腸管上皮細胞におけるヘパラン硫酸の硫酸化構造と機能に関する研究

Title

腸管上皮細胞におけるヘパラン硫酸の硫酸化構造と機能に

_{関する研究( 本文(Fulltext) )}

Author(s)

西田, 光貴

Report No.(Doctoral

Degree)

博士(農学) 甲第624号

Issue Date

2014-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/49104

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HS J 2 ̮̑^Ω5ɜγ*Ǐǯ3`_EЏƃɝG HS ˤψŵΞ˰χ̗H\^ 2 ̑ JʰƗJÞ̯*ˤψĬ3`_U8ĕXHЏN-ˆf~„§¯/N-ˤψŵΞ˰χ̗ ЌNDSTЍH\^ЏGlcNAc J N f~„©ŵ*̾f~„©Ĭ3`ЏŎȰH N ˤψĬ cń/ЏGlcNS *Ǐǯ3`_ɥ%DЏC 5 k•¢§¯* GlcNS HϨȌ7_ GlcA cIdoA QƁȑ7_;JǕЏHS 2-O-ˤψŵΞ˰χ̗Ќ2-OST-1Ѝ* GlcA/IdoA J C 2 ÞcˤψĬ7_ɥ%DЏHS 6-O-ˤψŵΞ˰χ̗Ќ6-OSTsЍ* GlcNAc J C 6 ÞcˤψĬ5CЏ˱HHS 3-O-ˤψŵΞ˰χ̗Ќ3-OSTsЍ* GlcNAc J C 3 Þcˤ ψĬ7_ЌVarki A, et al. 2008Ѝ1`]Jχ̗KЏ2-OST-1 E C 5 k•¢§¯cϡ

et al., 2001Ѝä'LЏŸi|K 6-OSTs J 3 AJfh€–l¯¡D$_ 6-OST-1, 6-OST-2, 6-OST-3 cːʷ7_1E*˞]`C%_ЌHabuchi H, et al., 1995; Habuchi H, et al., 1998; Habuchi H, et al., 2000; Habuchi H, et al., 2003ЍˤψŵΞ˰χ̗KЏ… rfi‰dzɿcʿ%_1EH\^Џɝ Gʽʹɣ̼HǞϷJǑģcǼ&1E*͵Ȯ

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ʎōʯc30%ʏĦ5C¸'=ŸōЏ̓ĉ̙͐JȺʦH))b]8Ƨ̓DJɇ̙̻J

ŽĦEƧ̓ŀNΨÞ̝̓H(%Cϣ˽JĒĬŽɬ*΂X]`Џ3]Hʦ͐§…‰J Ƨ̓HC̓ĉĒɾ̙̻Ƞ*߶5=ЌMcCullogh J S, et al., 1998ЍŒ¬‰¨H(% CYЏ›r„­ȕŃH\_Ƨ̓µ˓JǏǫƁĬ*ŷŖ3`=ЌLanghout D J, et al., 1999Ѝ1`]Jˡ˹KЏ›r„­*Ƨ̓H˗Ȍâʿ5CЏϣ˽J̙̻ŽɬcÉζ 5=̝ɍЏ̞ɱJŽɬ*êζ3`=Ň̼ǡc˨7E̴'=5)5G*]Џ›r„ ­GFJϼʯ̫̣KЏ¼Hƅ̓HƑū7_̓ĉ̙͐Jːχâʿcń/Cʽʹâʿc ːȒ7_E̴']`C%_=XЌBrown L, et al., 1999ЍЏ›r„­*Ƨ̓µ˓Qâʿ 7_ɣɜK͵Ȯ3`C%G%
›r„­KЏɍʯZό͑ϻGFJɗʯ̙̻žHƃύHƑū7_ƃ̑ϻD$_› r„­KЏɗʯ̛̭ºJɜǯǯĒZºϙƭJ¼ǯĒE5CЏ̛̭Z̙̻ϙHȌ˜Ĥ EƔƗǡcY=]5C%_ЌLunn J, et al., 2007Ѝ›r„­K˗ϔʱ(\NĒƯʱ Jɜγ)]ɜǯ3`_›r„­J¼ͯGɜγKЏα-D-o§r†«­ψĒƐ*α-1, 4-s¨vzŠ̝ō5=ϔʱɜγJœ£o§r†«‹­ЌHGЍD$^Џ›r„­J ̓65%H˘ǎ7_ЌMohnen D, 2008ЍU=Џ›r„­J̓10%K§¡o§r†« ‹­-IIЌRG-IIЍD̛ǯ3`Џ20-35%K§¡o§r†«‹­-IЌRG-IЍ)]ǯ_ ЌLeclere L, et al., 2013ЍRG-IIKƨG-EY²AJ1, 4-̝ō5=α-D-GalAɫŵ)]

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ŀNRG-II*¼ϔcÏ5C̬*@=ɜγcE_E̴']`C%_ЌMohnen D, 2008Ѝ ›r„­JõϔHKЏf§”¯|Џpz«¯|Џo§r‰¯|Џ–v¯|ЏŸ­ ¯|(\Ns©v¯|E%@=ºǡ̑*Ƒū7_ЌĹĵ]ЏЍ›r„­KЏ õϔ*ƟHĒƼ5=ɱʱϸŲЌhairy regionЍEõϔĒƼJƨG%ǀʚϸŲЌsmooth regionЍ)]ǯ_Ќ˚σЏЍƆʨHƑū7_›r„­KЏo§r†«­ψJn ©pzŵ*σĒ˒H¢„©k|‡©Ĭ3`C%_k|‡©ĬLJK¢‰pz©ŵ ЌOCH3ЍJŒȺύH\@Cͤ3`Џ¢‰pz©ŵ*7%Ќk|‡©ĬLJО42.9%Ѝ

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›r„­JȕŃKЏʋĬ̊Jɣ̼QŌ˳JʽʹĩɍcŀT7›r„­Jʽʹĩ ɍKЏ1) ʋĬ̊EJʯʹ˒®ĬƓ˒˘ÆâʿcÏ5CJʋĬ̊ɣ̼JΈ̋Џ2) Ñ JϼʯǯĒEJ˘ÆâʿЏ3) ʋĬ̊́ĉJĉσʻżЌpHЏ̓ĉ̙͐Jύ®ΕGFЍ JΈ̋Џ4) ›r„­*̓ĉ̙͐Jâʿcń/Cʽǯ3`_˟ϔ̷̽ψJK=]+ cÏ5C˨3`_ЌΥ]Џ Ѝ›r„­K̓ĉ̙͐H\@C90~100%Ē͵3` _=XЏʋĬ̊µσH(%CKĞ̵ÅAJâʿH\^ʽʹĩɍc˨5ЏʋĬ̊¶σ H(%CKǕ̵ÅAJâʿH\^ʽʹĩɍc˨7ЌΥ]Џ Ѝ1J\&Gʽʹ ĩɍH\^Џ›r„­Kvª|‡«¯©ß¶GFJĩ̼cȂAÑHЏϼʯfª©q ¯JǶĚâʿZǸ͂ˍʅǡЏ¶ˌZ̹̓JʣˋHƣ7_ĩɍY˨7ЌTerpstra A H, et al., 1998; Tazawa K, et al., 1999; Lee J C, et al., 2004; Olano-Martin E, et al., 2002; Ohkami H, et al., 1995; ƚƳЏ1990Ѝ

Ɂˡ˹DKЏɍʯJ˜©¯­)]̐ͫ5=›r„­cãʿ5=Ĉà˒HKЏ´ ŵŠÄЌɑЍ\^Ȑå3`=˜©¯­kp|Hk‚¯©cå5CЏf©v¯©· ʘǡ˃ĒЌAISЍcǻđ5=AISHK‚­’rΕ*̓50%ŒU`_=XЏAIScHCO3

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ǎˡ˹ƙDKЏ›r„­*Ƨ̞̓ɱJǏǫƁĬc΃Ʀ7_ɣɜc͵Ȯ7_=XHЏ in vitro“‰Ƨ̓µ˓£ˆ©D$_Caco-2̙̻H›r„­*¸'_ǐϴcˡ˹5C +=;J̝ɍЏĒĬCaco-2̙̻H›r„­cʏĦ7_EЏϵ̀Ќf•n©ЍõQ J‚­’rΕĒɾύ*ŽĦ7_1EZЌºɃЏ2009ЍЏ̙̻ĉJÓΊ*ƁĬ7_1 EЌºɃЏ2009; ˠƵЏ2011ЍЏ̙̻ƂŸ‰¨r|ɜǯ‚­’rΕD$_–g—« Žr„­JmRNAːʷύJÉζcːͰ5=ЌɁˀЏ2011ЍU=Џ›r„­K–g —«Žr„­JIII1CϸŲЌFNIII1CЍE̝ō7_1EEЏ–g—«Žr„­KĒĬ

Caco-2̙̻Jf•n©õHːʷ7_1E*Ͱđ3`=ЌɁˀЏ2011Ѝ3]HЏĒ ĬCaco-2̙̻ͤϲHSJɜγ*›r„­ʏĦH\^ƁĬ7_1EY˨ş3`C%_ ЌɃˀЏ2011Ѝ1`]J˞Ͱ)]Џ›r„­K̙̻ƂŸ‰¨r|J–g—«Žr „­E̝ō7_1EH\^ĒĬCaco-2̙̻Hěʝc¸'Џ̙̻ͤϲHSHǐϴcŀT 7EJÕΆc˾C=;1DɁˡ˹KЏĒĬCaco-2̙̻ͤϲJHSQ›r„­*¸' _ǐϴc͵Ȯ7_1EH\^Џ̓̊µ˓̙̻H(/_HSJɣ̼c͵Ȯ7_1Ec ˖˒E5=
;J=XHKЏ̙̻ͤϲHS*›r„­JěʝHƣ5CFJ\&HŁǠ7_)c ͵Ȯ7_Ǟͯ*$_*Џ̙̻ƂJěʝHƣ7_HSJǠ̆ɝLjKȿ͵ȮD$_1 `UDHЏHSJˤψĬɜγc΋ė5C̝ō7_FGF-1EFGF-2J˫̜̙̻ͤϲHSH ƣ7_̝ōǡ*Џ̿JːλȰȽHǠ6CƁĬ5=EJŷŖ*$_1E)] ЌNurcombe V, et al., 1993ЍЏ̙̻ͤϲHSK̙̻ƂJěʝHǠ̆5CˤψĬɜγcƁ Ĭ39_JDKG%)E̴'=3]HЏǎˡ˹ƙDKЏŸi|̨̣̙̻͌ͤϲ HSJˤψĬɜγ*fŠª‹¨­ěʝH\^ƁĬ7_1Ec˨ş7_̝ɍcǘ=1 E)]ЌͮˀЏ2005; ɶͰЏ2009ЍЏɁˡ˹DKЏU8ĕXHЏŸi|̨̣̙̻͌ cƘЂ£ˆ©H5CЏ̙̻ͤϲHSJ̙̻ƂěʝQJǠ̆ɣɜJ͵ȮHŃ^̛d> ;J˞ͰcŵH5CЏĒĬCaco-2̙̻ͤϲHSJɣ̼c͵Ȯ7_=XHЏĒĬCaco-2 ̙σͤϲHSQ›r„­*¸'_ǐϴcĒɋ5=

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̙̻ͤϲHS KЏ̙̻ƂʻżJƁĬHǠ̆5CˤψĬɜγcƁĬ39_1

E*˨ş3`=*ЌNurcombe V, et al., 1993ЍЏ1`UDJE1aȮ˥GͼȮ KG3`C%G%ǎˡ˹ƙDKЏL-M ̙̻ͤϲJ HS ɜγ*fŠª‹¨­ ěʝH\^ƁĬ7_1Ec˨ş7_˞ͰcǘC%_ЌͮˀЏ2005; ɶͰЏ 2009Ѝ°͊˒G̙̻ŴϿHK͟ʐcǞͯE7_*Џ͟ʐºJǯϖŦƐK̙ ̻ͤϲHS QǐϴcŀT71E*̴']`_5)5G*]ЏL-M ̙̻KŴ ϿH͟ʐcǞͯE5G%=XЌHiguchi K, 1970ЍЏŴŭHǷ¸7_ʯΕ* HS H¸'_ǐϴcȮ˥HͽçD+_;1DɁ˿DKЏfŠª‹¨­H*L-M ̙̻ͤϲHS H¸'_ǐϴcĒɋ5CЏ̙̻ͤϲ HS *̙̻Ƃ)]JěʝH Ǡ̆7_ɣɜc͵Ȯ7_1Ec˖˒E5= p£r Jœ\Y JœZb £-¢ Jœ\Y Ќ1ЍŸi|̨̣̙̻͌

Mus musculus (mouse)J˓¶̝ơ̭̄ˁɆ̨̣̙̻͌cɑĬŴϿ5= L-M ̙

̻cAmerican Type Culture Collection (ATCC)\^Ζă5=

Ќ2ЍŴŭ

Κ̔ɴ180 mL H‘r‰›˜‰­ЌBDЍ20 mg cĦ'Cm¯‰rª¯—5Џ 10%‘r‰›˜‰­cΈͫ5=Medium 199ЌLife TechnologiesЍ195 mL H 10% ‘r‰›˜‰­ 5 mL cʎō5CЏL-M ̙̻ŴϿʿJŴŭE5=

Ќ3ЍTE buffer, pH 8.0

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£-£ HS t… 2-2-1.
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υˠψɴ̗ L-fŠª‹¨­Ќ‹n§h‡|rЍcʙ͐ɴHʘ͵5CЏ50 mM J|‰…rc20HCìƑ5=̎ɀʱJfŠª‹¨­K 4HC 1 ǁϙČ͗ ìƑ7_EħĬ5==XЏǞͯύcĎ̝ìƑ5ЏĎ̝͞͵c̮^Ω3G%\& H5= (2) fŠª‹¨­ŒȺŴŭ
50 mM fŠª‹¨­ʘʌcŴŭDƽω5CЏ0.01, 0.05, 0.10, 0.20 mM fŠª ‹¨­ŒȺŴŭcΈͫ5= (3) PBS
KH2PO4 1 g, NaCl 40 g, Na2HPO4®12H2O 14.5 g, KCl 1 g cΚ̔ɴHʘ͵5Џ500 mL HƗƝ5C 10PBS cΈͫ5=1`c 10 ðƽω7_1EDЏ1PBS c Έͫ5= (4) –j¯©®r««œ©¡ʘʌ
–j¯©Er««œ©¡c1М1 Dʎō5Џ4HCìƑ5= (5) 3 M φψ‹‰¨i¡
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L-M ̙̻c 10.5105 _{cell/10 mL EG_\&H 75 cm}2_{–§|vHȖ˳5CЏ} 37Џ5% CO2Ʌ׶D3 ȪϙŴϿ5= 2-2-3.
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37Hìʒ5= PBS D̙̻cʄʇ5CЏfŠª‹¨­ŒȺŴŭ 10 mL cʏ Ħ5Џ37Џ5% CO2Ʌ׶D3 ȰϙU=K 6 ȰϙŴϿ5=U=Џv­‰«

