ஒᎦܭ6 cm dish ޑಒझϩձаᛰނೀࡕǴஒmedium ౽ନǴа ӇհPBS మࢱΒԛࡕܜଳǴӆуΕޑtrypsinஒಒझѺΠǴ٠ஒಒ
Ε100Pl CEA bufferǴமਗ਼ਁᕏΜࣾដǴᓉܭӇ10ϩដǴӆуΕ 4.4Pl CEB bufferமਗ਼ਁᕏΜࣾដǴᓉܭӇ1ϩដа10,000 rpm ӧ 4ʚΠᚆЈ5ϩដǴڗమనࣁಒझ፦ڗނǶќஒpellet уΕ50Plޑ NER bufferǴமਗ਼ਁᕏΜࣾដǴӧӇᓉ40ϩដЪ10ϩដமਗ਼ਁ
འΜࣾដǴ40ϩដࡕа14,500 rpm ӧ4ʚΠᚆЈ20ϩដǴ܌ளమన ࣁಒझਡڗނǶ
12. EMSA ( electrophoretic mobility shift assay )
ஒ4PgϐಒझਡܜڗނуΕreaction mixtureǴځಔԋࣁ 2Plޑ10७ binding bufferǵ glycerolǵMgCl2ǵ poly (dI-dC)ǵNP 40ǵlabeled probeǴ
ံНԿ20PlǴܭ࠻ྕΠϸᔈ20ϩដǴӧ࠻ྕΠуΕloading dyeǴ protein-DNA complexϷfree oligonucleotideǴаpolyacrylamidegelႝݚ ϩϩᚆ
13. ߃ભಒझᎦ
߃ભషӝጤಒझ(primary cultured mixed glial cells)Ꭶ
߃ભషӝጤಒझࢂڗԾܭрғΒϺϣޑSprague-Dawley ႵϐεတҜ
፦ಔᙃǶஒᓐᢅମӛΒୁᙌ໒Ǵ٬εတಔᙃᇘ៛рٰǴஒλတѐନ٠ ஒεတڗΠܫܭคϐᎦҝύǶӧό້֖Ϸᗔޑ+%66 +DQN¶V
ՈᆅǶ٬Ҕ1ml pipette аᐒఓޑБԄஒಒझᏃໆѺԋൂಒझǴ٠ӧ 37кΠа֖Ԗ 0.25% trypsin Ϸ1 mM EDTA ޑHBSS բҔ20~30ϩ ដǴஒ่ጌಔᙃϩᚆ໒ǴуΕᡏᑈ֖10% FBS ޑDMEM ಖЗ trypsin ޑբҔǴᚆЈ5 ϩដ1000 rpmǴѐନమనǴӆа֖10% FBS ޑDMEM/F12 ஒಒझλЈѺණǴၸ70 mM ޑᑔᆛǴஒಒझᎦܭа poly-D-lysine ೀၸޑT75 ϐᎦҝύǴܭ37к֖5% CO2 ϐᎦ ጃύǴࢃය׳ඤ1~2 ԛᎦనǶ
߃ભ༾ጤಒझᎦ (primary cultured microglia)
߃ભ༾ጤಒझࢂషӝጤಒझᎦ12~14 ϺѰѓǴஒಒझᎦҝа 180 rpm ޑᙯೲזೲའਗΒঁλਔǴ٬microgliaǵઓಒझаϷ oligodendrocytes ᝌੌǴԏಒझనǴᚆЈ5 ϩដ1000 rpmǴѐନ
మనǴஒಒझᅿӧؒԖೀޑᎦዬύǴᎦҝܭ37кᎦጃύ 20~30 ϩដࡕ׳ඤԛಒझᎦనǴஒmicroglia аѦޑځдಒझ౽ ନǶаanti-CD11b ޑלᡏխࣝᑻӀࢉՅǴᔠᡍmicrogliaޑપࡋǶ 14.ीБݤ
܌ԖჴᡍኧᏵаѳ֡ॶ ± ྗৡ (mean ± S.E.M) ߄ҢǴٿಔϐ໔ޑ
ಃΟക Peptidoglycan ӧ microglia ܌ᇨวޑ iNOS ک COX-2 ӝԋᐒ ᙯϐ
3-1 PGNቚу iNOS ک COX-2 ӧ microglia ޑ߄
BV-2 microglia๏ϒPGN (0.3-30Pg/ml) ڈᐟ24 λਔࡕǴቚуNO (nitric oxide)ޑញܫ(Fig. 3-1A)Ǵ٠ЪᒿPGNᐚࡋޑቚуǴNOޑౢ
ໆΨᒿቚуǴӧ๏ϒPGN 10 Pg/ml 24 λਔࡕNOޑౢໆቚуΟ७ (n=5)ǶԜѦךॺΨ٬ҔLPSԋpositive controlǹBV-2 micogliaӧ๏
ϒLPS (100 ng/ml)ځNOޑញܫऊ9७ǶךॺΨճҔWestern blottingٰ
ᢀჸPGNڈᐟiNOSکCOX-2ޑೈқ߄ǴPGNܴᡉޑቚуiNOSک COX-2ೈқޑ߄Ǵ߄ᒿᐚࡋቚуԶቚуೈқ߄ޑᖿ༈(Fig.
