ハウスダストマイトによって活性化されたイヌケラチノサイトにおけるGM-CSFの産生機序に関する研究

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(1)Title. ハウスダストマイトによって活性化されたイヌケラチノサイトにおけるGM-CSFの産生機序に関する研究( 本文 (Fulltext) ). Author(s). 木村, 剛. Report No.(Doctoral Degree). 博士(獣医学) 甲第412号. Issue Date. 2014-03-13. Type. 博士論文. Version. ETD. URL. http://hdl.handle.net/20.500.12099/49035. ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。.

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(7) AD: Atopic dermatitis̖DeqŲȪʀǻ̗ AP-1: Activator protein-1 AQP: Aquaporin CAD: Canine atopic dermatitis̖FiDeqŲȪʀǻ̗ CCL: CC chemokine ligand CCR: CC chemokine receptor CsA: Ciclosporin A Der f: Dermatophagoides farinae̖TgoGo_h̗ Der p: Dermatophagoides pteronyssinus̖RoGo_h̗ DPBS: Dulbecco’s phosphate-buffered saline E-64: Trans-Epoxysuccinyl-L-Leucylamido-̖4-Guanidino̗Butane ERK: Extracellular signal-regulated kinase GAPDH: Glyceraldehyde 3- phosphate dehydrogenase GM-CSF: Granulocyte macrophage colony-stimulating factor HDM: House dust mite̖lGY_YeyFe̗ Ig: Immunoglobulin̖ÅȝQs̗ IL: Interleukin INPPL: Inositol polyphosphate phosphatase-like 1 LPS: Lipopolysaccharide̖xħɓ̗ MHC: Major histocompatibility complex̖ʘɜɧ˕ûƅðʖû̗ MMP: Matrix metalloprotease̖ɚɹĦĚʶÔʞ˛ɖ̗ NFAT: Nuclear factor of activated T-cells̖ǥŲç T ɚɹǋËĎį̗. 5.

(8) NF-κB: Nuclear factor-kappa B NK: Natural killer NLR: Nucleotide binding oligomerization domain-like receptor̖NOD ǒöĺ ̗ PAR: Protease activated receptor̖tcD\ǥŲçöĺ̗ PCNA: Proliferation cell nuclear antigen PMA: Phorbol 12-myristate 13-acetate RTPCR̠Reverse transcription polymerase chain reaction SAA: Serum amyloid A SGPL: Sphingosine-1-phosphate lyase SPINK: Serine protease inhibitor Kazal SPL: Sphingosine 1-phosphate lyase S1P: Sphingosine 1-phosphate TEWL: Transepidermal water loss̖ɝʓȪǟÔʍƝˠ̗ TGF: Transforming growth factor Th: Helper T TIMP: Tissue inhibitor of metalloproteinase TLR: Toll like receptor̖Toll ǒöĺ̗ TNF: Tumor necrosis factor TRIF: Toll/IL-1 receptor/resistance domain-containing adaptor-inducing IFN-β TSLP: Thymic stromal lymphopoietin. 6.

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(188) '9üǒ-ǖɺBƶ'

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(199) [103]oe-D OŲǲȞǒȠĥ˙ ,

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(234) >)Ƒļ ?'

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(240) 25.

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(248) >9-)ű A?>[58, 105]ƥCAD Ƞĥ˙,

(249) '.ǔȇɚɹƞ-ġã-6ěā ?'

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(251) >)9ȸ?'

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(253) >øɺŲ > CCL20 . Der f 1 ǭã 24 Ʈ˪Ŧ;0 48 Ʈ˪Ŧ)9,ʽÍˠġã-ʦ8< ?!R}LFBTf>˗¨į( =CCR6 -Mf( >[107]AD Bþ7D OŲȪʀȟŴ-Ƞĥ˙,

(254) 'ġãʦ8<?'

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(258) >[82]Deqna`cYe,

(259) '˳Ų óůʦ8<?!oe-Ȫʀ(. CCL20 mRNA -ʽÍˠġã))9, CCR6+SlYɚɹ-ġãʦ8<?'

(260) >[38]oe;0Fi- AD Ƞĥ˙,

(261) '.ġã!SlYɚɹ, IgE ɞû'

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(264) >[9, 90]ƅð,;$'ǥŲç!Sl 26.