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KShowrak N W ]JȦɿcľ̴H5=ЌShworak N W, et al., 1996Ѝ

2-2-4.
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2-2-3Dťł5=µʐ)]‚­’rΕ̃cŃ^ϡ-=XHЏ–j¯©®r« «œ©¡ǻđc͠@=‚­’rΕcɜǯ7_f ψHKͲɴǡˉɴǡJ ¹˳JYJ*Ƒū7_*ЏɴʘʌºJ‚­’rΕĒƐDKf ψJˉɴŵc ĒƐJĉõHЏͲɴŵcɴHȌ7_ƂõHŐ/CƔƗĬ5C%_11H‚­ ’rΕƁǡâʿcȂA–j¯©câʿ39_EЏ˾àɜγ*ſ`Cˉɴŵ* –j¯©H‰§…˜3`ЏͲɴŵ*ɴƭH˅Ua&E7_;J̝ɍЏƁǡ 5=‚­’rΕĒƐKμǝĒϬH\@CɴƭE–j¯©ƭJ˄ϲHºϙƭ E5CĒϬ3`_1EEG_;1DЏ1.5 mL„¥¯—Hx­˜©ʘʌc750 µLÔ¶HG_\&Ēʂ5Џx­˜©ʘʌẼύJ–j¯©®r««œ©¡ʘ ʌcĦ'C©‡…r| px¯DȘǽ5=μǝĒϬ(ƙʒЏ2000gЏ10Ē) c͠%Џ‚­’rΕE–j¯©ƭcťł5G%\&HµƭJVcǬϋHťł 5=;JǕЏ–j¯©ƭJă@=„¥¯—Hʙ͐ɴ200 µLcĦ'CĊN ©‡…r| px¯DȚǽ5ЏμǝĒϬЌƙʒЏ2000gЏ10ĒЍc͠%Џµ ƭcťł5= 2-2-5.
f©v¯©ɹɮ
GAGKɚǡcȂAĒƐD$^ЏɴHʘ͵7_;JʘʌHƣ5Џɚǡ*Ƨ3 -GAGcʘ͵39G%f©v¯©cĦ'_1EDЏ7DHʘ/C%=GAG* ɋđ7_5)5GAGKˤψHˁɆ7_ΏJϮ͏cȂA=XÆ%HŁː5$% ɹɮ*D+H-%;1DЏφψ‹‰¨i¡GFcĦ'CϮ͏cºś5=ǕH f©v¯©cĦ'_1EDGAG*ɋđ5Џ1`cμǝĒϬ7_1EDɹɮ*ǘ ]`_ťł5=x­˜©ʘʌHƣ5CЏ̚ʟLJ0.2 MÔµEG_\&H3 M φ ψ‹‰¨i¡ʘʌcĦ'=x­˜©ʘʌЎφψ‹‰¨i¡ʘʌύHƣ5CЏ

-20DìƑ5=2-˜«’¯©c̃ύĦ'Џ„¥¯—cµ¶ŁΞ39CȘǽ5 =1`cμǝĒϬЌ4Џ15000gЏ60ĒЍ5Џɹɮ*ʈ%C-_ĞH̗ȫ -µʐcϡĽ5=

2-2-6.
v­Š«h„­ˤψЌCSЍJĒ͵

ťł5=GAG HŒU`_ CS cĒ͵7_1EH\^ЏHS J̐ͫc͠@= v­Š«h„‹¯ABCЌProteus vulgaris, EC 4.2.2.4ЏʽĬƓƷəЍKЏv­ Š«h„­ˤψЏˆ©Ÿ‚­ˤψЏ“f©«­ψJN-f~„©™p€x ŒŠ ̝ōc̾ϬŁǠ˒HēȤ5Џϱρýɀ̀H4-™p|«­ψɫŵcȂAÅ̑c ʽǯ7_t§‚­ˤψЏ™’¨­ЏHS HKâʿ5G%=XЏĩʵ˒H GAG ĉJv­Š«h„­ˤψ̑ϔcĒ͵D+_HS EćƑ7_v­Š«h„­ˤ ψЌCSЍ̑ϔcĒ͵7_=XЏ2-2-5 Dťł5=µʐcʿ%CЏTable 1 J\& HΈͫ5= Table 1 CS Ē͵JŁǠ̛ǯ
37D°ȲŁǠ39Џ;JǕ 2-2-4 E 2-2-5 JȦɿHǗ%Џ–j¯©®r ««œ©¡ǻđЏ˜«’¯©ɹɮc͠%Џχ̗‚­’rΕEĒ͵5=CS c ϡĽ5= £-¤ HS •dI
HScŒWGAGJƗύHKЏHPLCZn©‘¯©ˤψɿЏf©zf­—©¯ ɿ*ʿ%]`_HPLCcʿ%_EŻZDNAʎăJǐϴcń/8HGAGcƗύ 7_1E*D+Џn©‘¯©ˤψɿKŻJǐϴcń/_1EЏf©zf­— ©¯ɿDKˤψĬGAGJVcƗύD+_1E*ŷŖ3`C%_ЌFrazier, et al.,

2008Ѝ;1DɁˡ˹DKЏˤψĬGAGD$_HScƗύ7_=XHf©zf­ —©¯ɿcȊʿ5=
f©zf­—©¯K–‚«zfŒ­̗͋J°˳DЏϐļƐcŒWˉɴǡJv fcȂA4çJn„m­D$^Џ|©œ¡„­Zzf«¡„­Џψǡ¡vƃ̑ ϻcʰˈ˒HɏX_̛̭ɏ͋ɿE5CƦă3`=ÑJɏ͋ȦɿEɰΟ5C̍ èDɏ͋ȰϙY˟%̗͋*̝ō5=σÞKϯ͚͋HЇZ)Hɏ͋3`_ GAGHϚ5CKЏĄCJn©pz©ŵcºś7_HΛ_ßpHH(%CЏf ©zf­—©¯KˤψĬGAGEʰˈ˒H̝ō7_f©zf­—©¯ɿKЏ Zhang]Jdz϶cľ̴H5=ЌZhang, et al., 2001Ѝ 2-3-1.
;͙JΈͫ Ќ1Ѝ8 MsfŒ{­Żψ
sfŒ{­Żψ 76.4 gcĦʫ5CΚ̔ɴHʘ͵5Џ100 mLHƗƝ5= Ќ2ЍDye|‰…r

Alcian Blue 8GXЌFlukaЍ̓1 gcύ^Ń^Џ18 Mˤψ 20 µLE8 MsfŒ{­ Żψ 1 mLcĦ'ЏΚ̔ɴD20 mLHƗƝ5=;`c50 mLμǝ„¥¯—Hă `CμǝĒϬЌƙʒЏ3000rpmЏ1ĒЍ5CЏµʐcťł5=Κ̔ɴD100 ðH5=ϧHЏ600 nmJœĀLJ*1.4Ô¶D$_1Ec˥΂5= Ќ3ЍReagent 1
10% TritonX-100 750 µLH18 Mˤψ 20 µLcʏĦ5CЏΚ̔ɴD9.22 mLHƗ Ɲ5= Ќ4ЍReagent A
8 MsfŒ{­ŻψEReagent 1c1М1Dʎō5= Ќ5ЍWorking dye

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Ќ2ЍProteinase K
Ď̝Áʬ̎ɀЌ‹n§h‡|rЍc20 mg/mL EG_\&Hʙ͐ɴHʘ͵3 9Џ1`c|‰…rʘʌE5Џ-20DìƑ5= 2-4-2.
DNA ύʓƗɿ
2-2-3 Dťł5=̙̻Ǯʞʌc-20)]Ń^đ5Џ͵Ď5=Č PBS 500 mL cĦ'CǮʞ39ЏμǝĒϬЌ4Џ5000gЏ10 ĒЍc͠%Џ̙̻cɹɮ39 =µʐcϡĽ5Џɹɮ5=̙̻Hƣ5CDNA ǻ𑅖e¯ЌSDS-Ѝ1 mL cĦ'C̙̻cǮʞ39=;5CЏ10% SDS c 10 µL E 10 mg/mL ¨r ªf¯AЌ‹n§h‡|rЍc 1 µL Ħ'Џ37D 1 Ȱϙh­p¥š¯‰5 =ɥHЏ20 mg/mL Proteinase K c 5 µL Ħ'CЏ[@-^µ¶ŁΞ5C̩Z )Hʎō5Џ50D°Ȳh­p¥š¯‰5=̃ƝJ PCIЌ–j¯©®r« «œ©¡®h€f ©f©v¯©ЍЌ‹n§h‡|rЍcĦ'CЏ30 ˯ϙ© ‡…r|5=;JǕЏμǝĒϬЌƙʒЏ2000gЏ10 ĒϙЍc͠%Џÿ̀c ēȤ5CŅǒcƅ+-5=„…˜cʿ%Cµƭcťł5Џȥ5%„¥¯—H˰ 5=¶ƭHƣ5CTE buffer, pH 8.0 c 200 µL Ħ'Cʎō5ЏμǝĒϬЌƙʒЏ 2000gЏ10 ĒϙЍ5Cǘ]`=µʐcÿHťł5=µʐEʎō5=µʐ 600 µL Hƣ5CЏ3 M φψ‹‰¨i¡ 60 µLЏČ 2-˜«’¯© 660 µL cĦ'C

]`=ɹɮH70% k‚¯© 100 µL cĦ'=;`cʎō5=ǕHЏμǝĒ ϬЌ4Џ16000gЏ10 ĒϙЍc͠%ЏµʐcϡĽ5C)]p¡¬h˜µH„ ¥¯—cή3UH5C̯+Џ͇ʨÁʬ39=TE buffer, pH 8.0 c 300 µL Ħ' C©‡…r|5Џ 4D°Ȳț̯5=260 nm (\N 280 nm H(/_œĀ LJcʓƗ5ЏOD ɰЌA260 /A280Ѝ)]DNA J̔LJcʓƗ5=U= DNA K 1 A260

= 50 µg/mL E%&ʰǜcȂA1EH\^Џǘ]`=œĀLJ)] DNA ʟLJcʓ Ɨ5ЏDNA ύc̉đ5= £-¦ HS 5uzV;] 2-5-1.
Å̑|‚­ƒ¯Š
Ô¶H˨7Å̑|‚­ƒ¯ŠcʽĬƓƷə\^ădz5CЏHPLC J|‚­ƒ ¯ŠE5Cãʿ5= ΔDi-0S М 2-acetoamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-enopyranosyluronic acid)-D-glucose ΔDi-NS М 2-deoxy-2-sulfamino-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-D-glucose ΔDi-6S М 2-acetoamido-2-deoxy-4-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose ΔDi-diS1 М 2-deoxy-2-sulfamino-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose ΔDi-diS2 М 2-deoxy-2-sulfamino-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid)-D-glucose ΔDi-triS М 2-deoxy-2-sulfamino-(4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid)-6-O-sufo-D-glucose 2-5-2.
HS Ē͵χ̗H\_ HS JĒ͵
2.2-6 Dǘ]`=̐ͫ HS H 3 ˳ϻJ™’§­ˤψĒ͵χ̗ [™’¨‹¯ ЌFlavobacterium heparinum, EC 4.2.2.7ЏʽĬƓƷəЍЏ™’¨„‹¯ЌF. heparinum, EC 4.2.2.8ЏʽĬƓƷəЍЏ™’¨„‹¯ЌF. heparinum, no number

assignedЏʽĬƓƷəЍ]cʿ%CЏHS cÅ̑ĶÞHĒ͵5=χ̗ŁǠc͠ &=Xx­˜©cTable 2 J\&HΈͫ5=

Table 2 Composition of enzyme reaction solution

37D°ȲŁǠ39CЏ2-2-4 JȦɿHǗ%–j¯©®r««œ©¡ǻđ c͠@=ǘ]`=µʐcμǝʟ̪ɣЌCC-105, TOMYЍHCÁũ39CЏ16 mM ¨­ψÅɴ̗‹‰¨i¡ʘʌ 30 µL Hʘ͵39= 2-5-3.
HPLC
̾ɳ5=Κ̔ɴcʆβ1.0 mL/min HCʆΜ D H̓ 1 Ȱϙʆ5=ɥ%DЏ ̾ɳ5=16 mM ¨­ψÅɴ̗‹‰¨i¡ʘʌcʆβ 1.0 mL/min HCʆΜ D H ̓1 Ȱϙʆ5=;JǕЏ̾ɳ5= 538 mM ¨­ψÅɴ̗‹‰¨i¡ʘʌcž ­˜Hʆ5CЏž­˜*ɣ̼7_1Ec˥΂5=16 mM ¨­ψÅɴ̗‹‰¨ i¡ʘʌcʆβ1.0 mL/min HCʆΜHʆ5Cž­˜ĉJ 538 mM ¨­ψÅɴ ̗‹‰¨i¡ʘʌcʆđ39=100 µL z¨­{HE@=Κ̔ɴHCx­˜© ʂăŅcʄʇ5=HS Å̑|‚­ƒ¯ŠU=Kx­˜©c 10 µL z¨­{H E^Џf ˜«•©n§¡ЌYMC-Pack PA-GЏ¬hk¡z¯Ѝc÷'= HPLC H\@CĒɋ5=HS JÅ̛̑ǯĒɋKЏUV ɘđţE5C JASCO UV-2075 PlusЌJASCOЍc÷'= JASCO PU-2089 PlusЌJASCOЍcãʿ5C Table 3 H ˨5=˜«s§¡D͠@=G(Џr«Ÿ‰s§¡Jͺϓ(\N•¯rϲ˷J ̉đHKЏr«Ÿ‰s§¡ʿˆ¯‚Đʹ€–‰ijfD$_Chromato-PROЌ§ ­‚h¡h­|†©¢­†Ѝcʿ%=G(Џ1JȦɿKЏYoshida ]JȦɿ cľ̴H5=ЌYoshida K, et al., 1989Ѝ

Table 3 HPLC condition

£-§ 6-OSTs  mRNA ig•dI

2-6-1.
;͙JΈͫ Ќ1ЍDEPC ɴ
Š§–‰ĉDЏΚ̔ɴ400 mL H DEPCЌ‹n§hЍ400 µL cĦ'ЏįĒH ʎō39=1`c37D 1 Ȱϙh­p¥š¯‰5Џ°Ȳϰ̯5=3]Hm ¯‰rª¯—c2 ť͠@= Ќ2Ѝ70%k‚¯©
99%k‚¯©H DEPC ɴcĦ'CЏ70%Hƽω5= Ќ3Ѝ3M φψ‹‰¨i¡
φψ‹‰¨i¡cΚ̔ɴHʘ͵39Џ0.1%J DEPC cĦ'CЏ2 ťm¯‰r ª¯—5= Ќ4Ѝ1TAE
Tris 121 gЏφψ 28.55 mLЏEDTA®2Na 3.715 g cΚ̔ɴHʘ͵5CЏ500 mL HƗƝ5=1`cm¯‰rª¯—5CЏ50TAE cΈͫ5=1`cΚ̔ɴ D50 ðƽωC 1TAE cΈͫ5= 2-6-2.
̙̻ŴϿ
L-M ̙̻c 6 ˸˜ª¯‰H 1.6105_{ïȖ˳5CЏŴŭc3 mL EG_\&H}