3-1B&C)ǴԜѦճҔRT-PCRٰᢀჸPGNڈᐟiNOSکCOX-2ޑmRNA ߄ǴΨճҔreal time PCRՉۓໆǴ่݀ᒿ๏ϒPGNᐚࡋቚу ԶቚуRNAޑ߄(Fig. 3-1D&E)ǶԜѦךॺΨᔠࢂցPGNᇨᏤ iNOSکCOX-2ቚуࢂցቹៜBV-2ޑproliferationǴ่݀วPGN٠ όׯᡂmicogliaޑcell viability (MTT assay, data not shown)ǴךॺΨ ௨ ନ Α PGN ቚ у iNOS ک COX-2 ޑ ೈ қ ߄ ٠ ό ࢂ җ LPS contaminationǹ ך ॺ ٬ Ҕ LPS ڋ Ꮚ polymyxin B (1 PM) ǹ ว polymyxin B (1 PM)ёаֹӄڋLPS (100 ng/ml)ᇨᏤޑiNOSک
Pg/ml)ቚуiNOSکCOX-2 ೈқޑ߄ (Fig. 3-2A&B)Ƕ
3-2 PGNڈᐟ microglia ౢғ proinflammatory cytokine ޑ߄
ך ॺ Ψ གྷ ޕ ၰ ӧ BV-2 microglia ๏ ϒ PGN ࢂ ց ڈ ᐟ proinflammatory cytokine ޑ ౢ ғ Ǵ ך ॺ Ҕ RT-PCR ٰ ᢀ ჸ proinflammatory cytokineࢂցΨڙPGNፓǴ٠Ъ٬Ҕreal-time PCRٰۓໆǴᒿPGNᐚࡋޑቚуǴ IL-6ǵTNF-Dک IL-1EޑmRNA ߄Ψၟቚу(Fig. 3-3)ǶCD11bёаࣁmicrogliaࢲϯޑঁ
کઓଏϯຝԖࡐεޑᜢᖄ(Davis et al. 2000; Roy et al. 2008)Ǵճ Ҕ RT-PCR מ ೌ ך ॺ Ψ ว ๏ ϒ PGN ࡕ ቚ у CD11b ӧ BV-2 microgliaޑ߄(Fig. 3-4A)Ƕ
3-3 PGN ၸ TLR2 receptor/MyD88 ቚу iNOS ک COX-2 ޑ߄
ӭࣴزൔᡉҢ TLRs תᄽख़ाޑفՅӧᒣѦٰނᆶٛᑇ౦ ނΕߟ(Kirk and Bazan 2005; Creagh and O'Neill 2006; Kawai and Akira 2009)Ǵךॺགྷޕၰ PGN ࢂցၸ TLR receptors ௴Πෞޑૻ
৲ሀၡ৩ǶӃԖࣴزൔᏤࡰр PGN բҔӧ TLR2 receptor (Kielian et al. 2005; Ajuwon et al. 2009; Chiu et al. 2009)ǹԜѦԖࣴز
2008)ǶӢԜךॺෳ BV-2 microglia ύ PGN ࢂցΨࢂၸ TLR2 receptorሀૻ৲Ƕ२ӃճҔ real-time PCR ٰᔠຎ TLR ޑ߄Ǵӧ BV-2 microglia๏ϒ PGN ೀࡕΖλਔǴTLR2 mRNA ޑ߄ቚу(Fig.