(265) Yɚɹ. IL-1βIL-6IL-12TNF-α ;0F^rHBȔȓ> )oe;0yGY,

(266) 'ȸ?'

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(268) >)űA?>č-ɞǄ)? 5(-ěā<HDM ǥŲçR`kVFe.CCL20 BȔȓ>),; $'SlYɚɹ-Ƞĥ˙1-ˏʸBµˍVFeLF-Ȕȓ B'ȌŧÅȝ1-ȾʒBµˍ'

(269) >)ɮ<?! Der f 1 ǭã 48 Ʈ˪Ŧ,ʽÍˠ-ġãʦ8<?! TGF-2 .ƃÙŲVFe LF( =-3,9 4 %-DF]rJ{İē>)oe,

(270) 'ȸ?'

(271) >[36]TGF-β2 . TGF-β1 )ɕ 70̕-ȰüŲBƶǖɺ9 ̈ª>)oe;0yGY,

(272) 'ȸ?'

(273) >[36]<,oe,

(274) '.T ɚɹ- IL-4 ;0 IL-5 -Ȕȓ) B ɚɹ-ġǚBƃÙ>)9Ƭ <,+$'

(275) >[30, 62]oe;0Fi- AD ȠŹ,> TGF-β2 -ťà ,%

(276) '.Ƭ<,+$'

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(278) CAD Ƞĥ˙,> TGF-β -ȦȎ¬ ȸ?'

(279) >)<[87]T ɚɹǖɺ-ƃÙǖǑ-ȶɣ AD ȠŹ,˫ '

(280) >øɺŲ. >)űA?>[73]č-ȵɄɞǄ<HDM ,;$'ǥŲ ç?!R`kVFe.TGF-β2 BȔȓ>),;=T ;0 B ɚɹǖɺBƃÙȌŧÅȝɔ-ǻȡóůBƃÙ'

(281) >øɺŲȸ?! Der f 1 ǭãŦ,ʽÍˠġãʦ8<?! GM-CSF .ħɺŲˊʑŗɚɹB ĭȏìȏ5!.ĭ˜ȏ,?. ?Ôç>ǖɺBƈ%VFeLFBT. f>[1]GM-CSF .oe AD Ƞĥ˙(-ȦȎġãʦ8<?'=[12] Ƞĥ˙(ġã-ʦ8<?'

(282) >ĭ˜ȏ-Ôç;0ǥŲç,;>ɼ̆ɐ,˫ >3[59]źŲ-Ƞĥ˙,>yPrCX-DxeWYˮĸB 'źŲǻȡBɡƈ'

(283) >)ȸ?'

(284) >[12]5!GM-CSF .oe AD Ƞĥ˙,>R`kVFe<-Ȕȓġãȸ?'=Ƞĥ˙, >SlYɚɹ;0ǔȇɚɹ-ġã;0ǥŲç1-˫ȸć 27.

(285) ?'

(286) >[92]CAD Ƞĥ˙,> GM-CSF -ťà,˫>ʥɚ+Ǐʡ.ʒA ?'

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(289) >)ɮ<?>[47]<,Fi-ƻǍʑìȏB GM-CSF ( ÚǸ>)ʘɜɧ˕ûƅðʖû̖Major histocompatibility complexMHC̗ class II BȦȎ>ǔȇɚɹġã>)<[115]ƼÔį.ǔȇɚɹ1Ôçžȁ;0ǥŲçB'CAD Ƞĥ˙,>SlYɚɹġã,˫'

(290) >)ɮ<?'