Ħ'Џ37Џ5%CO2Ʌ׶D3 ȪϙŴϿ5=;JǕЏfŠª‹¨­ŒȺŴŭ HÊȑ5CЏ°ƗȰϙŴϿ5= 2-6-3.
RNA ̐ͫ
RNA ̐ͫJƷ˲KЏr¨¯­š­„ĉD͠G@=r¨¯­š­„ĉKЏ $])6XUV §­˜c 30 Ē˲LJʩƥ5ЏRNase QuitЌ‹n§h‡|rЍEk ‚¯©D\-ȁ%=
fŠª‹¨­cʏĦ5C°ƗȰϙŴϿ5=]Џ6 ˸˜ª¯‰cɵµH̯+Џ 10 Ēϙϰ̯5=;JǕЏŴŭcϡĽ5Џ1PBS 1 mL cĦ'Cʄʇ5=PBS DJʄʇK2 ť͠@=PBS cŃ^ϡ%=ǕЏRNAiso PlusЌ‚n§‘hmЍ c1 AJ well Hƣ5C 1.2 mL Ħ'=20 ť˲LJ•›…‡g­s5=ǕH 1.5 mL „¥¯—H˰5CЏƙʒD5 Ēϙϰ̯5=r¨¯­š­„ĉDr««œ©¡ 0.24 mL cĦ'CЏÀˑʱHG_UD\-Ȇ^ʎ:ЏμǝĒϬЌ4Џ12000gЏ 10 ĒЍc͠@=µƭcťł5Cȥ5%„¥¯—H˰5Џ̃ύJ 2-˜«’ ¯©cĦ'C\-ʎō5=ƙʒD 10 Ēϙϰ̯5=ǕHЏμǝĒϬЌ4Џ 12000×gЏ10 ĒЍc͠G@=µʐcŃ^ϡ+Џ70%Čk‚¯© 1 mL cĦ 'Cʄʇ5ЏμǝĒϬЌ4Џ12000×gЏ10 ĒЍc͠@=µʐcϡĽ5=„ ¥¯—cή3UH5CЏp¡¬h˜JµH˾CCƙʒDÁʬ39=;JǕH RNA c DEPC ɴ 43.5 µL Hʘ͵39=;1)] 1.0 µL cėJ 1.5 mL „¥¯ —HŃ^đ5ЏϮɳʃĪHʿ%_DNA ϡĽșâĞJx­˜©E5=ɫ^J

RNA ʘʌ 42.5 µL Hƣ5CЏTable 4 J\&HΈͫ5CЏ37D 30 Ēϙh­p ¥š¯‰5=

Table 4 Composition of DNase reaction

DEPC ɴ 350 mL E PCI 400 mL cĦ'Cʎō5ЏμǝĒϬЌƙʒЏ12000gЏ 5 ĒЍc͠@=µʐ 400 µL cȥ5%„¥¯—Hťł5Џr««œ©¡ 400 µl cĦ'Cʎō5ЏμǝĒϬЌ4Џ12000gЏ10 ĒЍc͠@=µʐ 350 mL

385 µL Ħ'CЏ-20D 1 ȰϙÔµϰ̯5=;JǕЏμǝĒϬЌ4Џ12000 gЏ10 ĒЍc͠%ЏµʐcŃ^ϡ%=Č 70%k‚¯©cĦ'Cʄʇ5Џ μǝĒϬЌ4Џ12000gЏ10 ĒЍc͠@=µʐcϡĽ5=ɹɮH DEPC ɴ c20 µL ˲LJĦ'Cʘ͵5= 2-6-4.
RNA J̔LJʓƗ
2-6-3 D̐ͫ5= RNA 1.0 µL cʿ%CЏ260 nm (\N 280 nm H(/_œĀ LJcʓƗ5=;JOD ɰЌA260/A280Ѝ)]ЏRNA J̔LJcʓƗ5= 2-6-5.
RNA JʟLJʓƗ
̐ͫ5=RNA 1.0 µL cʿ%C 260 nm H(/_œĀLJcʓƗ5=RNA K A260О40 µg/mL E%&ʰǜcȂA1E\^Џǘ]`=œĀLJ)] RNA ʟLJc ʓƗ5= 2-6-6.
RNA JϮɳʃĪ
U8Џ1.5%fo«¯|u©cΈͫ5=fo«¯|ЌϚɈĬƓЍ0.6 g c´ ʹ–§|vHă`Џ1TAE 40 mL cĦ'=´ʹ–§|vJŅJσĒH§… ˜c)/ЏƧ3G˸c 2Р3 ̈DzϘ/CϮƐª­{DĦʫ5=˻ɻ7_ĞH ϮƐª­{cɦXCЏ´ʹ–§|vc\-)+ʎ:C)]Џfo«¯|cƕĄ Hʘ͵39=1JʘʌcνLJHČU5CЏ0.44 mg/mL k„{i¡—«ŸhŠ Ќ‹n§h‡|rЍc1 ʛĦ'Cʎō5=
ϮɳʃĪ̯ͨcʗ÷5Џv¯¡cͻ̯5=ʃĪɞHfo«¯|u©cʆ5Φ VЏu©*ũU_UDϰ̯5=u©*ũU@=]Џv¯¡ců˗Hǹ+Ń@ =1TAE cʃĪɞHʆ5ΦVЏʌJІ3*u©\^Y 1Р2 mm µHG_U Dʂ%>2-6-3 Dťł5= DNA ϡĽĞJ RNA ĿK̐ͫ RNA JŌ 1.0 µL H 6 Loading BufferЌ‚n§‘hmЍ1 µL Eʎō5Џu©Hf˜§h5=Ϯɳ ʃĪKЏƗϮŪ100 VЏ30 ĒJɅ×D͠G@=30 ĒǕЏh¢¯{f‹§hy ¯LAS-3000 UV miniЌƠƀ–g©¡ЍE Multi Gauge €–‰ijfЌƠƀ–g ©¡ЍH\^˃û͵ɋc͠G@=

2-6-7.
cDNA ōǯ

1.5 mL „¥¯—H Table 5 H˨5=α^HŁǠʘʌcΈͫ5=cDNA ōǯ Hãʿ7_Total RNA ύK 1 µg E5==XЏDEPC ɴE total RNA Jō͸ύ*

4.5 µL EG_\&H5=

Table 5 Reaction composition of cDNA synthesis

1`c42D 30 Ēϙh­p¥š¯‰5Џ99D 5 Ēϙh­p¥š¯‰5C ͇ʨČĺ39C)]Џ-20DìƑ5=

2-6-8.
RT-PCR

RT-PCR Hãʿ5=˜§hŸ¯JŻŵτĔc Table 6 H˨7

Table 6 Sequences of primers for RT-PCR

x¯Ÿ©xhr§¯KiCyclerЌBio-RadЍcãʿ5=PCR ŁǠ„¥¯—KЏ 0.2 ml THIN-WALL TUBESЌMJResearchЍcãʿ5=

2-6-9.
PCR ʾʯJϮɳʃĪ

U8Џfo«¯|c1TAE HĦ'CĦʫʘ͵5Џk„{i¡—«ŸhŠc Ħ'C 2.0%fo«¯|u©câͫ5=1`cʃĪɞHă`CЏTAE cʊ5 =ɥHЏPCR ʾʯ 15 µL HЏ6Loading Dye 3 µL cĦ'Cʎō5=ǕЏu ©H18 µL f˜§h5=ĒƐύŸ¯n¯E5C Low MW DNA LadderЌNew

;JǕЏh¢¯{f‹§hy¯LAS-3000 UV miniЌƠƀ–g©¡ЍE Multi Gauge €–‰ijfЌƠƀ–g©¡ЍH\^˃û͵ɋc͠@=

2-6-10.
real time RT-PCR

ɣţKЏ¨f©‚h¡ƗύPCR ABI Step One PlusЌApplied BiosystemsЍcã ʿ5=¨f©‚h¡ RT-PCR ʿJŸhr«„¥¯—KЏFast Reaction Tube With CapЌApplied BiosystemsЍcãʿ5=ʓƗHKЏFAST SYBR Green Master MixЌApplied BiosystemsЍU=K THUNDERBIRD qPCR MixЌToyoboЍcãʿ 5=

¨f©‚h¡RT-PCR J Run method HA%CKЏTable 7 J\&HͻƗ5Џ xhr©ȠK40 cycles E5=U=Џ͝Āˆ¯‚ťłžh­‰KЏextend ȰE 5=ˆ¯‚͵ɋ€–‰KЏStepOne Software v2.2.2 cãʿ5=

πÛƐJːʷύJ̝ɍKЏɰΟ CTɿЌCTɿЍH\_˘ƣƗύD̉đ5

=CTɿEKЏŵʗE5=x­˜©EJCTó(Threshold Cycle)Jƹ)]˘

ƣócɷX_˘ƣƗύɿD$_‚¯u…‰Jócĉūǡv­‰«¯©JóD ͪɧc͠%Џ3]HŵʗH7_x­˜©EJ˘ƣóc̉đ7_1JɰΟ CT ɿcʿ%C˘ƣƗύc͠G&=XHKЏPCR Žƿĩʵ*ST 100%D$_E% &Ʌ×cʕ=5C%_Ǟͯ*$_;J=XHKЏRT-PCR H\^Џ˖˒Jπ ÛƐ*Ķ°HŽƿ5C%_1Ec˥)XC(-Ǟͯ*$_1JɅ×cʕ=5 C%`LЏCT ɿKЏmRNA Jːʷªš©cx­˜©ϙDɰΟ7_ŸōH ν5=ƗύɿD$_CT ɿDK,Ô¶J͸̉H\^˘ƣóJ̉đc͠@=
CT óEĕȽύŀN;J CT DJ PCR ʾʯύJϙHKЏCT DJ PCR ʾ ʯύ = ĕȽύ2CT E%&ϚéLj*ǯ^˾AU8ЏCT ó=‚¯u…‰J CT ó-ĉūǡv­‰«¯©J CT óE%&͸̉H\^Џ‚¯u…‰J CT ó cĉūǡv­‰«¯©J CT óDͪɧ5=ɥHCT ó=‚¯u…‰J CT ó-p¤¨—ª¯‚¯JCT óE%&͸̉H\^ЏŵʗH7_x­˜©(p ¤¨—ª¯‚¯) EJCT óJƹc̉đ5=;5CЏ˘ƣó=2-CT E %&͸̉Lj)]˘ƣócɷX=CT óK Threshold * 0.2 EG_xhr©Ƞ E5=G(ЏTable 7 J anneal ʒLJKЏ2-6-8 HCɅ×ɘ͹5=̝ɍJȸνʒ LJcŌπÛƐHA%Cνʿ5=6-OST-1 E GAPDH J anneal ʒLJK 62E5 =

Table 7 Reaction condition of real-time RT-PCR

£-¨ !0".2AL8—K=6-OST-1 ig—KJœ

271БƘЂɂȣ fŠª‹¨­ńƝàϝƛĠE5CЏÔ¶J4 ˳cʿ%= Ќ1ЍNicergolineЌα 1, 2 fŠª‹¨­ńƝàϝƛĠЏSigma-AldrichЍ
Nicergoline 4.84 mg c DMSO 200 µL Hʘ͵39CЏ0.22 µm –g©‚¯Dʡ κc5CЏ50 mM J|‰…rcΈͫ5= Ќ2ЍPropranolol hydrochlorideЌβ 1, 2 fŠª‹¨­ńƝàϝƛĠЏɈËĬǯƷ əЍ
Propranolol hydrochloride 14.79 mg cʙ͐ɴ 1 mL Hʘ͵5Џ0.22 µm –g© ‚¯Dʡκc5CЏ50 mM J|‰…rcΈͫ5=

Ќ3ЍICI 118,551 hydrochrolideЌβ 2 fŠª‹¨­ńƝàϝƛĠЏSanta Cruz BiotechnologyЍ

ICI 118,551 hydrochrolideЌÔ¶ЏICI 118,551 E˙ˆЍ4.43 mg cʙ͐ɴ 1412 µL Hʘ͵39CЏ0.22 µm –g©‚¯Dʡκc5CЏ10 mM J|‰…rcΈͫ 5=

κc5CЏ10 mM J|‰…rcΈͫ5= 272Б̙̻ŴϿ
L-M ̙̻c 6 ˸˜ª¯‰H 1.6105ïȖ˳5CЏŴŭc3 mL EG_\&H Ħ'Џ37Џ5% CO2Ʌ׶DЏ3 ȪϙŴϿ5=ŌfŠª‹¨­ńƝàϝƛĠ cʏĦ5CЏ2 ȰϙŴϿ5=ǕHЏ0.20 mM fŠª‹¨­ŒȺŴŭU=Kαƾ ŴŭHÊȑ5C1.5 ȰϙŴϿ5= 273БRNA J̐ͫ
́2 ˿Џ́ 2 ̋Џ2-6-3 EŎɝHƘȧ5= 274БRNA J̔LJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-4 EŎɝHƘȧ5= 275БRNA JʟLJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-5 EŎɝHƘȧ5= 276БRNA JϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-6 EŎɝHƘȧ5= 277БcDNA ōǯ
́2 ˿Џ́ 2 ̋Џ2-6-7 EŎɝHƘȧ5= 278БRT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-8 EŎɝHƘȧ5= 279БPCR ʾʯJϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-9 EŎɝHƘȧ5= 2710Бreal time RT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-10 EŎɝHƘȧ5=

£-© "/7’—K=h
6-OST-1 —KJœ 281БƘЂɂȣ zs‹©Ûλ̜ΜJϝƛĠE5CЏÔ¶J2 ˳cʿ%= Ќ1ЍPP2Ќc-Src ϝƛĠЏCalbiochemЍ
PP2 5 mg c DMSO 1655 µL Hʘ͵39CЏ0.22 µm –g©‚¯Dʡκc5CЏ 10 mM J|‰…rcΈͫ5= Ќ2ЍFR180204ЌERK1/2 ϝƛĠЏCalbiochemЍ
FR180204 1 mg c DMSO 305.5 µL Hʘ͵5Џ0.22 µm –g©‚¯Dʡκc5 CЏ10 mM J|‰…rcΈͫ5= 2-8-2Б̙̻ŴϿ
L-M ̙̻c 6 ˸˜ª¯‰H 1.6105_{ïȖ˳5CЏŴŭc3 mL EG_\&H} Ħ'Џ37Џ5% CO2Ʌ׶D3 ȪϙŴϿ5=PP2 U=K FR180204 cʏĦ5 CЏ2 ȰϙŴϿ5=ǕHЏ0.20 mM fŠª‹¨­ŒȺŴŭU=KαƾŴŭHÊ ȑ5C1.5 ȰϙŴϿ5= 283БRNA J̐ͫ
́2 ˿Џ́ 2 ̋Џ2-6-3 EŎɝHƘȧ5= 284БRNA J̔LJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-4 EŎɝHƘȧ5= 285БRNA JʟLJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-5 EŎɝHƘȧ5= 286БRNA JϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-6 EŎɝHƘȧ5= 287БcDNA ōǯ