3-4B)Ǵךॺ׳ࢂց PGN ቚу iNOS ک COX-2 ޑ߄ࢂ
ၸ TLR2 receptorǹךॺೀ TLR2 antibody(2 ȝJ)܈ࢂ IgG antibody ࡕӆ๏ϒ PGNǴ่݀ᡉҢճҔ TLR2 antibody ёаל PGN ڈᐟޑ iNOS ک COX-2 ޑ߄(Fig. 3-4C&D)Ǵ೭٤ᏵᡉҢ PGN ڈᐟ microglia ౢғ iNOS ک COX-2 ޑ߄ዴჴࢂҗ TLR2 receptor ܌൞ ϟǶԶ MyD88 ࢂ TLR receptor ޑ adaptor proteinǴΨୖᆶӧ TLRs ૻ
৲ሀύ(Sawaki et al. 2007; Xu et al. 2008)ǴӢԜךॺགྷޕၰ MyD88
ࢂցΨୖϒ PGN ڈᐟ iNOS ک COX-2 ޑ߄ǹϩձೀ Control peptide ک MyD88 ڋࠠ peptide ࡕǴӆ๏ϒ PGN ڈᐟ 24 λਔࡕǹ MyD88ڋࠠ peptide ڋ PGN ቚу iNOS ک COX-2 ޑೈқޑ߄
ǴЪևᒿڋࠠ peptide ᐚࡋቚуԶ׳ᡉޑڋբҔ(Fig.
3-4E)ǶӢԜ PGN ࢂҗ TLR2 receptor/MyD88 Զቚу iNOS ک COX-2 ޑ߄Ƕ
3-4 PGNၸ PI3K/AKT ૻ৲ሀቚу iNOS ک COX-2 ޑ߄
ӧၸѐޑЎύޕၰ PI3K/AKT pathway ୖᆶፓӭޑғނ
ᐟનޑౢғǶԶךॺ٬Ҕ AKT ڋᏊǵPI3K ڋᏊ LY294002 ک wortmannin ᢀჸ PGN ቚу iNOS ک COX-2 ޑೈқ߄ǶAKT ڋ ᏊǵPI3K ڋᏊ LY294002 ک wortmannin ڋ PGN ቚу iNOS ک COX-2 ೈқޑ߄ (Fig. 3-5A&B)ǶԜѦǴPGN ࢲϯ AKT ک p85
ૈᒿਔ໔ޑቚуԶԖࢲϯቚуޑᖿ༈(Fig. 3-5C&D)ǶԜѦǴךॺΨ
٬Ҕ MyD88 ڋࠠ peptide ዴᇡ MyD88 ࢂցΨୖᆶӧ PGN ፓࢲϯ AKT ک p85 ޑૻ৲ሀၡ৩ǶMyD88 ڋࠠ peptide ૈ
ڋ PGN ᇨᏤ AKT ک p85 ᕗለϯޑຝ(Fig. 3-5E&F)Ƕᆕӝа่
݀ǴPGN ቚу iNOS ک COX-2 ೈқޑ߄ࢂၸ PI3K/AKT ૻ৲
ሀǶ
3-5 NF-NB ୖᆶ PGN ቚу iNOS ک COX-2 ޑ߄
ӭЎᡉҢǴᙯᒵӢη NF-NB ӧխࣝวݹϸᔈǵಒझޑቚғǵ ϩϯᆶᕎϯғϸᔈύתᄽΑᜢᗖ܄ޑفՅ(Donadelli et al. 2000;
Roth et al. 2006; Pei et al. 2008; Reber et al. 2009)ǴЪΨᆶӭ੯ੰޑ วғڀԖ࣬ᜢ܄(Tu et al. 2003; Pieper et al. 2004; Brandi et al. 2008;
Pei et al. 2008)ǶԜѦΨԖӭࣴزൔᡉҢǴNF-NB ፓӭวݹ ϸᔈکಒझᐟનޑౢғ(Saldeen and Welsh 2004; Vandoros et al. 2006;
қޑ߄ǹೀ PDTCǵTPCK ک Bay 11-7082 ΟΜϩដڋ PGN ቚу iNOS ک COX-2 ೈқޑ߄(Fig. 3-6A&B)ǶԜѦΨճҔ reporter assayᢀჸ NF-NB ޑᙯᒵࢲ܄ǹPGN ቚу NF-NB luciferase ޑࢲ܄Ǵ Զಒझᙯ AKTǵp85 dominant-negative mutant ܈ IKKDǵIKKE kinase mutantૈڋ PGN ቚу NF-NB luciferase ޑࢲ܄(Fig. 3-6C)Ƕ 3-6 PGNࢲϯIKKDEکINBD௴Πෞૻ৲ሀ
NF-NBѳਔᆶځፓೈқ,N%่ӝǴԶԋঁόࢲϯޑፄӝ ᡏӸӧಒझ፦ύǴڙډѦٰޑڈᐟǴࢲϯIKK complex ᏤठINB