(291) >[91]č-ȵɄ-ɞǄ<HDM ,; $'ÚǸ?!R`kVFe.GM-CSF BȔȓ>),;$'ĭȏ ìȏ;0ĭ˜ȏ-ġãBʧŁ>))9,SlYɚɹ:ǔȇɚɹ -ǥŲçBˉ'ȌŧÅȝ1-ȾʒBµˍ'

(292) >)ɮ<?! CAD -ȠŹ,

(293) '.ƅðȅȜȨ IgE BŬ)> I ėːƛȡ˞ʘ+ť àBǄ!tZyɚɹ,>ƅðȅȜȨ IgE -Ȕȓ. Th2 ɚɹ<-V FeLFŭ̅( =- Th2 ɚɹ-Ôç;0ǥŲç,.Sl Yɚɹ,;>ƅðƓȸŭʘ)+>!$'CAD -ȦȡǖǑ-ǐ8'× Ƹ-Ǜ˴,

(294) '.SlYɚɹBŬ)!ȌŧÅȝɔ-Ȝœ+ǥ Ųçȓ'

(295) >øɺŲ >SlYɚɹ.ƺžȁ+ȇŹ(ʓȪË ,İē>ƅðƌƋÔç>)nɍ1ˏʸgFs T ɚɹ,ƅ ðBƓȸ>[66]ƼȵɄ,

(296) ' mRNA ʽÍˠġãʦ8<?! GM-CSF .SlYɚɹ-žȁǥŲç;0Û̏ɚɹ<-Ôç,˞ʘ+V FeLF( >)B˥6>)[20]HDM ǥŲçR`kVFe<Ȕȓ ?!ħˠ- GM-CSF ǐ8'×Ƹ- AD ÅȝȠŹ,˫'

(297) >)ɮ<?! !$'HDM ǥŲçFiR`kVFe,> GM-CSF -ȔȓǖǑ Ƭ<,+?/CAD ,Ŀ>ƤʚǡȣǢB˩Ȧ(>øɺŲ >)ɮ! (ǘ-ɉɇ(.FiR`kVFe,> GM-CSF ^nP-Ȕȓ 28.

(298) ,%

(299) 'ʣ³>))!. 29.

(300) 1 .5. ʓ. 30.

(301) kªÙÒµJ{´¬±LG®m·º¼±¬»z z. . AQP3 Carbonic anhydrase II CCL13/MCP4 CCL17/TARC CCL18/PARC CCL27/CTACK CD1a CD26 dipeptidylpeptidase IV CD45 Chondroitin sulphate proteoglican 2 Collagen VI a-1 C-terminal globular domain CXCL8/IL-8 CXCL10 Cysteine-rich protein 1 Cysteine-rich protein 2 Elafin Fibronectin 1 Filaggrin IL-1B IL-1F9 Involucrin Keratin 1 Keratin 2A Keratin 5 Keratin 6A Keratin 6B Keratin 10 Keratin 14 Keratin 16 Keratin 17 Loricrin Lymphocyte-specific protein 1 N-myc downstream regulated gene 1 Nel-Like 2 Osteoblast specific factor 2 Plasminogen activator inhibitor, type I PPP1R5 S100A2 S100A7 S100A8 S100A9 Serine protease inhibitor clade B, member 13 Small proline-rich protein 1A Small proline-rich protein 1B Small proline-rich protein 2B Small proline-rich protein 2C Small proline-rich protein 3 Tenascin precursor Trichohyalin VNN1 VNN3 β-defensin 2. :c >ÐàÕã B}d>|X MiHyq Zb¹¶Hµyq âåØH¹¶9EZbµyq ONZbµyq 9EZb¹¶ZbµfhM Zbk-éÍâåÛäÑ¿æÎ MiH v- Zbp«Q\ZbrYµ~:c ÉáæÈå½ÉæÓ°»3SHEÓÞÀå Hyq ÂÚÁÅÏæZbyq ZbpéZbËÆÔã ZbpéZbËÆÔã ÂáÌÏæÎ!é-(ID?) Zb.P«Zbµ*~«yq« ÇáÐÖÊÀÒµ«ÇáÐå_^µ AK)ÊÀÒÃÀå«âåØH?) Ú¾ÝâæµêR l#¹¶l#t$µ7whM 16#ÇáÐÖÊÀÒµZb 5çÇáÐåè NZb´LG°»ÇáÐå &#ÇáÐÖÊÀÒµZb 5çÇáÐåè +%NZbµZb 5çÇáÐåè +%NZbµZb 5çÇáÐåè 16#ÇáÐÖÊÀÒµZb 5çÇáÐåè &#ÇáÐÖÊÀÒµZb 5çÇáÐåè l#¹¶C´LG°»ÇáÐå #eNZb´LG°»ÇáÐå l#¹¶l#t$µ7whM ¨¢£³]«Zb 5½'* Zbp³4µµËÆÔã²< ;³Zb½oU N*~ NZbµ.P³yqé¡¦¢¤§¨¢£ ÛáÌÝÖæÈå?)! «_@½, hMdâå}|X Zb2xj«Zb âåØH¹¶Hµq) ¥§¤¦¢§¢£ Hµ?)«¿áÄÓå}su ë³³¸´WMiH¹¶H´LG ÄßÒâÛËå8ÍâåÛäÑ¿æÎ! ç0è ç0è ç0è l#t$µ7**«×â¿:c´ì ç0è Zb.P«<T´¯«[`