288БRT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-8 EŎɝHƘȧ5= 289БPCR ʾʯJϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-9 EŎɝHƘȧ5= 2810Бreal time RT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-10 EŎɝHƘȧ5= £-ª 2'12 2-9-1. ̙̻ŴϿ
L-M ̙̻c 75 cm2_{–§|vH10.510}5_{ïȖ˳5CЏŴŭc10 mL EG_\} &HĦ'=37Џ5% CO2Ʌ׶DЏ3 ȪϙŴϿ5= 2-9-2Б;͙JΈͫ Ќ1Ѝ̙̻ʘ͵‘…–e¯
1 M Tris-HCl (pH 7.4) 2 mL E 0.5 M EDTA 0.2 mL cʙ͐ɴD 100 mL HƗƝ 5CЏm¯‰rª¯—5= Ќ2ЍSDS Ĭx­˜©‘…–e¯Ќ2Ѝ
40 mM Tris-HCl (pH 6.8) 2.5 mL E 10% SDS 1.0 mLЏs¨~«¯©Ќ‹n§h ‡|rЍ2.0 mLЏEDTA-2Na®2H2OЌ‹n§h‡|rЍ18.6 mgЏ—«£–j ¯©—©¯̓1.0 mgЏΚ̔ɴ 4.0 mL cʎō5CЏƙʒDìƑ5=ãʿ˗Ğ HЏSDS Ĭx­˜©‘…–e¯E 2-¢©n˜‰k‚¯©ЌsigmaЍc 19М1 JģōDʎō5= 2-9-3. ‚­’rΕťł
–§|vcɵµD10 Ēϰ̯5=ǕЏŴŭcĄύϡĽ5CČ PBS DJʄʇș âc2 ť͠@=;JǕЏ˜«‡f¯h­“”‚¯nr‡©Ќ‹n§h‡| rЍcĦ'=̙̻ʘ͵‘…–e¯ 1 mL c–§|vHʏĦ5Џ~©|rªh’ ¯D̙̻cȏ+E^Џ1.5 mL Ÿhr«„¥¯—Hťł5=;`cɵµH̯

+ЏΚϳʁˣˢɣЌTAITECЏUltraS Homoginizer, VP-55Ѝcʿ%Cœ£{‹h }5=œ£{‹h}KЏ5 ˯ϙJΚϳʁĐʹǕH 30 ˯ϙϰ̯7_șâc 5 ť̮^Ω5=ΚϳʁĐʹ5=x­˜©cμǝЌ4Џ800gЏ10 ĒЍ5Џµ ʐcťł5=1`c3]HμǝЌ4Џ20000gЏ60 ĒЍ5CЏɹɮcťł 5=ɹɮc̙̻ʘ͵‘…–e¯HǮʞ5CЏ̈́‚­’rΕʘʌE5=̙̻ ̈́ ‚ ­ ’ r Ε c ť ł 7 _ Ȧ ɿ K Џ Chernogubova ] J Ȧ ɿ c ľ ̴ H 5 = ЌChernogubova E, et al., 2005Ѝ 2-9-4. BCA ɿH\_‚­’rΕʟLJʓƗ
BCAЌbicinchonic acidЍɿKЏf©n¨ǡJʘʌH(%CЏ›˜„Š̝ō( \NʰƗf ψɫŵЌCys, Cys-Cys, Trp, TyrЍH\@C 2 çϐhm­ЌCu2+_Ѝ

*ρý3`Cʽ6_1 çϐhm­ЌCu+_{ЍEÅĒƐJBCA *ŁǠ5CЏ562 nm}

HȸƅœĀLJcȺ7_ͬōàcǏǯ7_1EHŵB-ЌSmith P K, et al., 1985; Wiechelman K J, et al., 1988ЍƘϧJʓƗHKЏBCA Protein Assay KitЌThermo Fisher ScientificЍcʿ%=ÒƬŸŒ¥f©HǗ%ЏʟLJȩ˞Jʮ͟ʐf©—

 ­ЌBSAЍ\^âǯ5=ɘύ̨EJɰΟH\^Џx­˜©J‚­’rΕʟLJ

cɷX=Ĉà˒HKЏɟʗʘʌU=Kx­˜©J 25 µL HЏProtein Assay Reagent A ʌE B ʌc 50М1 Dʎō5= Working Reagent 200 µL cĦ'CЏ37 D30 ĒϙĦʒ5=;JǕЏ540 nm JœĀLJcʓƗ5= 2-9-5. x­˜©J SDS Ĭ
SDS Ĭx­˜©‘…–e¯Ë́‚­’rΕʘʌc̃ύʎō5CЏ©‡…r | px¯DȘǽǕHЏɻЃʉºD5 ĒϙĦʫĐʹ5= 2-9-6. ž¨fr¨©f Šu©Jâǯ
ɁƘЂDKЏ;ȣJĒϬc˖˒E5=ĒϬu©JµH;ȣJʟ̪c˖˒E 5=ʟ̪u©cϋƭ5=|§—u©cãʿ5=ĒϬu©(\Nʟ̪u©J fr¨©f ŠʟLJKЏ;`<`10%E 4%HͻƗ5= Ќ1Ѝ;͙JΈͫ A ʘʌМ30%fr¨©f Šʘʌ
fr¨©f ŠЌ£Ÿ¯Џ‹n§h‡|rЍ29.2 g E N, N’-¢„ª­”|

=ʡκǕЏãʿUD4HCξĀìƑ5=

B ʘʌЌĒϬu©̩͢ʌЍМ1.5 M Tris-HCl ‘…–e¯ЌpH 8.8Ѝ

Tris (hydroxymethyl) aminomethaneЌÔ¶ Tris EřPЏ‹n§h‡|rЍ18.15 g cΚ̔ɴHʘ͵5ЏHCl D pH 8.8 HΈȡǕЏ100 mL HƗƝ5=ʡκǕЏ ãʿUD4HCìƑ5=

C ʘʌЌʟ̪u©̩͢ʌЍМ0.5 M Tris-HCl ‘…–e¯ЌpH 6.8Ѝ

Tris 6.06 g cΚ̔ɴHʘ͵5ЏHCl D pH 6.8 HΈȡǕЏ100 mL HƗƝ5= ʡκǕЏãʿUD4HCìƑ5= D ʘʌМ10%κˤψf­£Œi¡ʘʌ
κˤψf­£Œi¡Ќ‹n§h‡|rЍ100 mg cΚ̔ɴ 1 mL Hʘ͵5= ʿÄΈͫE5= 10% SDS

Sodium Dodecyl SulfateЌSDSЏBIO-RADЍ5 g cΚ̔ɴ 5 mL Hʘ͵5=

ɴϽśh€—‚¯©
2-Methyl-propanol HνύJΚ̔ɴcĦ'CƾʒDìƑ5=ãʿ˗ĞH\-ʎō5CЏĒϬ5=µʐcãʿ5= Ќ2ЍĒϬu©JâǯЌ2 ɌЍ
u©JâǯHʿ%_;͙cĄCƙʒHǰ5Џʿ%_ţĈcĄC¢‚¯© Dȁ%=o§|ɊJƅƧcϋICu©n~…‰HũƗ5Џo§|ɊJϙH ͔˅ɴcʂ,Џ10 Ēϰ̯5CYɴʜ`*G%1Ec˥΂5Џʂ%>͔˅ɴc ȈC=A ʘʌ 4.0 mLЏB ʘʌ 3.0 mLЏ10% SDS 0.12 mLЏΚ̔ɴ 4.9 mL c 50 mL μǝ„¥¯—Hʂ,ЏʑŪ¶D 15 Ēϙ̾ɳ5=ɥ%DЏD ʘʌ 60 µL ETEMEDЌ‹n§h‡|rЍ6 µL cĦ'Cʎō5=;`cЏo§|ɊJº Ɖ)]ʆ5ΦVЏ;JµσHɴϽśh€—‚¯©̓100 µL cϰ)Hϋƭ5 CЏ1 ȰϙSFϰ̯5Cfr¨©f ŠcϋōĬ39= Ќ3Ѝʟ̪u©JâǯЌ2 ɌЍ

A ʘʌ 0.4 mLЏC ʘʌ 1.0 mLЏ10% SDS 0.04 mLЏΚ̔ɴ 2.6 mL c 50 mL μǝ„¥¯—Hʂ,ЏʑŪ¶D 15 Ēϙ̾ɳ5=ɥ%DЏD ʘʌ 20 µL E TEMEDЌ‹n§h‡|rЍ4 µL cĦCʎō5=;`cЏɴϽśh€—‚ ¯©cΚ̔ɴDϡĽ5=ĒϬu©JµHϋƭ5CЏx­˜©v¯¡cƹ5Φ d>u©JǏǯc˥΂ǕHx­˜©v¯¡cǹ+Ń^ЏãʿUDΚ̔ɴº DìƑ5= 2-9-7. ϮɳʃĪ Ќ1Ѝ;͙JΈͫ ʃĪ‘…–e¯

s¨z­ЌBIO-RADЍ14.4 gЏTris 3 gЏSDSЌBIO-RADЍ1 g c 1 L JΚ̔ɴ Hʘ͵5Џ\-ʎō5=

Ќ2ЍϮɳʃĪ

ʃĪ‘…–e¯cʕ=5=ʃĪɞЌ Œ˜«‡gf­3ЏBIO-RADЍH|§

—u©cͻ̯5CЏij©ĉcʄʇ5=ǕHЏSDS Ĭx­˜©cνύf˜§h

5=G(ЏĒƐύŸ¯n¯HKЏPrecision Plus Protein Standards (BIO-RAD) cãʿ5=;JǕЏƙʒЏ20 mA ƗʆJɅ×HCЏʃĪÿ̀Ќ—«£–j ¯©—©¯Ѝ*u©¶̀ÒΨHęɆ7_UDʃĪc͠@= 2-9-8.
ij|‚­—«…‰ Ќ1Ѝ;͙JΈͫ Ξċ‘…–e¯
s¨z­ЌSigmaЍ14.4 gЏTris 3.6 g cΚ̔ɴHʘ͵39CЏ¢‚¯© 40 mL cĦ'=ǕHΚ̔ɴD800 mL HƗƝ5=ãʿUD 4HCìƑ5=G(Џ Ʌ×ɘ͹J̝ɍЏɁƘЂJ¢‚¯©ʟLJK10%E5= 20% Tween 20
Tween 20 (BIO-RAD) 20 g cΚ̔ɴHʘ͵ǕЏ100 mL HƗƝ5= TBST (10)
Tris 6.057 g E NaCl 45 g cΚ̔ɴHʘ͵ǕЏHCl D pH 7.4 HΈȡ5=;1

CЏ4DČ͗ìƑ5=

1Ћ BSA/TBSTЌ—«…p­s‘…–e¯Ѝ

BSA (Roche) 0.3 g c TBST 30 mL Hʘ͵39CЏ–g©‚¯ЌPore Size 0.22 μmЍDʡκc͠@=

0.2Ћ BSA/TBSTЌǸàƽω‘…–e¯Ѝ

BSA (Roche) 0.02 g c TBST 10 mL Hʘ͵39CЏ–g©‚¯ЌPore Size 0.22 µmЍDʡκc͠@= CBB ɏ͋ʌ
CBB 0.5 gЏ¢‚¯© 225 mLЏφψ 50 mL cΚ̔ɴD 500 mL HƗƝ5= CBB ̾͋ʌ
¢‚¯©225 mLЏφψ 35 mL cΚ̔ɴD 500 mL HƗƝ5= Ќ2Ѝ̈́Ξċ
ϮɳʃĪHCĒϬ5=;ȣ‚­’rΕJ̈́QJΞċKЏij…‰Lj Œ‰§ ­|—«…‰~©ЌBIO-RADЍÒƬJŸŒ¥f©HǗ%͠@=
U8KЏ‚…’¯H PVDF ̈́ЌMILLIPOREЍcă`CЏ¢‚¯©Hʊ5 CʅǡĬ39=Κ̔ɴDʄʇǕHΞċ‘…–e¯Hʊ5CЏ10 ĒÔµȆ˕ 39=U=Џ|ž­{2 ɌEa̕ 2 Ɍc 1 ïJ‚…’¯Hă`CЏΞċ‘… –e¯Hʊ5CЏ10 ĒÔµȆ˕39=
ϮɳʃĪǕHЏo§|ɊJz¦¯‰˜ª¯‰cƂ5Cʟ̪u©cĝ^Ń^Џ ʃĪ‘…–e¯că`=‚…’¯HĒϬu©cʊ5=1`c 10 ĒÔµȆ˕ 39=
¢‚¯©Dȁ%=‘…‰HΞċ‘…–e¯că`CЏu©œ©ƒ¯JЊ c¶H5CЏ|ž­{Џa̕Џu©ЏPVDF ̈́Џa̕Џ|ž­{J϶HϋIЏ —«…‰«¯§¯cʿ%C˺ɳcǹ%=u©œ©ƒ¯DũƗ5CЏ‰§­ |—«…‰~©Hͻ̯5=U=ЏČĺɞHɵcͿXCЏ‰§­|—«…E ~©Hͻ̯5=|‚¯§¯µH|‚¯§¯‘¯că`=‰§­|—«…‰ ~©c̯+Џ‘…‰JΞċ‘…–e¯cʂ,ă`CЏƙʒЏ200 mA ƗʆHC 1 ȰϙαϮ5=

u©œ©ƒ¯Jˑc¶H5CЏ|ž­{Ea̕c±ƢHK*5ЏϏ̂Du ©JÞ̯*b)_\&HĹcA/ЏΞċϲ*b)_\&HΞċϲH!µ"Eͤ ͺ5=ĒƐύŸ¯n¯Jª¯­Kē^Ϭ5CÁʬ39=U=ЏΞċǕJ ž¨fr¨©f Šu©cCBB ɏ͋ʌHʊ5CЏ10 Ēϙʊ˕5=;JǕЏ CBB ̾͋ʌHʊ5C°Ȳʊ˕5CЏΞċĩʵc˥΂5= Ќ3ЍΞċ‚­’rΕJɘđ
˖˒‚­’rΕJɘđKЏ‚­’rΕāˊɘđ̯ͨ SNAP id (MILLIPORE) JΆȮȶHǗ@=7Gb?ЏSNAP i.d ̯ͨcɴǀGijµH̯+Џ‘p¥¯ ¡„¥¯—̀J̝ōσĒc̯ͨɁàJǕaH$_rh…rˆg|vŽr‰– g…‡­sHƹ5Φd>„¥¯—JY&°ȦJ̀KœljʖHȌ̢5=ɥ %DЏ—«…‰œ©ƒ¯J͖cϘ/CЏĉõJˑ%ͤϲ*ʢ͋HG_UDΚ ̔ɴDʠ]5=PVDF ̈́J‚­’rΕcΞċ5=ϲ*ˑ%ͤϲEȌ7_\& H—«…‰œ©ƒ¯JºƉH̯+Џ—«…‡g­s¢­—ª­cJ9C—« …‰œ©ƒ¯DɳʀcŃ^ϡ%=—«…‰œ©ƒ¯JO=cϗXCЏSNAP i.d.̯ͨH—«…‰œ©ƒ¯cͻ̯5=—«…p­s‘…–e¯c 30 mL ʆ 5C7.Hœlj5=;JǕ—«…‰œ©ƒ¯*ƕĄH˺HG@=]ЏǸà ƽω‘…–e¯D300 ðHƽω5=Ǹ3 fŠª‹¨­ńƝàǸàЌM20, Santa Cruz BiotechnologyЍ1 mL cʏĦ5CЏ15 ĒϙŁǠ39=;JǕœlj5CЏ —«…‰œ©ƒ¯*ƕĄH˺HG@=]œljcON JUU 1TBST 30 mL D 3 ťʄʇ5=œljc OFF H5ЏǸàƽω‘…–e¯D 2000 ðHƽω5= HRP-conjugated rabbit anti-goat IgG heavy and light chain secondary antibody ЌBethyl LaboratoriesЍ1 mL cʏĦ5Џ15 ĒŁǠ39=;JǕœlj5Џ—« …‰œ©ƒ¯*ƕĄH˺HG@=]œljcON JUU TBST 30 mL D 3 ťʄʇ 5=