ᕗለϯԶफ़ှǴ٬ளNF-NBёаΕಒझਡǴԶࢲϯΠෞ୷Ӣ ޑᙯᒵǶBV-2 microglia๏ϒPGNόӕਔ໔ڈᐟࡕǴᒿPGNբҔ ਔ໔ቚуԶቚуIKKDEǵINBޑᕗለϯຝǴԶINBDΨᒿPGNբҔ ਔ ໔ ޑ ᡂ ϯ ᅌ फ़ ှ ( ӧ Fig. 3-7A&C&E) Ƕ Ԝ Ѧ Ǵ ך ॺ ೀ LY29400ǵwortmannin 30ϩដǴௗ๏ϒPGN 2λਔǴIKKDEǵpINB ޑᕗለϯکIN% Dޑफ़ှຝ߾Ԗᙯޑຝ(Fig. 3-7B&D&F)Ƕ NF-NBࢂҗp65/p50ፄӝೈқᄬԋǴࢲϯޑp65௴ΠෞޑᙯᒵӢ ηǴ๏ϒPGNǴᒿբҔਔ໔ቚуΨቚуp65ޑᕗለϯຝ(Fig.
3-7G)Ƕҗаޑ่݀ёޕǴPGNբҔӧTLR2/MyD88җࢲϯ PI3K/AKTૻ৲ၡ৩ǴԶᕗለϯIKKDEکࢲϯINBDǴԋINBDޑϩ
Fig. 3-1 PGN increases iNOS and COX-2 expression in BV-2 microglia. (A) BV-2 microglia cells were incubated in concentrations dependent of PGN or 100 ng/ml of LPS for 24 h. The culture medium were collected and analyzed by Griess reaction. Whole cell lysis protein were extracted and subjected to Western blot for iNOS (B) and COX-2 (C) after 24 h incubation with PGN or LPS. Note that PGN increases iNOS or COX-2 protein expression in a concentration-dependent manner. Cells were stimulated with PGN in various concentrations for 6 h and the mRNA levels of iNOS (D) and COX-2 (E) were analyzed by real-time PCR. Results are expressed as the mean ± S.E.M. of five independent experiments
Fig. 3- 2 Effects of polymyxin B on LPS- and PGN-induces iNOS and COX-2 production in BV-2 microglia. Cells were pretreated with SRO\P\[LQ % 3% ȝ0 IRU PLQ EHIRUH LQFXEDWLRQ ZLWK ng/ml /36RUȝJPO3*1IRUK:KROHFHOOO\VLVprotein were subjected to Western blot for iNOS (A) and COX-2 (B). The quantitative data were showed in lower panel. Results are expressed as the mean ± S.E.M. of four independent experiments. *, p<0.05 as compared with vehicle treatment group. #, p<0.05 as compared with the LPS or PGN treatment groups, respectively.
Fig. 3-3 PGN increases proinflammatory cytokine expression in BV-2 microglia. Cells were stimulated with PGN (10 Pg/ml) for 6h and the mRNA levels of IL-6 (A) IL-ȕ (B) and TNF-Į (C) were analyzed by real-time PCR. Note that PGN increases proinflammatory cytokine mRNA expression concentration-dependently. Results are expressed as the mean ± S.E.M. of four independent experiments.