(302) 7V½ x =´LG«=µ< ©¹¶½n" ©¹¶½n" -gÜÛÐÓ. 31. a/F. [82] [52, 82] [82] [52] [82] [82] [82] [82] [52] [109] [82] [52] [52] [82] [82] [52, 109] [109] [109] [52] [109] [109] [109] [109] [109] [109] [109] [109] [109] [109] [109] [109] [60] [60] [52, 109] [82] [82] [82] [109] [109] [109] [109] [82] [109] [109] [109] [109] [109] [82] [109] [52] [52] [52, 109].

(303) Ja_buecf^5Wyx}72 zO }xV V. +F. B$1. ARTS-1. " © %. [74]. POSTN. 9?E#:UQ`jmqnprsno`jiklh. [74, 121]. Down-regulated in ovarian cancer 1 isoform 2. «&¬. [74]. eIF-5B. ANZL. [74]. Kinectin 1. £ªªI8P~w©. [74]. Myosin Va. £ªªI8P. [74]. A-kinase anchor protein 9 isoform 2. «&¬. [74]. Canis familiaris ret proto-oncogene (RET). «&¬. [74]. GOLGA4 subfamily a, 5. ¥¡RS. [74]. GOLGA4 subfamily a, 4. ¥¡RS. [74]. GOLGA4 subfamily a, 5. ¥¡RS. [74]. EEA1. (©ª¢L=@{,. [74]. CG1574-PA. «&¬. [74]. chromosome 6 open reading frame 142. «&¬. [74]. sushi-repeat-. «&¬. [74]. Hypothetical protein LOC55086. «&¬. [74]. S100A8. 3- v¥©XRS. [74, 121]. INPPL1. d^?EC/?Ev¥©§©?E-. [74, 121]. SCCA-2. £¦©*¦©¨ª\. [74]. SAA3. I8. [74]. Serum amyloid A protin (Canis familiaris). I8. [74]. SAA. I8. [74, 121]. TIMP1. ?EP M!

(304). [74]. similar to RAD50 homologue isoform 1. «&¬. [74]. CIDE-3. GD?E . [74]. sperm-associated antigen 5 (Astrin). )H'< K>Y[TI8P. [74]. Mucin-15. ;. . [74]. hypoxia-induced gene 1. «&¬. [74]. DPP4/CD26. l^?EJ]"®¦©¨ª. [121]. SPINK5. ¦©¨ª\. [121]. SGPL1. ?EUQ?E [|^kaj^ %

(305) [121]. PPAR. hg_td^- !

(306) "064. [121]. Cullin4A. ¤©¦ª. [121]. 32.

(307) 1-3

(308)

(309) . TNF-α IFN-γ IL-1 IL-12 IL-6 IL-8/CXCL8 IL-7 IL-10 IL-15 IL-18 TGF-β CCL2/MIP-1 CCL5/RANTES CCL7/MCP-3 CCL8/MCP-2 CCL11/Eotaxin CCL12/MCP-5 CCL13/MCP-4 CCL17/TARC CCL20/MIP-3α CCL22/MDC CCL27/CTACK CXCL9/Mig CXCL10/IP10 CXCL11/IP9 α-MSH PGE2 GM-CSF SCF. 7 "+jeohewX/36_'\ X36\dsoxkm9 tivqcxlY`]$36\ "+jeohewX/36\ UNEB36^\ 9XSPB36Y`]BUB36[ZaBOMSDW