—«…‰œ©ƒ¯c̯ͨ)]Ń^đ5ЏPVDF ̈́cŃ^đ5ЏÅΤcƫϘ5 =”Œ¯©ͥJµH(+Џz¯§¯cʿ%C PVDF ̈́*̅ʱHG_\&H” Œ¯©ͥcȌ˜5=̈́ĄàH ImmobilonTM Western Chemiluminescent HRP SubstrateЌMerck MilliporeЍ*%+b=_ύcʏĦ5Џh¢¯{f‹§hy¯ LAS-3000 UV miniЌƠƀ–g©¡ЍE Multi Gauge €–‰ijfЌƠƀ–g© ¡ Ѝ H \ ^ ˃ û ͵ ɋ c ͠ @ =  ; J Ǖ Џ ʃ Ī Ÿ ¯ n ¯ c ˃ û Ĭ 3 9 C Џ Photoshop HCōǯ5CЏ˖˒‚­’rΕJĒƐύc˥΂5=

£-¢¡ Phostag 2'12 2-10-1. ̙̻ŴϿ
L-M ̙̻c 6 well ˜ª¯‰H 1.6105ïȖ˳5CЏŴŭc3 mL EG_\& HĦ'Џ37Џ5% CO2Ʌ׶D3 ȪϙŴϿ5=0.20 mM fŠª‹¨­ŒȺ ŴŭHÊȑ5CЏ0Ќv­‰«¯©ЍЏ5, 10, 20, 30, 40, 60 ĒϙŴϿ5= 2-10-2. x­˜©Έͫ Ќ1Ѝ‚­’rΕťł ́2 ˿Џ́ 2 ̋Џ2-9-3 EŎɝHƘȧ5= Ќ2ЍBCA ɿH\_‚­’rΕʟLJʓƗ ́2 ˿Џ́ 2 ̋Џ2-9-4 EŎɝHƘȧ5= Ќ3Ѝx­˜©J SDS Ĭ ́2 ˿Џ́ 2 ̋Џ2-9-5 EŎɝHƘȧ5= 2-10-3. Phostag ŒȺž¨fr¨©f Šu©Jâǯ
ɁƘЂDKЏKinoshita ]JȦɿHŵB+ЏPhostag ŒȺž¨fr¨©f Š u©câǯ5=ЌKinoshita E, et al., 2011ЍĒϬu©(\Nʟ̪u©Jfr¨ ©f ŠʟLJKЏ;`<`12ЋE 4%HͻƗ5= Ќ1Ѝ;͙JΈͫ A ʘʌМ30%fr¨©f Šʘʌ
́2 ˿Џ́ 2 ̋Џ2-9-6 (1)EŎɝHΈͫ5= D ʘʌМ10%κˤψf­£Œi¡ʘʌ
́2 ˿Џ́ 2 ̋Џ2-9-6 (1)EŎɝHΈͫ5= 2 M Bis Tris-HCl (pH 6.8)
Bis TrisЌŎÍŶЍ41.85 g cΚ̔ɴHʘ͵39CЏHCl D pH c 6.8 HΈȡ5 =1`cΚ̔ɴHC100 mL HƗƝ5=

10 mM Zn(NO3)2
Zn(NO3)2 148.74 mg cΚ̔ɴHʘ͵39CЏ50 mL HƗƝ5= 5.0 mM Phostag
PhostagЌśĀЍ2.0 mg cʰ̖¢‚¯© 20 µL Hʘ͵39=;1HΚ̔ɴ 640 µL cĦ'Cʎō5=1`cf© œh©DξĀ5CЏ4DìƑ5= ɴϽśh€—‚¯©
́2 ˿Џ́ 2 ̋Џ2-9-6 (1)EŎɝHΈͫ5= Ќ2ЍĒϬu©JâǯЌ1 ɌЍ
u©JâǯHʿ%_;͙cĄCƙʒHǰ5Џʿ%_ţĈcĄC¢‚¯© Dȁ%=o§|ɊJƅƧcϋICu©n~…‰HũƗ5Џo§|ɊJϙH ͔˅ɴcʂ,Џ10 Ēϰ̯5CYɴʜ`*G%1Ec˥΂5Џʂ%>͔˅ɴc ȈC=A ʘʌ 2.4 mLЏ2M Bis Tris-HCl (pH 6.8) 1.07 mLЏΚ̔ɴ 2.45 mL c 50 mL μǝ„¥¯—Hʂ,ЏʑŪ¶D 15 Ēϙ̾ɳ5=ɥ%DЏ5.0 mM Phostag 24 µL E 10 mM Zn(NO3)2 24 µL cĦ'Cʎō5=;1H D ʘʌ 30 µL ETEMEDЌ‹n§h‡|rЍ3 µL cĦCʎō5=;`cЏo§|ɊJºƉ )]ʆ5ΦVЏ;JµσHɴϽśh€—‚¯©̓100 µL cϰ)Hϋƭ5CЏ 1 ȰϙSFϰ̯5Cfr¨©f ŠcϋōĬ39= Ќ3Ѝʟ̪u©JâǯЌ1 ɌЍ
A ʘʌ 0.33 mLЏ2 M Bis Tris-HCl (pH 6.8) 0.45 mLЏΚ̔ɴ 1.71 mL c 50 mL μǝ„¥¯—Hʂ,ЏʑŪ¶D15 Ēϙ̾ɳ5=ɥ%DЏD ʘʌ 12.5 µL E TEMEDЌ‹n§h‡|rЍ2.5 µL cĦCʎō5=;`cЏɴϽśh€—‚ ¯©cΚ̔ɴDϡĽ5=ĒϬu©JµHϋƭ5CЏx­˜©v¯¡cƹ5 Φd>u©JǏǯc˥΂ǕHx­˜©v¯¡cǹ+Ń^ЏãʿUDΚ̔ɴ ºDìƑ5=G(ЏPhostag u©KϖȽìƑ*D+G%JDЏPhostag u©K âǯ5=ǎȪU=KɥJȪHãʿ5=

2-10-4. ϮɳʃĪ Ќ1Ѝ;͙JΈͫ ʃĪ‘…–e¯
Tris 6.06 g, SDS 0.5 g, MOPSЌ‹n§h‡|rЍ10.46 g, Èˤψɴ̗ Na 0.26 g cΚ̔ɴHʘ͵5CЏ500 mL HƗƝ5= Ќ2ЍϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-9-7 EŎɝHƘȧ5=G(Џ2-9-7 EˈG^ЏĒƐύŸ ¯n¯Kf˜§h98Џ3]H SDS Ĭx­˜©cf˜§h5G%ij©HK x­˜©‘…–e¯cf˜§h5= 2-10-5. ij|‚­—«…‰ Ќ1Ѝ;͙JΈͫ Zn ϡĽ‘…–e¯
s¨z­3.6 g E Tris 0.76 g cΚ̔ɴHʘ͵39CЏ500 mM EDTA 4 mL E ¢‚¯©10 mL cĦ'=ǕHЏ200 mL HƗƝ5= ;JÑJ;͙KЏ́2 ˿Џ́ 2 ̋Џ2-9-8 EŎɝHΈͫ5= Ќ2Ѝ̈́Ξċ
ϮɳʃĪǕHЏZn ϡĽ‘…–e¯Hu©cʊ5CЏ10 ĒϙȆ˕5=1J șâc3 ť̮^Ω5=;JǕKЏ́ 2 ˿Џ́ 2 ̋Џ2-9-8 (2)EŎɝHƘȧ5 = Ќ3ЍΞċ‚­’rΕJɘđ
́2 ˿Џ́ 2 ̋Џ2-9-8 (3)EŎɝHƘȧ5=G(Џ°ɥǸàE5CǸ ERK1/2 ǸàЌ137F5ЏCell Signaling TechnologyЍЏÅɥǸàE5C ECL peroxidase-labelled anti-rabbit IgG secondary antibodyЌGE HealthcareЍcãʿ5CЏ;`<` 300 ð E3000 ðHƽω5=

£-¢¢ ERK1/2 —K=h
 HS _‘H?—KJœ

FR180204ЌERK1/2 ϝƛĠЏCalbiochemЍ 2-11-2. ̙̻ŴϿ
L-M ̙̻c 75cm2–§|vH10.5105ïȖ˳5CЏŴŭc10 mL EG_\ &HĦ'=37Џ5% CO2Ʌ׶DЏ3 ȪϙŴϿ5=20 µM FR180204 cʏ Ħ5C2 ȰϙŴϿ5=ǕHЏ0.20 mM fŠª‹¨­ŒȺŴŭU=KαƾŴŭH Êȑ5C3 ȰϙŴϿ5= 2-11-3. HS J̐ͫ
́2 ˿Џ́ 2 ̋Џ2-2 EŎɝHƘȧ5= 2-11-4. HS JÅ̛̑ǯĒɋ
́2 ˿Џ́ 2 ̋Џ2-5 EŎɝHƘȧ5= £-¢£ „˜(-,2:h
 HS NGF k69hdI

Surface Plasmon Resonance (SPR) cĘʿ5=ĒƐϙ˘Æâʿ͵ɋz|‡¡E 5C BIAcore J (GEHealthcare) cʿ%=BIAcore z|‡¡K¨o­ŠEf‹ §h‰J˘Æâʿc¨f©‚h¡HʓƗD+_

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2-12-1. ̙̻ŴϿ

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2-12-2. HS J̐ͫ ́2 ˿Џ́ 2 ̋Џ2-2 EŎɝHƘȧ5=G(ЏfŠª‹¨­Děʝ5=̙̻ )]̐ͫ5=HS c aHS E5CЏfŠª‹¨­Děʝ5G%̙̻)]ťł5= HS c cHS E5= 2-12-3. HS E NGF J˘ÆâʿʓƗ
~­x¯„…˜KЏǎˡ˹ƙH(%C2009 ǁHâǯ3`=Ǹ NGF ǸàũƗ Ĭ„…˜cãʿ5=ǸNGF ǸàЌsc-549; Santa Cruz BiotechnologyЍũƗĬ„ …˜Hƣ5C0.77 µM NGFЌmNGF 2.5 S; Alomone LabsЍЏ0.77µM NGF E 0.77 µM cHS JʎōʯЏU=K 0.77µM NGF E 0.77 µM aHS JʎōʯcʏĦ5=ϧ J~­x¯s§¡cɰΟ5=G(ЏHS J£©ʟLJKЏHS JĒƐύc 20,000 EÕƗ5C̉đ5= £-¢¤ NGF  6-OST-1 ig4R™ 2-13-1 ̙̻ŴϿ
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́2 ˿Џ́ 2 ̋Џ2-6-3 EŎɝHƘȧ5= 2133БRNA J̔LJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-4 EŎɝHƘȧ5= 2134БRNA JʟLJʓƗ
́2 ˿Џ́ 2 ̋Џ2-6-5 EŎɝHƘȧ5= 2135БRNA JϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-6 EŎɝHƘȧ5= 2136БcDNA ōǯ

́2 ˿Џ́ 2 ̋Џ2-6-7 EŎɝHƘȧ5= 2137БRT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-8 EŎɝHƘȧ5= 2138БPCR ʾʯJϮɳʃĪ
́2 ˿Џ́ 2 ̋Џ2-6-9 EŎɝHƘȧ5= 2139Бreal time RT-PCR
́2 ˿Џ́ 2 ̋Џ2-6-10 EŎɝHƘȧ5=

p¤r {^ ¤-¢ L-M y„˜ HS l”?_‘!0".24R™
ǎˡ˹ƙJ1`UDJˡ˹H\^ЏL-M ̙̻HfŠª‹¨­c¸'= 24 Ȱ ϙǕHЏ̙̻ͤϲHS JˤψĬɜγ*ƁĬ7_1E*˨ş3`=ЌͮˀЏ2005Н ɶͰЏ2009Ѝ;1DЏL-M ̙̻ͤϲ HS JˤψĬɜγHfŠª‹¨­*¸' _ǐϴcȮ˥H7_=XHЏfŠª‹¨­c¸'=6 ȰϙǕJ̙̻ͤϲ HS J ɜγcЏfŠª‹¨­c¸'G%v­‰«¯©EɰΟ5=HS ƗύJϧHť ł5=HS cχ̗DÅ̑ĶÞHĒ͵5Џ̑ϻJĒϬHƃʿ3`_n§¡cͨ˜ 5=HPLC cʿ%CÅ̑JģōcĒɋ5=HPLC DĒϬ5=ŌÅ̑KЏÅ̑ J|‚­ƒ¯ŠJʘđȰϙEɰΟ7_1EH\^ŎƗ5=ЌFig. 2-1Ѝ;J̝ ɍЏfŠª‹¨­c¸'_EЏ̙̻ͤϲHS JˤψĬɜγ*ƁĬ7_1E*Ȯ ])EG@=AU^ЏfŠª‹¨­H\@CЏHS JÅ̛̑ǯKЏ6 Þˤψ ĬÅ̑(Di-6S)Jģō* 15%)] 30%QŽĦ5Џ°ȦDϱˤψĬÅ̑(Di-0S) Jģō*55%)] 35%Qʑƨ7_1EcːͰ5=ЌFig. 2-2ЍU=Џ0.01 mM JfŠª‹¨­KЏˤψĬɜγcƁĬ39G%1EYȮ])EG@=
U=ЏfŠª‹¨­cʏĦ5=3 ȰϙǕJ L-M ̙̻)] HS c̐ͫ5CЏÅ ̛̑ǯĒɋc͠@=ЌFig. 2-3Ѝ;J̝ɍЏfŠª‹¨­cʏĦ5G%v­‰ «¯©JHS KЏ¼HϱˤψĬÅ̑ЌDi-0SЏ̓ 45%ЍE 6 ÞˤψĬÅ̑( Di-6SЏ̓ 27%)Dɜǯ3`C%=N ÞˤψĬÅ̑ЌDi-NSЍE N ÞE C 6 ÞˤψĬÅ̑ЌDi-diS1ЍJģōKß)@=0.05 mM JfŠª‹¨­cʏĦ 5CYЏÅ̛̑ǯKƁĬ5G)@=*Џ0.10 mM E 0.20 mM JfŠª‹¨­c ʏĦ7_EЏDi-0S Jģō*;`<`̓ 37%E 29%Hʑƨ5=3]HЏ Di-6S JģōKЏ0.10 mM E 0.20 mM JfŠª‹¨­cʏĦH\^Џ̓ 31%E 35%HŽĦ5=0.10 mM E 0.20 mM JfŠª‹¨­cʏĦ5CYЏ;JÑJ Å̑JģōKƁĬ5G)@=

Fig. 2-1
Å̑ĶÞHĒ͵5= HS J HPLC Ēɋ

HS Å̑|‚­ƒ¯ŠЌAЍU=K 0.10 mM fŠª‹¨­ʏĦ 6 ȰϙǕJ HSЌBЍ

cYMC-Pack NH2PA n§¡Hå5CЏ16 mM )] 538 mM ¨­ψÅɴ̗‹‰¨i

Fig. 2-2
fŠª‹¨­ʏĦ 6 ȰϙǕJ L-M ̙̻ͤϲ HS JÅ̛̑ǯ
fŠª‹¨­ŒȺŴŭU=Kv­‰«¯©Ŵŭc L-M ̙̻HʏĦ5CЏ6 ȰϙŴ Ͽ5=;JǕЏ̙̻ͤϲ HS cťł5CЏHS Ē͵χ̗H\@CÅ̑ĶÞHĒ͵ 5C)]ЏHPLC cʿ%CŌÅ̑Jģōc̉đ5=ǘ]`=óЌ%ЍKf¯rx h­Ɓȑc͠%ЏTukey ɿcʿ%C̟͸Ēɋc͠@=*: P < 0.05, **: P< 0.01
Ũ ºJ 0.01 mM Ad.̃KЏʏĦ5=fŠª‹¨­JʟLJc˨7

**

**

**

**

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ΔDi-0S ΔDi-NS ΔDi-6S ΔDi-diS1 ΔDi-diS2 ΔDi-triS

(%)

Control

0.05 mM Ad.

