޑ‬ᜢ‫܄‬εӭᗋࢂ๱Ꮐӧ ODC‫ک‬polyaminesჹTಒझϐғߏว‫ػ‬Бय़‫ޑ‬௖૸ۚӭǶODCࣁ΋ঁ ಒझғߏѸሡ‫܄‬ሇનǴ‫ځ‬ೈқ፦߄౜Ϸࢲ‫ܴ܄‬ᡉӦё࿶җ೚ӭғߏӢ ηᇨᏤԶϲଯǹpolyamines߾ࢂୖᆶಒझϣDNAǵRNAǵೈқ፦Ϸᕗ ለિ፦‫ޑ‬ғӝԋǴࣁಒझғߏ‫ک‬ว‫܌ػ‬Ѹሡ‫ޑ‬Ǵჹ‫ܭ‬ಒझғߏǵϩϯ ϷԝΫ‫ת‬ᄽख़ा‫ف‬ՅǶၸѐЎ᝘ςࡰрODCᆶpolyaminesჹ‫ځܭ‬Tಒ झ‫ޑ‬ቚғ‫ک‬ว‫ୖػ‬ᆶ‫ځ‬ύǴՠёெ‫ࢂޑ‬ჹ‫ܭ‬Tಒझ‫ځ‬дғ౛фૈ‫ޑ‬ፓ ௓‫ٯ‬ӵࢲϯǵϩϯ‫܈‬ಒझ঒Ϋ฻࣬ᜢࣴ‫ز‬ϝࢂό‫ޑى‬Ǵ‫׳‬ձග‫ډ‬ antizymeፓ௓Tಒझғ౛фૈ‫ޑ‬ᐒ‫ڋ‬ǶฅԶǴ߈൳ԃٰҁჴᡍ࠻ᙖҗ ᄬᑐ߄ၲΓᜪantizyme୷Ӣၩᡏᆶᙯࢉ੿ਡಒझჴᡍѳѠഌុ᛾ܴΑ ӄߏ‫ڀ‬фૈ‫ޑ܄‬antizyme‫ޑ‬ғԋዴჴቹៜΑTಒझ‫ޑ‬ғ౛բҔǶӧ 2006ԃ‫܌‬ว߄‫ޑ‬Ў᝘ࡰрӄߏ‫ڀ‬фૈ‫ޑ܄‬antizymeᙖҗಈጕᡏጢႝ Տ‫ޑ‬ѐཱུϯǴᐟวBax‫ޑ‬ಕᑈᆶBcl-xL‫߄ޑ‬౜ໆΠफ़Ǵ຾Զߦ຾Jurkat. 40.

(57) Tಒझ‫ޑ‬ीฝ‫܄‬ԝΫ‫و‬ӛಒझ঒Ϋ೼৩ (Liu et al., 2006)ǶԜ่݀Ψӑ ᛾ΑӃ߻Ў᝘‫܌‬ගр‫ޑ‬antizyme‫ޑ‬ၸࡋ߄౜ዴჴ཮Ꮴठಒझ‫و‬ӛಒ झ঒Ϋ೼৩ (Hoshino et al., 2005; Jiang et al., 2007)ǶନΑantizyme཮ ᏤठTಒझ‫ޑ‬ಒझ঒ΫѦǴ຾΃‫؁‬Ψว౜Α΋ঁ੝ਸ౜ຝջTಒझ‫ޑ‬ ࢲϯ཮ගϲantizyme +1 frameshiftingբҔ (ߕᒵΜ)ǶਥᏵ೭٤ࣴ‫่ز‬ ݀ᡉҢǴantizymeዴჴӧTಒझ‫ޑ‬ғ౛фૈ΢‫ת‬ᄽࢌᅿำࡋፓ࿯‫ޑޣ‬ ‫ف‬ՅǶය೚ҁࣴ‫ૈ่݀ز‬ග‫ٮ‬നཥ‫ૻޑ‬৲ᆶ‫ޔ‬ௗ‫ޑ‬᛾Ᏽ᛾ܴantizyme ፓ࿯‫ޑ‬ϩηᆶಒझቫԛϐᐒ‫ڋ‬Ǵஒٰૈ‫׳‬౛ှӧTಒझࢲϯ‫ࢂ܈‬ว‫ػ‬ ύantizyme‫ܴޑ‬ዴ‫ف܄‬ՅǶ ၸѐЎ᝘ࡰрTಒझ‫ࢲޑ‬ϯёϩࣁ‫ٿ‬ᅿᜪࠠǺ΋ࢂ‫ל‬চ٩ᒘࠠǹ ќ΋ᅿ߾ࢂߚ‫ל‬চ٩ᒘࠠǶ‫ל‬চ٩ᒘࠠΏࡰTಒझ߄य़‫לޑ‬চௗ‫ڙ‬Ꮤ! (TCR)!