(310) 9 )(36\< '=; KB36\A36\< tivqcxlY`]$36\BOQDEFB)(XRNLBOOB-&< SPB36Y`]BUB36\< SPB36Y`]BUB36\BOMSDW

(311) 9 UB36\Y`]7\y:36\BOVJB)( LLTFBb-&Za '\=; LLTECBLLTGCBLLTIBb-&Za.8'\=; LLTECBLLTFCBLLTGBb-&Za 'X>'Y`]'\=; LLTECBLLTFBb-&Za5!36X>'X'X 'XU36\=; LLTGBb-&Za>'X'yU36\=; LLTFBb-&Za 'X>'\=; LLTFCBLLTGBb-&Za 'X>'X'XU36\=; LLTHBb-&ZaBUNFB36Y`]'\=;. '236\=; LLTHBb-&Za '_rupxU36\=; 0/36\=; gqfinx36\=; gqfinx36\=; gqfinx36\=; "+* "+X-#X-,. 'X@1'Y`]$36\ X Y`] 36?. 33. 4% [1,21,29,37] [1,29] [1,21,29,37] [1,21,29,37] [1,21,29,37] [1,21,26] [21,29,37] [1,21,29,37] [1,21,29,37] [1,21,29,37] [1,21,29,37] [29] [1,21,37] [29] [29] [29] [29] [29] [1,21,29] [1,21,29] [29] [1,21,29] [1,21,29] [1,21,29] [1,21,29] [21] [1,21,29] [21,29] [1,21,29].

(312) 34.

(313) 1-5

(314)

(315)

(316) . . 26 58 603 687. 65 179 1566 1810. . 623 29 5. 1659 247 32. 657. 1938. 1344. 3748. . . 35.

(317) 36. 523:192. Aquaporin 3. 718426()'$(. < ./. ! ! 2.263 ** 14.152 ** 2.168 3.382 ** 1.296 NA NA 1.257 0.127 * 0.474 * 0.132 0.672 NA NA 4.882 ** 1.055 1.406 1.066 NA NA 11.486 ** 3.084 ** 19.973 12.149 ** 8.562 ** 11.860 ** 1.102 NA NA 0.964 NA NA 0.604 0.817 0.800 NA NA 0.375 * 0.288 * 0.373 * 4.198 ** NA NA 0.771 NA NA 0.627 NA NA 1.411 NA NA 1.321 0.629 NA NA 0.567 0.318 * 0.831 0.279 0.462 * 0.468 * 2.223 ** 0.559 0.766 NA NA 0.771 1.065 NA NA ND NA NA ";.

(318) &%"+0,**&#"-,. 8.725 ** 9.657 ** PREDICTED CA2 XM_535117 0.530 Carbonic anhydrase II 0.543 NM_001003051 0.547 CCL17 CCL17 NM_001003968 1.144 CCL27 CCL27 PREDICTED DPP4 XM_535933 ND Dipeptidyl peptidase 4 / CD26 1.076 partial Collagen VI-alpha1 COL6A1 AJ388539 NM_001003200 2.143 ** IL-8 IL8 2.389 ** 2.185 ** 2.123 ** DR104583 0.988 Cysteine-rich protein 1 CRIP1 U52105 0.836 Fibronectin (Fragment) U16207 0.831 XM_536059 0.859 PREDICTED KRT5 XM_855013 1.157 Keratin, type II cytoskeletal 5 1.101 1.007 1.063 NM_001013425 1.666 keratin 10 KRT10 PREDICTED KRT16 XM_548101 0.919 Keratin, type I cytoskeletal 16 XM_849236 0.795 lymphocyte-specific protein 1 isoform 1 PREDICTED LSP1 PREDICTED NDRG1 XM_539170 1.421 N-myc downstream regulated gene 1 1.388 PREDICTED SERBP1 XM_536673 0.989 Plasminogen activator inhibitor 1 RNA-binding protein 0.657 PREDICTED S100A2 XM_850065 0.751 S100 calcium-binding protein A2 1.104 1.113 S100 calcium binding protein A8 S100A8 DR104918 PREDICTED S100A9 XM_547585 1.102 S100 calcium-binding protein A9 1.123 XM_538811 ND Tenascin precursor, transcript variant 1 PREDICTED TNC NM_001003372 0.757 vanin 1 VNN1 ".. Genbank Accession Number PREDICTED AQP3 XM_849503. 1-6 AD

(319) . *. **. *. **.