0.10 mM Ad.

0.20 mM Ad.

60

50

40

30

20

10

0

0.01 mM Ad.

Fig. 2-3
fŠª‹¨­ʏĦ 3 ȰϙǕJ L-M ̙̻ͤϲ HS JÅ̛̑ǯ
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Ũº J0.01 mM Ad.̃KЏʏĦ5=fŠª‹¨­JʟLJc˨7

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ΔDi-0S ΔDi-NS ΔDi-6S ΔDi-diS1 ΔDi-diS2 ΔDi-triS

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0.10 mM Ad.

0.20 mM Ad.

60

50

40

30

20

10

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fŠª‹¨­*L-M ̙̻ͤϲJ HS ύH¸'_ǐϴcΈR_=XHЏfŠª ‹¨­c¸'=6 ȰϙǕJ̙̻ͤϲ HS ύcЏfŠª‹¨­c¸'G%v­‰ «¯©EɰΟ5=L-M ̙̻)]‰¨˜z­ĐʹH\@Cē^Ϭ5=̙̻ͤϲ J̑ϔc̐ͫ7_1EH\^ HS cťł5ЏHS GFJˤψĬ̑ϔcʰˈ˒H ɏ͋7_̗͋(Alcian Blue)Dɏ͋7_1EH\^ЏHS ύcƗύ5=ŎȰH HS cťł5=̙̻J DNA ύYƗύ5ЏDNA ύ)]ȑ̉3`_̙̻ȠD HS ύcͪɧ5=;J̝ɍЏ̙̻ͤϲHS ύKЏfŠª‹¨­Ƿ¸JȺʦH\] 8Џ25.19.7 pg/cell D$^ЏfŠª‹¨­c¸'CYƁĬ5G%1E*Ȯ]) EG@=ЌFig. 2-4ЍU=ЏfŠª‹¨­cʏĦ5= 3 ȰϙǕJ̙̻ͤϲ HS ύcŎɝJȦɿH\^Ēɋ7_EЏfŠª‹¨­Ƿ¸JȺʦH\]821.22.1 pg/cell D$^ЏfŠª‹¨­c¸'CYƁĬ5G%1E*Ȯ])EG@=ЌFig. 2-5Ѝ

Fig. 2-4
fŠª‹¨­ʏĦ 6 ȰϙǕJ L-M ̙̻ͤϲ HS ύ
fŠª‹¨­ŒȺŴŭU=Kv­‰«¯©ŴŭcL-M̙̻HʏĦ5CЏ6ȰϙŴ Ͽ5=;JǕЏ̙̻ͤϲHScťł5CЏHSύcAlcian Blueɏ͋H\^̉đ5= U=ЏHScťł5=̙̻)]DNAcǻđ5CЏDNAύ)]̙̻Ƞc̉đ7_1E DЏ̙̻ǎ^JHSύcȑ̉5=Tukeyɿcʿ%C̟͸Ēɋc͠@=

Adrenaline (mM)

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0.05

0.10

0.20

Amount of HS

( pg/ cell )

0

10

20

30

40

50

Fig. 2-5
fŠª‹¨­ʏĦ 3 ȰϙǕJ L-M ̙̻ͤϲ HS ύ

fŠª‹¨­ŒȺŴŭU=Kv­‰«¯©Ŵŭc L-M ̙̻HʏĦ5CЏ3 ȰϙŴ Ͽ5=;JǕЏ̙̻ͤϲ HS cťł5CЏHS ύc Alcian Blue ɏ͋H\^̉đ5 =U=ЏHS cťł5=̙̻)] DNA cǻđ5CЏDNA ύ)]̙̻Ƞc̉đ7 _1EDЏ̙̻ǎ^J HS ύcȑ̉5=Tukey ɿcʿ%C̟͸Ēɋc͠@=

Adrenaline (mM)

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0.05

0.10

0.20

Amount of HS

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30

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0

¤-¤ 6-OST-1 ig!0".24R™

fŠª‹¨­ěʝH\@C̙̻ͤϲHS J 6 ÞˤψŵÅ̑JģōJŽĦc˥ ΂5=1E)]ЌFig. 2-3ЍЏHS cɜǯ7_Å̑J GlcNAc J C 6 ÞHˤψŵc Ξ˰7_χ̗Ќ6-OSTsЍJπÛƐːʷQfŠª‹¨­*¸'_ǐϴcΈR= L-M ̙̻GFJŸi|KЏ6-OSTs HA%CЏ6-OST-1, 2, 3 E 6-OST-2 J splice variant D$_ 6-OST-2S cȂA1E*˞]`C%_L-M ̙̻H(/_1`] JːʷcΈR=E1aЏ6-OST-1 *Іːʷ5C(^Џ6-OST-2 E 3 Kːʷ5C %G%1EcːͰ5=ЌFig. 2-6, AЍ;1DЏfŠª‹¨­* 6-OST-1 JπÛ ƐːʷH¸'_ǐϴcĒɋ5=

°ƗȰϙfŠª‹¨­c¸'=L-M ̙̻)] total RNA cťł5C cDNA c ōǯ5Џ6-OST-1 J mRNA ːʷύc real time PCR DΈR=ŌȰϙH(/_ 6-OST-1 JːʷύKЏ GAPDH cĉσɟʗπÛƐE5Cͪɧ5=;J̝ɍЏ 0.2 mM fŠª‹¨­c¸'= 90 ĒǕHЏ6-OST-1 J mRNA ːʷύ* 1.5 ± 0.3 ðHŽĦ7_1E*Ȯ])EG@=ЌFig. 2-6, BЍ

L-M ̙̻HfŠª‹¨­cʏĦ7_EЏ˫̜ǯϖŦƐЌNGFЍJĒɾύ*Ž Ħ7_1E*ŷŖ3`C%_ЌFurukawa S, et al., 1986Ѝ3]HЏNGF *§… ‰Ģ̀ЅΕ̙̻ͭ͋͂ЌPC12Ѝ̙̻ͤϲ HS JˤψĬɜγcƁĬ39_1E* ŷŖ3`C%_1E)]ЌMargolis R K, et al., 1987ЍЏL-M ̙̻ͤϲ HS YĒɾ 3`=NGF H\^ɜγ*ƁĬ5=Ň̼ǡ*˨ş3`_;1DЏNGF * L-M ̙̻ͤϲ HS H¸'_ǐϴcΈR_=XHЏNGF cʏĦ5= 1.5 ȰϙǕJ 6-OST-1 J mRNA ːʷύc real time RT-PCR H\^Ēɋ5=;J̝ɍЏ20 pM )]400 pM J NGF cʏĦ5=*Џ6-OST-1 J mRNA ːʷύKƁĬ5G)@ =ЌFig. 2-7Ѝ

Fig. 2-6
6-OSTs J mRNA ːʷύHfŠª‹¨­*¸'_ǐϴ

ЌAЍ0.20 mM fŠª‹¨­cʏĦ5= 90 ĒǕJ L-M ̙̻)] total RNA cťł 5CЏ6-OST-1Џ6-OST-2Џ6-OST-3 J mRNA ːʷc RT-PCR H\^˥΂5=U =Џ«¯ˆg­sv­‰«¯©E5CЏGAPDH J PCR ʾʯcʃĪ5=ŨºƸõ JȠóKĒƐύ|‚­ƒ¯ŠJĒƐύc˨7ЌBЍfŠª‹¨­ʏĦ 1.5 hЏ3.0 hЏ 6.0 h ǕH(/_ 6-OST-1 J mRNA ːʷύc real time RT-PCR H\^ʓƗ5=f Šª‹¨­ϱʏĦȰЌ0 hЍH(/_ 6-OST-1 Jːʷύc 1.0 E5=ŸōJ˘ƣóc ˨7Tukey ɿcʿ%C̟͸Ēɋc͠@=*: P < 0.05, **: P< 0.01

Relative mRNA levels of 6-OST-1

TimeK

0 90 0 90 0 90

6-OST-1 6-OST-2 6-OST-3

200 250 500 500 (min) GAPDH 150

A

B

Fig. 2-7
NGF * 6-OST-1 J mRNA ːʷH¸'_ǐϴ

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Adrenaline

NGF(pM)

Relative mRNA levels of 6-OST-1

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㸩

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20

100

200

400

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¤-¥ Sulf1/2 ig!0".24R™

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Fig. 2-8
fŠª‹¨­* Sulf-1/2 ːʷH¸'_ǐϴ

0.20 mM fŠª‹¨­ʏĦ 1.5 h ǕH(/_ Sulf-1 E Sulf-2 J mRNA ːʷύc real time RT-PCR H\^ʓƗ5=

¤-¦ L-M yig!0".2AL8nšeI

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fŠª‹¨­ńƝàHKЏ1, 2 E1, 2, 3 ńƝà*Ƒū7_1E*˞]` _;1DŌ˳fŠª‹¨­ńƝàϝƛĠЌNicergolineМ1, 2 ńƝàϱοǺ ˒ϝƛĠЏPropranololМ1, 2 ńƝàϱοǺ˒ϝƛĠЏICI 118,551М2 ńƝà οǺ˒ϝƛĠЏL 748,337М3 ńƝàοǺ˒ϝƛĠЍcŴŭHǷ¸5C 2 Ȱϙ ŴϿ5=ǕЏ0.20 mM fŠª‹¨­c¸'= 90 ĒǕJ̙̻)] total RNA cť ł5Џ6-OST-1 J mRNA ːʷύc real time RT-PCR H\^͵ɋ5=ЌFig. 2-9, A, BЍ;J̝ɍЏ3 ńƝàοǺ˒ϝƛĠc¸'=ŸōJVЏ0.20 mM fŠª‹ ¨­H\_6-OST-1 J mRNA ːʷŽĦcƕĄHǶĚ5=1E)]ЌFig. 2-9, BЍЏ L-M ̙̻DK3 fŠª‹¨­ńƝà*fŠª‹¨­c̝ō5CЏ6-OST-1 J

ːʷcĚǙ5C%_1E*˨ş3`=U=ЏL-M ̙̻)]̈́‚­’rΕcť

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Rv: 5’-TCACAGCAGAAAGGTCCAAG-3’Ѝ cʿ%=RT-PCR J̝ɍЏβ2 fŠª‹¨­ńƝàJ mRNA * L-M ̙̻Dːʷ 7_1Ec˥΂5=5)5G*]Џ̈́‚­’rΕ̐ͫp…‰ЌDUALXtract total Membrane Protein Extraction KitЏDualsystems BiotechЍcʿ%C L-M ̙̻) ]̈́‚­’rΕcťł5CЏβ2 fŠª‹¨­ńƝàǸàЌab 61778ЏabcamЍ H\^β2 fŠª‹¨­ńƝàJːʷcΈR=*Џɘđ7_1EKD+G)@ =

Fig. 2-9
fŠª‹¨­ńƝàϝƛĠ* 6-OST-1 ːʷH¸'_ǐϴ

ЌAЍNicergolineЌα1, 2 fŠª‹¨­ńƝàϝƛĠЍU=K PropranololЌβ1, 2 fŠª‹¨­ńƝàϝƛĠЍЏICI 118,551Ќβ2 fŠª‹¨­ńƝàϝƛĠЍcŴŭ HʏĦ5C2 ȰϙŴϿ5CЏ0.20 mM fŠª‹¨­ŒȺŴŭHÊȑ5= 1.5 h ǕJ 6-OST-1 ːʷύc real time RT-PCR H\^ʓƗ5=ЌBЍL-748,337Ќβ3 fŠª‹ ¨­ńƝàϝƛĠЍcŴŭHʏĦ5C2 ȰϙŴϿ5CЏ0.20 mM fŠª‹¨­ŒȺ ŴŭHÊȑ5=1.5 h ǕJ 6-OST-1 ːʷύc real time RT-PCR H\^ʓƗ5=ЌCЍ L-M ̙̻)]̈́‚­’rΕcťł5CЏǸβ3 fŠª‹¨­ńƝàǸàcʿ%=i j|‚­—«…‡g­sc͠@=β3AR Kβ3 fŠª‹¨­ńƝàc˨5ЏŨºƸ õJȠóKĒƐύŸ¯n¯JĒƐύc˨7Tukey ɿcʿ%C̟͸Ēɋc͠@= **: P< 0.01 Adrenaline Nicergoline Propranolol ICI 118,551 Adrenaline ࣮ 㸩 ࣮ 㸩 L-748,337 ࣮ ࣮ 㸩 㸩 ** ** ** ** **

A

B

Relative mRNA levels of 6-OST-1

Relative mRNA levels of 6-OST-1

0 0.5 1.0 1.5 2.0 㸫 㸩 㸫 㸩 㸫 㸩 㸫 㸩 㸫 㸫 㸩 㸩 㸫 㸫 㸫 㸫 㸫 㸫 㸫 㸫 㸩 㸩 㸫 㸫 㸫 㸫 㸫 㸫 㸫 㸫 㸩 㸩 50 100 37 25 200 kD β3-AR 2.0 1.5 1.0 0.5 0

C

¤-§ 6-OST-1 ig<S"/7’`_;]