ёаᒣᇡ੝‫לޑۓ‬চǴ྽TCR‫לډڙ‬চ‫ڈ‬ᐟਔǴ཮Їଆҗಒझ ጢǵಒझ፦Կಒझਡ΋ೱՍ‫ૻޑ‬৲໺ሀၡ৩‫ࢲޑ‬ϯǴ຾Զࢲϯಒझ୺ ՉфૈǹԶߚ‫ל‬চ٩ᒘࠠ߾ࣁTಒझ೸ၸࢌ٤ಒझ߄य़‫ל‬চ (ӵ CD2ǹCD28฻) ‫ޑ‬բҔǴ຾ԶࢲϯTಒझǶTಒझ‫ࢲޑ‬ϯϷϩϯၸำ ۳۳౐ੋ‫ډ‬೚ӭಒझᐟન‫߄ޑ‬౜аϷፓ௓Ƕ྽Tಒझ‫לډڙ‬চև౜ಒ झAPCௗ᝻‫ڈ‬ᐟϐࡕ཮٩όӕ‫ૻޑ‬৲ϸᔈԶϩϯԋόӕᜪࠠ‫ޑ‬Tಒ झǴ೭྽ύߡ཮Ԗ೚ӭόӕ‫ޑ‬ಒझᐟનୖᆶ‫ځ‬ύǴхࡴΑᆶTh1ಒझ ࣬ᜢ‫ޑ‬IL-2ǵIL-12ǵIFN-JϷTNF-DǹᆶTh2ಒझ࣬ᜢ‫ޑ‬IL-4ǵIL-5ǵ. 41.

(58) IL-6ǵIL-10‫ک‬IL-13ǹᆶTh17ಒझ࣬ᜢ‫ޑ‬IL-17‫ک‬ᆶTregಒझ࣬ᜢ‫ޑ‬ TGF-E฻฻όӕಒझᐟનǶ‫ځ‬ύǴಒझᐟનIL-2ࣁ‫ࢲډڙ‬ϯTಒझԾᡏ ϩ‫܌ݜ‬ౢғ‫ޑ‬ख़ाಒझᐟનǴΨࢂTಒझ‫ܭ‬ϟᏤ᏾ᡏխࣝፓ࿯ϐಒझ ᐟનǶѬёբࣁTಒझࢲϯ‫ޑ‬ғ‫ࡰނ‬኱ǴЬљTಒझϐғߏፓ௓ǵࢲ ϯǵϩϯаϷஎЬ᏾ᡏխࣝٛᑇ฻фૈǶIL-2‫ޑ‬ғԋЬाΨࢂ࿶җT ಒझᇨᏤࢲϯϐࡕ࿶ၸ΋‫س‬ӈૻ৲໺ሀၡ৩‫ࢲޑ‬ϯനࡕӕਔࢲϯኧ ঁख़ा‫ޑ‬ᙯᒵӢη‫܄ࢲޑ‬Ǵ຾Զ௴୏୷Ӣ‫߄ޑ‬౜ǶӧIL-2୷Ӣ‫ޑ‬ promoter΢ёаว౜ኧঁᙯᒵӢηǴхࡴΑNFATǵAP-1ǵOct1ϷNF-NB ฻Ǵ೭٤ᙯᒵӢη཮࿶җ΢ෞόӕ‫ૻޑ‬৲໺ሀၡ৩‫ࢲޑ‬ϯԶѐቹៜ IL-2୷Ӣ‫߄ޑ‬౜ (Good et al., 1996; Ward et al., 1998)ǶҞ߻ᇨᏤTಒझ ࢲϯ‫ޑ‬ჴᡍኳԄԖаΠ൳ᅿБԄǺಃ΋ᅿБ‫ࢂݤ‬ճҔTಒझ߄य़੝‫ۓ‬ ‫ޑ‬ௗ‫ڙ‬ᏔTCR/CD3 complexǴуΕAnti-human CD3ߦ٬Tಒझࢲϯᇨ วIL-2ញрǹಃΒᅿБ‫ࢂ߾ݤ‬ճҔPhytohemagglutinin (PHA) ٰᇨว ࢲϯTಒझǶPHAࣁ‫ࣽل‬෌‫ނ‬ϩᚆрٰ‫ޑ‬ᗐೈқǴឦ‫ܭ‬෌‫ނ‬ન (Lectin) ‫ޑ‬΋ᅿǴҗ‫ܭ‬Ѭ‫ڀ‬ԖआՈౚᏉ໣ϐ੝‫܄‬ǴࡺύЎӜᆀΞᆀࣁआՈౚᏉ ໣નǶҗ‫ܭ‬TಒझࢲϯቚғၸำύǴಒझϣѦ້ᚆηᐚࡋ‫ޑ‬ӭჲࣁ΋ ߚதख़ा‫ޑ‬ಒझૻ৲ (Feske, 2007; Gardner, 1989)ǴӢԜPHAᙖҗፓ ௓ಒझϣѦ້ᚆηᐚࡋ‫ڈ‬ᐟTಒझࢲϯቚғǹಃΟᅿБ‫ࢂ߾ݤ‬ӝ‫ٳ‬٬ ҔPhorbol 12-myristate 13-acetate (PMAǹTPA) ϷIonomycin (Ion)Ζၸ. 42.

(59) ѐࣴ‫ز‬ᡉҢऩࢂൂᐱߦ٬ಒझϣ້ᚆηᐚࡋ΢ϲό‫ى‬аЇଆTಒझ‫ޑ‬ ࢲ ϯ ቚ ғ Ǵ ᗋ Ѹ ໪ ा Ԗ PKC ೈ қ ፦ ‫ ୖ ޑ‬ᆶ ω ૈ ᇨ ว ࢲ ϯ T ಒ झ (Altman et al., 1992)ǶIonࣁ້‫ޑ‬ឫ஥‫ޣ‬ǴЬाፓ࿯ಒझϣ້ᚆη‫ޑ‬ၮ ଌǴԶPMA߾ࣁPKC‫ࢲޑ‬ϯᏊǴ‫ޣٿ‬Ӆӕ٬ҔΠ཮ߦ٬ಒझϣ້ᚆη ᐚࡋ΢ϲаϷPKC‫ࢲޑ‬ϯǴനಖᏤठTಒझࢲϯቚғ (Dadsetan et al., 2008; Kerschbaum and Cahalan, 1999)Ƕ! ӧҁፕЎჴᡍύࢂճҔPhorbol 12-myristate 13-acetate (PMAǹ TPA) ϷIonomycin (Ion) ‫ޑ‬ჴᡍኳԄٰᇨᏤJurkat Tಒझࢲϯ٠ញ‫ܫ‬ рIL-2Ƕҗ‫ܭ‬ၸѐࣴ‫ࡰز‬рԜኳԄࢂ೸ၸፓ࿯ಒझϣ້ᚆη‫ޑ‬ᐚࡋа ϷPKC‫ࢲޑ‬ϯٰၲ‫ډ‬Tಒझ‫ࢲޑ‬ϯǴ‫܌‬аӧԜҁፕЎ௢ෳIL-2ࣗԿࢂ ‫ځ‬дᆶTಒझࢲϯ࣬ᜢ‫ޑ‬ಒझᐟન‫߄ޑ‬౜೿ࢂ೸ၸPKCǵIKKǵINBԿ NF-NBࢲϯᙯՏ຾Εಒझਡϣ೭చૻ৲໺ሀၡ৩‫܌‬຾Չ‫ޑ‬ǶԜѦǴၸ ѐࣴ‫ز‬ว౜྽Tಒझ‫ډڙ‬ᇨวࢲϯ‫ޑ‬௃‫ݩ‬ΠǴNF-NBᕗለϯ‫߄ޑ‬౜཮ ፓ௓NF-NBᙯՏ຾ΕಒझਡǴ੝ձࢂӧNF-NB p65 Ser536‫ޑ‬Տ࿼Ǵ‫ځ‬ ᕗለϯ‫߄ޑ‬౜཮ቹៜNF-NBᙯՏ຾Εಒझਡϣ຾Զቹៜ୷Ӣ‫߄ޑ‬౜ (Mattioli et al., 2004; Perkins, 2007)ǶӢԜӧҁჴᡍύǴ‫׆‬ఈᙖҗ೭చ ၡ৩ٰΑှ‫ز‬ഖantizymeࢂց཮ᙖҗቹៜ೭٤ૻ৲໺ሀೈқ፦‫߄ޑ‬ ౜຾ԶቹៜᆶTಒझࢲϯ࣬ᜢ‫ޑ‬ಒझᐟન‫߄ޑ‬౜ǶԶԖ፪‫ࢂޑ‬Ǵၸѐ Ў᝘ςࡰрODC‫ޑ‬ၸࡋ߄౜Ϸಒझϣpolyaminesᐚࡋࣣ཮ቹៜNF-NB. 43.