(320) 37. PREDICTED SGPL1 Sphingosine-1-phosphate lyase 1 Peroxisome proliferator-activated receptor gamma PPARG cullin 4A isoform 1 PREDICTED CUL4A. DPP4 SPINK5. Dipeptidyl peptidase 4 / CD26 Serine peptidase inhibitor, Kazal type 5. PREDICTED SRPX2 PREDICTED INPPL1. sushi-repeat-containing protein Inositol polyphosphate phosphatase-like 1. SAA1 S100A8 TIMP1. PREDICTED MYO5A PREDICTED AKAP9 PREDICTED EEA1. Myosin-5A (Myosin Va) A-kinase anchor protein 9 isoform 2 Early endosome antigen 1. Serum amyloid A protein S100 calcium binding protein A8 TIMP metallopeptidase inhibitor 1. PREDICTED KTN1. Kinectin 1. PREDICTED FILIP1L. Downregulated in ovarian cancer 1 isoform 2 ** ** **. ** **. **. **. ** ** ** **. ** ** *. **. **. *. 2.075 ** NA NA. 1.055 0.166 *. 1.08 NA NA. 1.406 172.01 **. ND 0.559 1.162. NA NA. NA NA. ND 2.223 ** 3.178 **. NA NA NA. NA. NA. NA NA NA. ! NA. NA. < ./. XM_535715. &%"+0,**&#"-,. ! 15.024 15.136 15.542 1.766 1.621 1.655 1.209 0.834 0.467 0.717 1.203 1.019 0.512 2.177 1.216 2.446 3.651 2.398 5.664 0.468 0.765 0.841 4.882 2.220 2.448 2.390 2.269 3.407 0.755 1.083 1.023 1.044. 718426()'$(. ";.

(321) . ** ** **. 523:192 2.195 2.277 2.640 1.035 XM_537455 1.493 1.141 1.165 0.870 XM_535487 1.883 XM_532456 0.876 XM_532649 1.113 1.030 0.633 XM_549134 1.782 XM_542327 1.155 2.672 0.537 1.609 3.679 NM_001003050 1.113 DR104918 1.379 NM_001003182 1.236 ND XM_535933 1.992 NM_001025397 1.719 3.890 2.017 1.323 XM_546150 0.735 NM_001024632 0.880 XM_844449 0.916 0.977 ".. Genbank Accession Number. 1-7CAD

(322)

(323) .

(324) 38 TGFB2 CSF2. Transforming growth factor beta 2 (Fragment). Granulocyte-macrophage colony stimulating factor. IL15 IL15 IL18. Interleukin-15 (Fragment) Interleukin 15 Interleukin 18 CCL2 CCL17 CCL20 CCL27 TGFB1. IL7. Interleukin 7. CCL2 CCL17 CCL20 CCL27 Transforming growth factor, beta 1. IL12A IL12B IL8. Interleukin 12A (IL-12, p35) Interleukin 12B (IL-12, p40) Interleukin 8. PREDICTED. TNF. . Tumor necrosis factor. 523:192 ! 0.823 1.890 1.775 ND 11.486 12.149 8.562 11.860 ND 0.870 2.025 1.751 0.622 0.601 4.388 0.127 19.046 0.672 1.020 1.028 2.050 2.170 6.806 4.213. &%"+0,**&#"-,. NM_001003244 ND ND NM_001003293 1.747 NM_001003292 ND NM_001003200 2.143 ** 2.389 ** 2.185 ** 2.123 ** NM_001048138 ND 1.144 AF479882 1.985 XM_844053 1.642 NM_001003169 0.835 0.840 NM_001003297 3.755 ** NM_001003051 0.547 NM_001005254 ND NM_001003968 1.144 NM_001003309 1.041 1.043 XM_853503 1.180 DQ525400 1.530 NM_001003245 2.474 ** ND ".. Genbank Accession Number. 6.376 ** 0.474 * 9.530 ** NA NA. 2.199 ** NA. 28.470 ** 0.132 * 12.424 ** NA NA. 1.096 NA. NA. NA. < ./. ** 1.806 3.778 ** ** ** 2.278 ** 9.996 ** ** ";.