3 fŠª‹¨­ńƝàK¼H̷̙̻̽HƑū5CЏ̷̽ψJÓΊHϚ¸7

_1E*˞]`C%_ЌRobidoux J, et al., 2006ЍŸi|J̷̙̻̽H(%CЏ ̷̽ψJÓΊ*͠b`_ϧKЏ3 fŠª‹¨­ńƝàH c-Src *̝ō5CЏ ̙̻Ƃzs‹©Έ̋p‹¯D$_ERK1/2 *ʅǡĬ3`_1E*˞]`C% _ЌSoeder K J, et al., 1999Ѝ;1DЏL-M ̙̻* 6-OST-1 JːʷcΈ̋7_z s‹©Ûλ̜ΜHSrc E ERK1/2 *Ϛ¸5C%_)cɘͼ5=
PP2ЌSrc ϝƛĠЍU=K FR180204ЌERK1/2 ϝƛĠЍcŴŭHĦ'C 2 Ȱϙ ŴϿ5Џ;JǕ0.2 mM fŠª‹¨­c¸'= 90 ĒǕJ̙̻)] total RNA c ťł5CЏ6-OST-1 ːʷH¸'_ǐϴcΈR=;J̝ɍЏPP2 E FR180204 EYH, 0.20 mM fŠª‹¨­H\_ 6-OST-1 JːʷύŽĦcƕĄHǶĚ5= 1E)]ЌFig. 2-10, A, BЍЏfŠª‹¨­H\@C΃Ʀ3`_ 6-OST-1 Jːʷ KЏSrc E ERK1/2 *Ϛ¸5C%_1E*˨ş3`=U=ЏfŠª‹¨­ʏ ĦH\@CERK1/2 *ʅǡĬ3`_)cΈR_=XHЏPhostag ij|‚­—« … ‡ g ­ s c ͠ @ =  ; J ̝ ɍ ЏERK1/2 J ƃ - K ϱ ¨ ­ ψ Ĭ ʱ ǫ Ќnp-ERK1/np-ERK2ЍDƑū5=*ЏfŠª‹¨­cʏĦ7_E¨­ψĬ ERK1/2 *ͳơ3`=ЌFig. 2-10, CЍERK1/2 * 2 ̈Dz¨­ψĬ3`= pTpY-ERK1/2 KЏfŠª‹¨­ʏĦ 5 ĒǕH•¯rcΧ'Џ60 ĒǕH)/C ǖ Hʑƨ5=U=Џ1 ̈Dz¨­ψĬ3`= pT/pY-ERK1/2 KfŠª‹¨­ ʏĦ5 ĒǕJVHͳơ3`=
αƾERK1/2 K̙̻ΕHƑū7_*Џ¨­ψĬcń/CʅǡĬ3`= ERK1/2 KɒĉH˰͠5CЏΞċŦƐE˘Æâʿ7_1EH\^ΞċJĚǙc͠&1E *˞]`C%_;1DЏfŠª‹¨­*΃Ʀ7_6-OST-1 Jːʷ* ERK1/2 H\@CĚǙ3`C%_G]LЏERK1/2 K̙̻ͤϲ HS JˤψĬɜγYĚǙ 5C%_E̴']`_;1DЏERK1/2 ϝƛĠc¸'=̙̻HfŠª‹¨­ c¸'_EЏfŠª‹¨­*΃Ʀ7_HS JɜγƁĬ*ǶĚ3`_J)cɘͼ 5=;J̝ɍЏFR180204ЌERK1/2 ϝƛĠЍc¸'=̙̻H 0.20 mM fŠª ‹¨­c¸'= 3 ȰϙǕJ̙̻ͤϲ HS JÅ̛̑ǯKЏERK1/2 ϝƛĠEfŠ ª‹¨­c¸'G%v­‰«¯©J HS JÅ̛̑ǯESTŎ6D$@=ЌFig. 2-11Ѝ1J̝ɍ)]ЏfŠª‹¨­*΃Ʀ7_ 6-OST-1 JːʷK ERK1/2 *Ě Ǚ5C%_1E*˥΂3`=

Fig. 2-10
6-OST-1 ːʷcĚǙ7_zs‹©Ûλ̜ΜJɘ͹

ЌAЍPP2Ќc-Src ϝƛĠЍcŴŭHʏĦ5C 2 ȰϙŴϿ5CЏ0.20 mM fŠª‹¨ ­ŒȺŴŭHÊȑ5=1.5 h ǕJ 6-OST-1 ːʷύc real time RT-PCR H\^ʓƗ5 =ЌBЍFR180204ЌERK1/2 ϝƛĠЍcŴŭHʏĦ5C 2 ȰϙŴϿ5CЏ0.20 mM fŠª‹¨­ŒȺŴŭHÊȑ5=1.5 h ǕJ 6-OST-1 ːʷύc real time RT-PCR H \^ʓƗ5=ЌCЍ0.20 mM fŠª‹¨­c 0, 5, 10, 20, 30, 40 ĒʏĦ5= L-M ̙ ̻)]‚­’rΕcťł5ЏǸERK1/2 Ǹàcʿ%= Phostag ij|‚­—«…‡ g­sH\^¨­ψĬERK1/2 cɘđ5=«¯ˆg­sv­‰«¯©E5CЏi j|‚­—«…‡g­sH\^β-fr„­Jɘđc͠@=Tukey ɿcʿ%C̟͸ Ēɋc͠@= **: P< 0.01

A

B

Relative mRNA levels of 6-OST-1

Relative mRNA levels of 6-OST-1

0 0.5 1.0 1.5 2.0 Adrenaline PP2 (μM) ࣮ 㸩 ࣮ 㸩 ࣮ 㸩 ࣮ ࣮ 10 10 50 50 Adrenaline FR180204 (μM) 0 0.5 1.0 1.5 2.0 ࣮ 㸩 ࣮ 㸩 ࣮ 㸩 ࣮ ࣮ 10 10 ࣮ 㸩 20 20 30 30 0 5 10 20 30 40 60 actin np-ERK1 pTpY-ERK1/2 adrenaline stimulation (min)

pT/pY-ERK1/2

np-ERK2 Phos-tag western blotting

Fig. 2-11
fŠª‹¨­H\_ HS JɜγƁĬH ERK1/2 ϝƛĠ*¸'_ǐϴ
FR180204ЌERK1/2ϝƛĠЍcŴŭHʏĦ5C2ȰϙŴϿ5Џ0.20 mMfŠª‹¨ ­ŒȺŴŭU=Kv­‰«¯©ŴŭcHÊȑ5C3ȰϙŴϿ5=;JǕЏ̙̻ͤ ϲHScťł5CЏHSĒ͵χ̗H\@CÅ̑ĶÞHĒ͵5C)]ЏHPLCcʿ%CŌ Å̑Jģōc̉đ5=ǘ]`=óЌ%ЍKf¯rxh­Ɓȑc͠%ЏTukeyɿc ʿ%C̟͸Ēɋc͠@= **: P< 0.01

ŨºJ”Ad.”K0.20 mMfŠª‹¨­cʏĦ5=Ÿōc˨5Џ”Inh. + Ad.”K FR180204DĞŴϿ5=ǕH0.20 mMfŠª‹¨­cʏĦ5=Ÿōc˨7 0 10 20 30 40 50 Control Ad. Inh. + Ad.

**

**

**

ΔDi-0S ΔDi-NS ΔDi-6S ΔDi-diS1 ΔDi-diS2 ΔDi-triS

¤-¨ !0".2HS _‘H? NGF k69h4R™

L-M ̙̻K NGF cĒɾ7_*ЏfŠª‹¨­cʏĦ7_E NGF JĒɾύ* Ž'_1E*ŷŖ3`C%_=XЌFurukawa S, et al., 1986ЍЏfŠª‹¨­H\ _HS JɜγƁĬ* NGF EJ˘ÆâʿH¸'_ǐϴcΈR=U8ЏǸ NGF ǸàũƗĬ~­x¯„…˜HNGF cå5ЏNGF J̝ōc˥΂5=1EH\^Џ 2009 ǁHâǯ3`=„…˜*ɣ̼7_1Ec˥΂5=ЌFig. 2-12, AЍɥHЏ NGF E HS cʎō5CǸ NGF Ǹà̝ō„…˜Hå5CЏNGF ĶʲŸōEɰΟ 7_1EH\^ЏNGF E HS J˘ÆâʿJɘđc;V=;1DЏcHS E NGF J̃ʟLJʎōʯcʏĦ7_EЏʏĦ10 ĒǕJ RU ó* NGF ĶʲJŸōEɰΟ 5CȺǨHʑƨ5=ЌFig. 2-12, BЍ3]HЏaHS E NGF J̃ʟLJʎōʯcʏ Ħ7_EЏʏĦ10 ĒǕJ RU ó* NGF ĶʲJŸōEɰΟ5CȺǨHʑƨ5= µЏcHS E NGF J̃ʟLJʎōʯcʏĦ5=ŸōEɰΟ5CYȺǨHʑƨ7_ 1EcͰđ5=ЌFig. 2-12, BЍ

Fig. 2-12
L-M ̙̻ͤϲ HS E NGF J˘ÆâʿʓƗ
ЌAЍv­‰«¯©ŴŭU=K 0.20 mM fŠª‹¨­cʏĦ5= 6 ȰϙǕJ L-M ̙̻)]HS cťł5=Ќ;`<` cHSЏaHSЍHS JĒƐύc 20,000 EÕƗ5C ʟLJc͸̉5ЏNGF Eʎō5=Ō HS cǸ NGF ǸàũƗĬ~­x¯„…˜Hf˜ §h5CЏNGF ĶʲJ̝ɍEɰΟ5=ЌBЍA H(%CЏx­˜©ʏĦ 10 ĒǕЌ˝ ƩЍJRU óc˘ƣͽç5=Tukey ɿcʿ%C̟͸Ēɋc͠@= #: P < 0.01 vs NGF ĶʲЏЏ**: P< 0.01 0 20 40 60 80 100 120 ratio (%) 0 50 100 150 200 response (RU) Time (min) 0 4 10

A

B

p¥r €M
Ɂ˿DKЏ̙̻ͤϲHS *̙̻Ƃ)]JěʝHǠ̆7_ɣɜc͵Ȯ7_=X HЏfŠª‹¨­JěʝH\^NGF ĒɾύcŽĦ7_ L-M ̙̻c£ˆ©H5 CЏL-M ̙̻ͤϲ HS HfŠª‹¨­*¸'_ǐϴHA%Cɘ͹5=
L-M ̙̻ͤϲ HS JˤψĬɜγHfŠª‹¨­*¸'_ǐϴcΈR_EЏf Šª‹¨­JʏĦȰϙc6 ȰϙE5=ŸōЏ0.05 mMЏ0.10 mMЏ0.20 mM J7 RCJʟLJH(%CЏ6 ÞˤψĬÅ̑Jģō*Ŏ˲LJHŽĦ5=°ȦDЏϱ ˤψĬÅ̑JģōKȺǨHʑƨ5=1`]J̝ɍH\^ЏL-M ̙̻ͤϲ HS KЏfŠª‹¨­JěʝHǠ̆5CˤψĬɜγcƁĬ39_1E*˨ş3`= 5)5G*]Џ6 ÞˤψĬÅ̑JŽĦ*fŠª‹¨­ʟLJHæƑ5G)@=1 E)]ЏfŠª‹¨­ʏĦ6 ȰϙǕHK 0.05 mM GFJNj%ěʝD$@CYЏ 6 ȰϙǕHKfŠª‹¨­JěʝH\_ɜγQJǐϴ*įĒHŁȯ3`_)] DKG%)E̴'=U=Џ0.01 mM JfŠª‹¨­KЏHS JˤψĬɜγc ƁĬ39G)@=1E)]ЏL-M ̙̻ͤϲJ HS ɜγcƁĬ39_=XHKЏ 0.05 mM ÔµJʟLJJfŠª‹¨­*ǞͯHG_1E*˨ş3`=;1DЏ fŠª‹¨­ʏĦ3 ȰϙǕJ L-M ̙̻ͤϲ HS ɜγHfŠª‹¨­*¸'_ǐ ϴcΈR_EЏ0.05 mM fŠª‹¨­Kǐϴc¸'G)@=*Џ6 ÞˤψĬÅ ̑KfŠª‹¨­JʟLJæƑ˒HŽĦ5=U=Џ0.10 mM E 0.20 mM fŠª ‹¨­ʏĦH\@CϱˤψĬÅ̑Jģō*ʑƨ5=1E)]ЏL-M ̙̻ͤϲ HS KЏfŠª‹¨­JěʝHǠ̆5CϱˤψĬÅ̑J C 6 ÞHˤψ*ÒĦ3 `=̝ɍЏ6 ÞˤψĬÅ̑*ŽĦ5=E˨ş3`=
fŠª‹¨­H\_L-M ̙̻ͤϲ HS JɜγƁĬcėJȦɿH\^˥΂7_ =XHЏǎˡ˹ƙJƧŹH\^ЏHS JʰƗJˤψĬɜγc΋ė5C̝ō7_ ̨̣̙̻͌ŽɬŦƐЌFGFЍE HS J̝ōύ* SPR ɿH\^ʓƗ3`=;J ̝ɍЏ0.20 mM fŠª‹¨­Děʝ5= 6 ȰϙǕJ̙̻)]ťł5= HSЌaHSЍ EFGF-1 J͵ϬƗȠЌ151 ± 9 nMЍKЏfŠª‹¨­Děʝ5G% L-M ̙̻) ]ťł5=HSЌcHSЍEJ͵ϬƗȠЌ104 ± 4 nMЍ\^ȺǨHʑƨ7_1E* Ȯ])HG@=1`H\^ЏL-M ̙̻ͤϲ HS JˤψĬɜγ*ƁĬ5=1E *˥΂3`=U=Џ1 mol J cHS H̝ō5= FGF-1, 2, 10 J̝ōύ*Џ0.21, 2.2, 2.3 mol D$@=1E)]ЏfŠª‹¨­Děʝ5G%ŸōJ L-M ̙̻ͤϲ HS