Fig. 3-4 Involvement of TLR2 receptor in PGN-mediated iNOS and COX-2 production in BV-2 microglia. (A) BV-2 cells were treated with various concentrations of PGN. Total RNA was extracted and subjected to RT-3&5 IRU WKH ȕ-integrin marker of microglia, CD11b. Cells were stimulated with PGN (10 Pg/ml) for various time periods and the mRNA and protein levels of TLR2 were then analyzed by real-time PCR (6 h, B) and Western blot (0, 2 and 8h, C). Note that CD11b and TLR2 expression are increased in response to PGN application. Cells were preincubated with anti-7/5 DQWLERG\ 7/5 $E ȝJ RU FRQWURO ,J* IRU PLQ
and WKHQ VWLPXODWHG ZLWK ȝJPO 3*1 IRU K DQG VXEMHFWHG WR UHDO
time-PCR for iNOS (D) or COX-2 (E) BV-2 microglia were pretreated with MyD88 inhobitory peptide (50 and 100PM) and then stimulated ZLWK3*1 ȝJPO IRUK Results are representative of at least three independent experiments. *, p<0.05 as compared with control group. #, p<0.05 as compared with the PGN treatment groups.
Fig. 3-5 PI3K/AKT pathway involves in PGN-induced iNOS and COX-2 production in BV-2 cells. BV-2 microglia were pretreated with
$.7 LQKLELWRU ȝ0 /< ȝ0 RU ZRUWPDQQLQ ȝ0 IRU
30 min and then stimulated with PGN (10 ȝJPO IRU K :KROH FHOO
lysis protein were subjected to Western blot for iNOS (A) and COX-2 (B).
BV- PLFURJOLD ZHUH LQFXEDWHG ZLWK 3*1 ȝJPO IRU WKH LQGLFDWHG
time periods, and cell lysates were separated by SDS-PAGE and immunoblotted with anti-phosphoPI3K (p85) (C) or anti-phosphoAkt antibody (D). Note that PGN-induced iNOS and COX-2 expression are significantly antagonized by AKT inhibitor, LY294002 or wortmannin, and PGN induces PI3K (p85) or AKT phosphorylation in a time-dependent manner. (E) BV-2 microglia were pretreated with MyD88 inhobitory peptide (50 and 100PM) and then stimulated with PGN (10 ȝJPO IRU 1 h , and cell lysates were separated by SDS-PAGE and
Fig. 3-6 NF-NB is involved in the potentiation of iNOS and COX-2 production by PGN. BV- FHOOV ZHUH SUHWUHDWHG ZLWK 3'7& ȝ0
73&. ȝ0 RU%D\- ȝ0 IROORZHGE\VWLPXODWLRQZLWK3*1
ȝJPO IRUK:KROHFHOOO\VLVSURWHLQZHUHVXEMHFWHGWR:HVWHUQ
blot for iNOS (A) and COX-2 (B). (C) Cells were co-transfected with ț%-OXFLIHUDVH H[SUHVVLRQ SODVPLG DQG SĮ $.7 IKKD, IKKE kinase mutant or empty vector for 24 h. Luciferase activity was then assayed after PGN stimulated for another 24 h. Results are expressed as the mean
± S.E.M. of four independent experiments. *, p<0.05 as compared with control group. #, p<0.05 as compared with the PGN treatment groups.
Fig. 3-7 PGN induces IKKĮ/ȕ activation, IțBĮ phosphorylation, IțBĮdegradation and p65 Ser536 phosphorylation in BV-2 microglia.
BV-2 ceOOV ZHUH LQFXEDWHG ZLWK 3*1 ȝJPO IRU LQGLFDWHG WLPH
periods, and cell lysates were separated by SDS-PAGE and immunoblotted with anti-SKRVSKR,..Įȕ (A)SKRVSKR,ț%Į (C) ,ț%Į (E), and phospho-p65 Ser536 antibodies (G). Note that PGN induces,..Įȕ
phosSKRU\ODWLRQ ,ț%Į SKRVSKRU\ODWLRQ ,ț%Į GHJUDGDWLRQ DQG S
6HUSKRVSKRU\ODWLRQ &HOOV ZHUH SUHWUHDWHG ZLWK /< ȝ0 RUZRUWPDQQLQ ȝ0 IRUPLQIROORZHGE\VWLPXODWLRQZLWK3*1IRU
60 min, and cell lysates were then evaluated using immunoblotting with antibody specific for phospho-,..Įȕ % SKRVSKR,ț%Į ' DQG ,ț%Į
(F). Results are the representative of at least three independent experiments.