(325) . ** * **. **. ** ** ** **. NA NA 19.973 **. ! NA. NA NA 3.084 **. NA. 718426()'$(. 1-8

(326) .

(327) ɉ ̛ɇ. " %!$&

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(330) 'ĭ˜ȏ-ǥŲçBµˍ>)9Ƭ<)+$'=[116]ǻȡŲVFeL F)'ǒ +ȟŴ-ȠŹ,˫'

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(333) >)ɮ<?'

(334) >[76]˫ɍǷʁǻB Ƞĥ)>ʄŏÅȝŲȟŴ( >oe-Gy`,

(335) '.ǷʁËʑɌĂĐ, ƅðƓȸɺBƶ>ǔȇɚɹÓȎ̡ɚɹ;0̣ɚɹ-ǥŲçȓ'

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(338) >Ǽ(Gy`-ȠŹ,̈ª'

(339) >ǔȇɚɹ-Ôç žȁ;0ǥŲç,;>ƅðƓȸɺ-ʵǥ,GM-CSF˞ʘ( >)< [120]?<-ȟŴ,

(340) 'ġã>GM-CSF.ƅȔȓ,

(341) !>ǐ8'×ƸȠŹ,˫'

(342) >øɺŲ >GM-CSF-ʘ+Ȕȓɚɹ)'̣ɚɹ ;0yPrCXİē>oeADŴɯ<Ǝõ!ʓȪR`kVFe -×£ę̍,

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(344) >)<[92]R `kVFeȔȓ>GM-CSFAD-ȠŹ,˫'

(345) >øɺŲ.̒

(346) Der f;0Der p.oe;0Fi-AD,>ʘ+ȑĠD S( >ȅ,Der f.Fi-AD,

(347) 'Ƶ9ħŸ¯ʦ8<?>HDM( > )ȸ?'

(348) >[46]Der f-ʘ+D S^nP( >Der f 1.nnF 40.

(349) ǒWYcFtcD\( =[28]ƼÔį-ƶ>^nPʶÔʞǥŲ. HDM-D SŲ)˫ˋ>)ȸć?'

(350) >[75]oe-ǙœȪʀ-R `kVFe,

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(352) >ɉ1ɇ-ɞ Ǆ;=Ȇ,

(353) '9HDMD S,;$'R`kVFe,>GM-CSF mRNA-ʽÍʧŁ?!)<R`kVFe<Ȕȓ?!GM-CSF Ȇ-D OŲǻȡBʧŁ'

(354) >øɺŲɮ<?!+< GM-CSF-^nP v,>ȦȎ,%

(355) '.Ǐʡ?'

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(358) 'Ǐʡ!. 41.

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(360) >9-Bȕ

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(365) 'ɚɹȓİȍBǏʡ!. 2.2.4. ę̍Ǯ,>GM-CSFǺŜ CPEKBDer f5!.E-64;0Der f-İē(24Ʈ˪5!.48Ʈ˪ę̍! Ŧę̍ǮBčô!"#,-80(Ïɞ·İ!ę̍Ǯ-GM-CSFǺ Ŝ. DuoSet® Canine GM-CSF ELISA kit̖R&D System, Minneapolis, MN̗ Bȕ

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(367) !tcD\ˮĸĶ̐(.E-64Bǭã'

(368) +

(369) Der fǭ ãɬBĿǿ)!Dunnet ǏĵBȕ

(370) !ɟʠĲȨƶŷŎ. P < 0.01 )ĵɭ 43.

(371) !ʞǃ,.ɟʠ]re JMP version 5.1.2̖SAS institute, Cary, NC̗B° ȕ!. 44.

(372) 2 .3. ɞǄ. 2.3.1. CPEK-ɚɹţŹ)ȓİȍ Der f;0E-64-İē,

(373) '9ɚɹ-ţŹ.ǽǭãɬ)Ȱ˓+ɗˢţ Bȸɚɹ˪;0W~ ś˙1-ƐȳŲ9ɡƈ?'