=3]HЏ FGF-1, 2, 10 E aHS J̝ōύc cHS EɰΟ7_EЏ̝ōύ*; ` < ` ̓ 85%, 62%, 45% H ʑ ƨ 7 _ 1 E * ŷ Ŗ 3 ` =  FGF-1 K Џ IdoA2S-GlcNS6S-IdoA2S cŒWʰƗJ 8 ̑c΂΋7_1E*ŷŖ3`C(^ ЌKreuger J, et al., 1999; Kreuger J, et al., 2001ЍЏFGF-2 K N ˤψĬs©vx ­ HϨ^ō&h}«­ψJC 2 ÞJˤψĬHĦ'C 5 ̑ÔµJϔϖc΂΋7_1 E*˞]`_ЌJemth PЏet al., 2002; Ashikari-Hada S, et al., 2004ЍFGF-10 KЏ3 AJHexA2S-GlcNS6S cYA 8 ̑H̝ō7_ЌAshikari-Hada S, et al., 2004Ѝ5 )5G*]ЏFGF-1 E HS J˘Æâʿ* HS JˤψŵJȠHæƑ5G)@=E JŷŖ*$_1E)]ЌKreuger J, et al., 1999ЍЏFGF E HS J̝ōKˤψŵJȠ DKG-ЏHS JˤψĬɜγЌ’‚¯­ЍHæƑ7_E̴']`_Ǘ@CЏ 0.20 mM E%&ІʟLJJfŠª‹¨­cʏĦ5=1EH\^Џ6 ÞˤψĬÅ̑ *ŽĦ5=̝ɍЏL-M ̙̻ͤϲ HS JˤψĬ’‚¯­*ƴ`==XЏFGF J̝ ōύ*ʑƨ5=JDKG%)E̴'=
fŠª‹¨­ʏĦ3 ȰϙǕH HS ɜγ*ƁĬ5=*Џ1`K HS JÓΊȰϙ cΈR=ˡ˹E°͉7_7Gb?Џȥ=Hōǯ3`=HS *w©{à)]̙ ̻ͤϲHΡέ3`_UDH̓12 Ēͯ7_ЌYanagishita M, 1992Ѝ;5CЏHS *̙̻ͤϲHęλ7_EЏ̓4 ȰϙǕHēȤ3`C̙̻ƂŸ‰¨…r|HĒɾ 3`_)Џk­Šxh‰¯z|H\^̙̻ĉHŃ^ΦU`_ЌYanagishita M, 1992ЍHS JÓΊЌıʑȽЍ*̓ 4 ȰϙDΙ+_1Ec̴'_EЏfŠª‹¨ ­ʏĦ3 ȰϙǕH HS JɜγƁĬ*ʽ6=1EKƌǎD$_E̴']`_
fŠª‹¨­H\^6 ÞˤψĬÅ̑*ŽĦ5=1E)]ЏGlcNAc J C 6 Þ HˤψŵcΞ˰7_ 6-O-sulfotransferasesЌ6-OSTsЍJϚ¸*˨ş3`= RT-PCR cʿ%C mRNA JːʷcĒɋ7_EЏL-M ̙̻HK 6-OST Jfh€ –l¯¡Ќ6-OST-1, 6-OST-2, 6-OST-3ЍJ&?Џ6-OST-1 JV*ːʷ7_1E *Ȯ])EG@=;1DЏfŠª‹¨­*6-OST-1 J mRNA ːʷH¸'_

ǐϴcΈR_EЏfŠª‹¨­ʏĦ1.5 ȰϙǕHfŠª‹¨­JʟLJæƑ˒H

6-OST-1 ːʷ*ŽĦ5=fŠª‹¨­ʏĦ 3 ȰϙǕE 6 ȰϙǕJ 6-OST-1 ː

ʷKƁĬ5G)@=1E)]ЏfŠª‹¨­JěʝKʏĦ1.5 Ȱϙ˲LJȂ̢7

_1E*ȍƗ3`=U=ЏfŠª‹¨­cʏĦ5G%v­‰«¯©J1.5 Ȱ

ϙǕJ6-OST-1 Jːʷ*ŽĦ5=JKЏŴŭÊȑH\@C̙̻ĉJÓΊ*ǐϴ 3`=EJŷŖ*$_1E)]ЌCaldwell J, et al., 1991ЍЏŴŭÊȑH\_E̴

'=U=ЏSulf-1/2 K̙̻ͤϲH(%CDi-diS1 ZDi-TriS J GlcNAc C 6 ÞJˤψŵc̾ˤψĬ7_=XЏ6 ÞˤψĬÅ̑JǏǯHϚ¸7_E̴']` _1E)]fŠª‹¨­ʏĦ1.5 ȰϙǕJ Sulf-1/2 ːʷcΈR=*ЏƁĬ5G )@=Ǘ%ЏfŠª‹¨­H\_6S ɜγJŽĦKЏ6S c̾ˤψĬ7_ Sulf ːʷJǶĚDKG-Џ6-OST-1 JːʷÉζH\_E̴'=

Margolis ]JŷŖH\_EЏPC12 ̙̻H NGF cʏĦ7_E̙̻ͤϲ HS J ˤψĬɜγ*ƁĬ5=ЌMargolis R K, et al., 1987ЍL-M ̙̻YfŠª‹¨­J ěʝH\^NGF JĒɾύ*ŽĦ7_=XЏĒɾ3`= NGF H\@C L-M ̙ ̻ͤϲHS Jɜγ*ƁĬ5=Ň̼ǡ*Ȅ0]`=3 ȪϙŴϿ5= 3T3 Ÿi| ̨̣̙̻͌KŴŭ1 mL ºH 0.49 ng J NGF cĒɾ7_1E*ŷŖ3`C(^ ЌOger J, et al., 1974ЍЏǎˡ˹ƙDY L-M ̙̻*Ŵŭ 1 mL ºH̓ 0.45 ng J NGF cĒɾ7_1Ec˥΂5=3]HЏL-M ̙̻H 0.20 mM fŠª‹¨­cʏĦ 7_EЏNGF Ēɾύ*ȸƅD 20 ðHŽĦ5=EJŷŖ*$_1E)] ЌFurukawa S, et al., 1986ЍЏαƾʱǫЌ̓ 20 pMЍ)]ȸƅóЌ400 pMЍUD JNGF c L-M ̙̻HʏĦ5CЏ1.5 ȰϙǕJ 6-OST-1 ːʷH¸'_ǐϴcΈ R=;J̝ɍЏ%8`JʟLJJNGF Y 6-OST-1 ːʷHǐϴ5G)@=U =ЏɁˡ˹H(%CЏfŠª‹¨­cʏĦ5=1.5 ȰϙǕH 6-OST-1 ːʷ*f Šª‹¨­JʟLJæƑ˒H΃Ʀ3`Џ3 ȰϙǕH 6 ÞˤψĬÅ̑*ŽĦ5=1

EEЏfŠª‹¨­ʏĦ 4 ȰϙǕH NGF JĒɾύŽĦ*Ͱ]`=1E

ЌFurukawa S, et al., 1986ЍEōb9C̴'_EЏL-M ̙̻ͤϲ HS JɜγƁĬ

KЏfŠª‹¨­ěʝH\^Ēɾ3`= NGF DKG-ЏʏĦ5=fŠª‹¨ ­H\_E̝ΉÒ/=1`]J̝ɍ\^ЏL-M ̙̻Hːʷ7_ 6-OST-1 Kf Šª‹¨­H\@C΃Ʀ3`_1E*Ȯ])EG@=0.20 mM fŠª‹¨­ Kʽʹ˒GʟLJ\^ІʟLJD$_*ЏHS ʽōǯχ̗Jːʷc΃Ʀ7_ěʝK 1`UDHŷŖ3`C%G%1E)]ЏfŠª‹¨­H\@C6-OST-1 Jːʷ *΃Ʀ3`_1EE6 ÞˤψĬÅ̑*ŽĦ7_EJːͰKЏHS JˤψĬɜγ *Ǐǯ3`_Ð̛Vc͵Ȯ7_=XJƅ+G°ɨEG_;1DЏ1JʷΎc ƘЂ£ˆ©H5C 6-OST-1 JːʷcĚǙ7_zs‹©Ûλ̜ΜJʰƗc͠@ =
6-OST-1 ːʷ*fŠª‹¨­H\^΃Ʀ3`=1E)]ЏfŠª‹¨­ńƝ

Šª‹¨­ńƝàKЏα1, α2, β1, β2, β3 Hƅė3`_ЌMolinoff P B, 1984Ѝ1

`UDJE1aЏL-M ̙̻HKfŠª‹¨­ńƝàcːʷ5G%1E*ŷŖ3

`C+=*ЌSchroeder F, et al., 1981; Furukawa S, et al., 1986ЍЏɁˡ˹DK L-M ̙̻*β3 fŠª‹¨­ńƝàcȂA1EcːͰ5=ЌFig. 2-9 CЍβ3 fŠª‹ ¨­ńƝàJπÛƐK̶ʕπÛƐEYřL`ЏπÛƐƁˈH\^̶ʕcƦ-1 E*˞]`Џβ3 fŠª‹¨­ńƝàK¼H̷̙̻̽Hːʷ7_1E*˞]` _ЌLi H, et al., 2010ЍL-M ̙̻K L-929 ̙̻)]ɡ˾3`=*ЏL-929 ̙̻J ˁɆEG@=L ̙̻K˓¶̷̛̭̽)]ɡ˾3`=1Ec̴'_EЏL-M ̙̻ *̷̙̻̽Hːʷ7_β3 fŠª‹¨­ńƝàcȂA1EKƌǎD$_Eͷ' _
β3 fŠª‹¨­ńƝàcÏ5=zs‹©Ûλ̜ΜK̷̙̻̽H(%Cˡ˹ *ζdD%_Ќ_{Robidoux J, et al., 2006Ѝβ3 fŠª‹¨­ńƝàKЏ™‡«´} ύàJG ‚­’rΕD$_ Gs E Gi H̝ō5C%_ЌSoeder K J, et al., 1999Ѝ Gi KЏcAMP E PKA ̜ΜHϚ¸5CЏGs K c-Src E ERK1/2 ̜ΜcĚǙ7_ ЌSoeder K J, et al., 1999ЍņƵ]* L-M ̙̻HK cAMP E PKA ̜Μ*Ƒū5 G%1EcŷŖ5C%_1E)]ЌFurukawa S, et al., 1986ЍЏfŠª‹¨­H\ _6-OST-1 ːʷJ΃ƦHЏSrc E ERK1/2 *Ϛ¸7_)cΈR=;J=XHЏ Src JϝƛĠD$_ PP2 E ERK1/2 JϝƛĠD$_ FR180204 cãʿ5=;J ̝ɍЏPP2 E FR180204 *EYHfŠª‹¨­H\_ 6-OST-1 Jːʷ΃ƦcǶ Ě5=1E)]ЏfŠª‹¨­Děʝ5=6-OST-1 K Src-ERK1/2 ̜ΜDĚǙ

3`_1E*˨ş3`=fŠª‹¨­Děʝ5G%ʱǫD PP2 U=K

FR180204 câʿ39CY 6-OST-1 JːʷKƁĬ5G)@=1E)]Џ̙̻* Ǣƾ˒Hːʷ7_6-OST-1 K Src-ERK1/2 ÔƂJ̜ΜDĚǙ3`_1EY˨ş 3`=U=ЏfŠª‹¨­H\@CERK1/2 *¨­ψĬ3`_1EY˥΂5 =ERK1/2 KЏMitogen-activated protein kinase kinaseЌMEKЍH\@C ERK1 JThr-202 E Tyr-204ЏERK2 J Thr-183 E Tyr-185 *¨­ψĬ3`_1EH\ ^ʅǡĬ7_ЌCrews C M, et al., 1992; Seger R, et al., 1992Ѝ3]HЏFR180204 cŁǠ39=L-M ̙̻HfŠª‹¨­cʏĦ5CY HS JˤψĬɜγ*ƁĬ5 G)@=1E)]ЏfŠª‹¨­H\@Cěʝ3`_6-OST-1 K ERK1/2 H\ @CĚǙ3`_1Ec˥΂5=6-OST-1 cǍĚːʷ39_E 6 ÞˤψĬÅ̑ Jģō*ŽĦ5=EJŷŖ*$_\&HЌDo A T, et al., 2006ЍЏɁˡ˹J̝ɍЏ fŠª‹¨­K6-OST-1 Jːʷc΃Ʀ5CЏ;J̝ɍ 6 ÞˤψĬÅ̑Jģō*

ŽĦ5=E̴']`=

Ɂˡ˹DKЏERK1/2 ϝƛĠD$_ FR180204 cʿ%_1EDЏfŠª‹¨­ H\_HS JˤψĬɜγƁĬcϝƛ7_1EHǯĥ5=ЌFig. 2-11Ѝ1J̝ɍ KЏˤψŵΞ˰χ̗D$_6-OST-1 JːʷcǶĚ7_1EH\^ЏHS Jˤψ

ĬɜγcÌƷ˒HĚǙ5=EY̴']`_̙̻Ko­Ĭ7_EHS JˤψĬ

ɜγ*ƁĬ7_1E*˞]`_=XЌMolist A, et al., 1998ЍЏHS JˤψĬɜγ cÌƷ˒HĚǙ7_1E*D+`LЏo­ĬJζ͠cǶĚ7_ȥ=GɼˎɿJ ϘːHƞ¸7_Ň̼ǡ*$_HS JˤψĬɜγK̙̻Jo­ĬJVG]8Џ ƃ˳ƃɝGʽʹɣ̼HϚ¸7_=XЌBishop J R, et al., 2007ЍЏɁˡ˹ǯɍcΙ ʥE5CЏHS JˤψĬɜγcÌƷ˒HĚǙ7_1EH\^ HS *Ϛ¸7_ʽ ʹɣ̼J͵Ȯ*ζW1E*ȽǓ3`_
Ɂˡ˹DKЏHS ʽōǯHϚ¸7_ͬȠJχ̗J&?Џ6-OST-1 JːʷcĚ Ǚ7_zs‹©Ûλ̜ΜcĕXCʰƗ5=*Џ6-OST-1 ːʷ̜ΜcƕĄH͵Ȯ 7_=XHKЏ6-OST-1 JΞċŦƐcʰƗ7_Ǟͯ*$_1`UDJE1aЏ HS JʽōǯHϚ¸7_ˤψŵΞ˰χ̗JΞċŦƐK°AYʰƗ3`C%G% *ЏΞċŦƐAP-2 E Runx2 *z­ˆn­Zs¨•n­JōǯHϚ¸7_1E *˨ş3`C%_ЌBrucato S, et al., 2002; Jackson R A, et al., 2007; Haupt L M, et al., 2009; Teplyuk N M, et al., 2009; Araujo A P, et al., 2010Ѝ;1DЏ6-OST-1 J ΞċŦƐJñͪcȋ7=XHЏ6-OST-1 J˜«£¯‚¯ϸŲHƑū7_ΞċŦ Ɛ̝ōσÞcȋ̘5=U8ЏNational Center for Biotechnology Information (NCBI))] mouse 6-OST-1 J reference sequence ǥŷЌNM_015818.2Ѝcădz5 =ɥHЏnon-coding region EΞċϘƎÞ̯ЏΞċŦƐ̝ōŦƐJǥŷcŒW ˆ¯‚š¯|D$_Mammalian Promoter/Enhanced DatabaseЌPEDBЍH reference sequence ǥŷcăĤ5CЏ˜«£¯‚¯ϸŲJŻŵτĔǥŷcădz5=ɥHЏ JASPAR database H˜«£¯‚¯ǥŷcăĤ5CЏ6-OST-1 J˜«£¯‚¯ϸ Ų H ̝ ō 7 _ Ξ ċ Ŧ Ɛ J ñ ͪ c ǘ =  ; J ̝ ɍ ЏAP-1Ќc-FosЏc-JunЍZ NF-kappaBЏSox17ЏKlf4 GF* 10 ÔµJІ%|vfc˨5=Ɂˡ˹DKЏ 6-OST-1 JΞċŦƐcʰƗ7_1EKD+G)@=*Џ1`]J̝ɍcŵH5 CΞċŦƐcʰƗ7_1E*ȽǓ3`_