Fig. 3-8 Schematic diagram of the signaling pathways involved in PGN-induced inflammatory mediators expression in microglia. PGN binds with TLR2 receptor/MyD88 and activates PI3 kinase/Akt pathway, ZKLFK LQ WXUQ LQGXFHV ,..Įȕ SKRVSKRU\ODWLRQ ,ț%Į SKRVSKRU\ODWLRQ
ಃѤക Peptidoglycan ӧ microglia ᇨว IL-6 ߄ޑᐒᙯϐ
4-1 PGNڈᐟ microglia ౢғ IL-6 ޑೈқک mRNA ޑ߄
ӧךॺӃޑࣴزวǴPGN ߦ٬ iNOS ک COX-2 ߄ቚуǴ ԜѦΨቚу IL-1EǵTNF-Dک IL-6 ޑ߄ǹԖࣴزൔᏤࡰр IL-6 ޑ
ౢғߦޔဉᕎޑวғ(Becker et al. 2005; Li et al. 2009)ǴԶ glia cells܌ౢғޑ IL-6 ্ઓಒझǴԶቹៜځӸࢲ(Li et al. 2009)Ǵ ӧۦߎහМੱੰΓတϣεໆޑ߄ IL-6 (Palhagen et al. ; Shen et al.
2005)ǹฅԶჹܭॠើМ܄ಒझᏛԋҽ PGN ቹៜ IL-6 ޑૻ৲ሀ ၡ৩ӧ microglia ޑբҔҞ٠όΑှǶךॺགྷޕၰ PGN ڈᐟ microglia ܌ౢғ IL-6 ޑሀၡ৩ϷځЇଆϐઓวݹϐᐒᙯǶ२Ӄ PGN (10 Pg/ml)ڈᐟ microglia ౢғ IL-6 ޑ mRNA ߄کೈқញܫޑ ໆᒿೀ PGN ޑਔ໔ቚуԶቚуǹӕਔ microglia ౢғޑ IL-6 ޑ mRNAᒿ PGN բҔޑᐚࡋቚуԶቚу(Fig. 4-1A&B&C)ǶԜѦךॺ Ψ٬Ҕ߃ભ༾ጤಒझᎦӑ೭ঁຝǶPGN ڈᐟ primary cultured microgliaΨቚу IL-6 ޑ mRNA ޑ߄ǴЪԜቚуޑຝុډ 24 λ ਔ ࡕ (Fig. 4-1D) Ƕ ࢂ ց PGN ቚ у IL-6 ޑ ߄ Ԗ LPS contaminationޑቹៜǴךॺ٬Ҕ LPS ޑڋᏊ polymyxin Bǹךॺޑ
่݀ว polymyxin B (1PM)ёаֹӄڋ LPS (300 ng/ml)ᇨᏤ IL-6
ғ IL-6 ޑೈқԋቹៜ(Fig. 4-2A)Ƕ
4-2 PGNၸ TLR2 receptor ቚу IL-6 ೈқޑ߄
ೈ қ ޑ ғ ԋ ࢂ җ ᙯ (translation) ౢ ԋ Ǵ ٬ Ҕ ᙯ ڋ Ꮚ cyclohexamideёаԖਏڋ PGN ڈᐟ microglia ౢғ IL-6 ೈқ (Fig.