(60) ‫܄ࢲޑ‬Ϸफ़եᆶ௴୏η่ӝ‫ૈޑ‬Κ (Babbar et al., 2006; Liao et al., 2008; Merentie et al., 2007)Ƕ ନΑNF-NBૻ৲໺ሀၡ৩ϐѦҁፕЎό௨ନᗋԖ‫ځ‬дၡ৩‫ޑ‬ፓ ௓Ǵ‫ځ‬ύхࡴΑCBP/P300ೈқ፦‫߄ޑ‬౜ǶCBP/P300ࣁ΋ᙯᒵӢηߦ ຾ࢲϯ‫ޑ‬ೈқ፦ǴӧTಒझਡϣЬљፓ௓ᙯᒵӢη‫܄ࢲޑ‬Ǵ຾Զߦ٬ ᙯᒵբҔ‫ޑ‬຾ՉǶၸѐЎ᝘ࡰрSnip1ࣁCBP/P300‫ڋ׭ޑ‬ೈқǴ཮ᙖ җᆶSmad1/HsN3/antizyme‫׎‬ԋፄӝᡏ೏ឫ஥Կ26s proteasome຾Չफ़ ှբҔǴ຾ԶቹៜCBP/P300ೈқ፦‫߄ޑ‬౜ԶቹៜΑᙯᒵӢη‫ࢲޑ‬ ‫܄‬ǴӢԜҁፕЎᇡࣁantizymeΨёૈ೸ၸ೭చၡ৩ԶቹៜΑTಒझ‫ޑ‬ ࢲϯǴѝࢂ೭٤௢ፕۘࡑሡा‫׳‬ӭ‫زࣴޑ‬᛾Ᏽٰӑ᛾Ƕ !. ಃΒ࿯ǵჴᡍ่݀ញፕ ӧ߻‫ق‬ᆣፕക࿯ύଞჹҁࣴ‫ز‬Ьᚒ‫ु܌‬р‫ޑ‬ჴᡍ฼ౣǴᕴӅϩࣁ ΟεБӛǴ౜ӧ٩‫܌‬ள‫ޑ‬ჴᡍ่݀΋΋૸ፕǶ २Ӄӧಃ΋೽ҽǴࣁΑ‫ޔ‬ௗ௖૸ӄߏ‫ڀ‬фૈ‫ ޑ܄‬antizyme ᆶ T ಒझϐ໔‫࣬ޑ‬ᜢࣴ‫ز‬ǴӢԜҁჴᡍ࠻ࡌҥᄬᑐ߄ၲΓᜪ antizyme ୷Ӣ ၩᡏᆶᙯࢉ੿ਡಒझ‫ޑ‬ჴᡍѳѠǴஒ஥Ԗ Vector Ϸӄߏ‫ ޑ‬Antizyme (‫ڮ‬Ӝࣁँᡂࠠ Antizymeǹm-AZ)‫ ޑ‬Tet-On ‫س‬಍ pTRE2/hyg ၩᡏଌԿ Jurkat T cell ύǴբࣁࣴ‫ز‬ኳԄǶࣁΑዴߥ JK Tet-On System ໩ճၮ 44.