4-2B) Ƕ Toll ኬ ڙ ᡏ (Toll-like receptors, TLR) ឦ ܭ ၠ ጢ ೈ қ (transmembrane protein)ǴڀԖձߟΕᡏϣޑ༾ғނԶᐟࢲխࣝಒ झޑբҔǴӧӃϺ܄խࣝسύתᄽᜢᗖفՅ(Takeda and Akira 2004)Ƕόӕᅿᜪޑಒझύ PGN ૈၸ TLR2 receptor ࢲϯૻ৲ሀ ၡ৩ǴԶ LPS Ьाࢂၸ TLR4 receptor ࢲϯૻ৲ሀၡ৩(Akira and Hemmi 2003; Yang et al. 2007)ǴӧךॺӃޑࣴزჴ PGN ၸ TLR2ቚу iNOS ک COX-2 ޑೈқک mRNA ޑ߄(Lin et al.)ǶTLR2 ޑ mRNA ک TLR2 ೈқᒿೀ PGN ޑਔ໔ቚуԶቚуǴՠࢂ TLR4
߾ؒԖᒿ PGN բҔਔ໔ԶԖܴᡉᡂϯޑຝ(Fig. 4-3A&B)ǴԜѦ
٬Ҕ TLR2 ک TLR4 ޑύک܄לᡏᢀჸځჹ PGN ڈᐟ microglia ౢғ
IL-6 ೈқޑቹៜǹӧуΕ TLR2 ύک܄לᡏёа෧Ͽ PGN ڈᐟ
microglia ౢғ IL-6 ೈқǴӕਔךॺஒಒझᙯ TLR2 siRNA ӆԛዴ ᇡԜຝǴӧಒझᙯ TLR2 siRNA 24 ࡕԖਏޑڋ PGN ڈᐟ
4-3 PGNፓ IL-6 ޑ߄ࢂၸ JNK/c-Jun ሀૻ৲
ӭࣴزൔᡉҢǴIL-6 ӧፓխࣝфૈ໔תᄽख़ाޑفՅ (Oleksowicz and Dutcher 1994; Williams et al. 1998; Knupfer and Preiss 2007)ǴٯӵѮᏘಒझޑࢲϯǵB ಒझޑวػǵวݹϸᔈǵՈфૈ
ک࡚܄ϸᔈǶԶ IL-6 ЬाࢂҗѮᏘಒझǵT ಒझǵB ಒझǵᠼᆢ҆ಒ झ«܌ϩݜ(Pulliam et al. 1995)ǹӧךॺޑჴᡍύǴPGN ڈᐟ microglia ౢғ IL-6ǴௗΠٰךॺाૻ৲ሀၡ৩ޑፓᐒᙯǹ Mitogen-activated protein kinase (MAPK) ࣁಒझૻ৲ሀޑख़ाၡ ৩ǴᆶፓಒझғߏǵϩϯکԝΫख़ाфૈԖᜢǴЬाёаϩࣁΟ ᜪǴextracellular signal-regulated kinase (ERK)ǵp38 mitogen-activated protein kinases (p38 MAPK)Ϸ c-Jun N-terminal kinase (JNK)ǴϐԖ
ࣴزൔᏤࡰр MAPK ୖᆶ PGN ᇨᏤ proinflammatory cytokines ޑ
ౢғ(Kumar et al. 2004; Wang et al. 2005; Cheon et al. 2008)ǴӢԜ٬Ҕ ERKڋᏊ(PD98059)ǵP38 ڋᏊ(SB203580)Ϸ JNK ڋ(SP600125) ᢀჸځჹ PGN ڈᐟ microglia ౢғ IL-6 ޑቹៜǶMAPK ڋᏊ
ڋ PGN ڈᐟ microglia ౢғ IL-6 ೈқޑ߄Ǵځύа JNK ڋᏊ ޑբҔനࣁܴᡉ(Fig. 4-4A)ǶJNK ڋ(SP600125)ёаԖਏڋ IL-6
ೈқک mRNA ޑ߄(Fig. 4-4D&E) ฅԶ PGN ჹܭ JNK ૻ৲৩ӧ
ғ IL-6 ޑ߄ӧ JNK ૻ৲ፓǶPGN ࢲϯ JNK ک c-Jun ᒿ PGN բҔਔ໔ޑቚуԶቚуǴ٠Ъவ 10 ϩដ໒ۈёаុډ 120ϩដǶԜѦӧ primary cultured microglia Ψӑ PGN ࢲϯ JNK ک c-Junޑຝ(Fig. 4-4B)ǶךॺΨ٬Ҕ JNK assay kit ٰෳໆ JNK/SAPK (c-Jun N-terminal kinases/stress-activated protein kinases)ޑࢲ܄Ƕᒿ
ೀ PGN ޑਔ໔ቚуёаቚу JNK ჹܭ c-Jun ᕗለϯޑբҔ(Fig.
4-4C)ǴӢԜ PGN ڈᐟ(c-Jun N-terminal kinases)ᕗለϯǴԶ٬ JNK ࢲ܄ቚуǴԶᕗለϯΠෞ c-JunǶᆕӝа่݀ǴPGN ڈᐟ microglia
ౢғ IL-6 ޑ߄ࢂၸࢲϯ JNK/c-Jun ޑૻ৲ሀၡ৩Ƕ