(374) !̖đ2-1̗5!

(375) ?-ǀ¥,

(376) '9ɚɹȓİȍ. 79.7̤84.6% ( =Der f;0E-64 ǭã-Ť́.ʦ8<?+$!̖đ2-2̗. 2.3.2. Der fǭã,;>R`kVFe-GM-CSFȔȓ ę̍24Ʈ˪Ŧ,>ę̍Ǯ-GM-CSFǺŜ.ǽǭãɬ 2.042 ±2.095 pg/mL Der f 1ǭã 1 μg/mLɬ 5.273 ±6.371 pg/mL Der f 1ǭã 10 μg/mL ɬ(. 21.733 ±18.551 pg/mL ( $!ę̍48Ʈ˪Ŧ,

(377) 'úɬ-ę̍Ǯ -GM-CSFǺŜ.ġãÀýBȸǽǭãɬ 5.214 ±3.985 pg/mLDer f 1 ǭã 1μg/mLɬ(. 40.156 ±11.131 pg/mL10μg/mLɬ(. 70.415 ±19.169 pg/mL( $!ɟʠʞǃ-ɞǄCPEK-GM-CSFȔȓ.Der f 1-ǺŜ; 0ę̍Ʈ˪,²İ'ƶŷ,ġã>)Ƭ<)+$!̖đ2-3̗. 2.3.3. WYcFtcD\,;>GM-CSFȔȓ-ƃÙ Der f 1B!R`kVFe-GM-CSFȔȓ.E-64B10-7 M-ǺŜ(ǭã !Ĝû(-6ƃÙ?10-8 M¤-ǺŜ(.Ŀǿɬ̖Der f 1-6̗)-ƶŷ Ŏ.ʦ8<?+$!̖đ2-4̗. 45.

(378) 2 .4. ɮļ. ƼȵɄ-ɞǄ<Der f .Fi-R`kVFe,Ŀ'9 GM-CSF -Ȕ ȓBʧŁ>)Ƭ<)+$!5!GM-CSF -Ȕȓ.WYcFt cD\ˮĸÞ,;$'ƃÙ?!)<ȔȓǖǑ,. Der f -tcD \ǥŲ˫'

(379) >)űA?!Der f ,.WYcFtcD\( > Der f 1 þ5?'=CPEK ,> GM-CSF -Ȕȓ,

(380) 'ʘ+ťà BǄ!'

(381) >)ɮ<?!HDM -ƅð^nP( > Der f 1 .ńËĘ 1 g ,

(382) '. ŕĖ 2.25 ±4.97 μg İē'=ħ

(383) )@(. 30 μg ¤İē>Ĝû9 >[99]5!CAD ɩŴȆB̌ɵ>ĹńË-Ę,İē > Der f .̌ɵȆ CAD ,ɩŴ'

(384) +

(385) Ĺŝ)<2'ħ

(386) )ȸ ?'

(387) >)<[31]ƼȵɄ(ȕ

(388) ! Der f 1 ǺŜ̖10 μg/mL̗.Ķ˶-ȑ Ġ)34üȿŜ-ǺŜ( >)ɮ<?! HDM ȅȜȨ IgE -Ȕȓ,ÃɆ#ƅðƓȸɚɹ,;>ƅðƌƋ)nȏ 1-ƅðƓȸŭʘ( >ʓȪ,>ƅðƓȸɚɹ( >SlY ɚɹ.ƺÔçȇŹ(ʓȪ,İēÚǸ,;$'ǥŲçMHC class II B ȦȎ'ƅðBƓȸ>[69]ǥŲç!SlYɚɹ.ʓȪ-^Fe X~PWBʦʭÔǅBʝŇƥý,©˦>),;$'ƅðBƌƋ >)yGY,

(389) 'Ƭ<,?'

(390) >[69]-; , GM-CSF . SlYɚɹ-Ôç;0ƅðƌƋɺ-ǥŲç,˞ʘ+Ôį( > GM-CSF -ʘȔȓɚɹ. T ɚɹ:yPrCX+*( >ƼȵɄ-ɞ Ǆ<ƅð´Æ-ǐ8'ƪƸ-Ǜ˴,

(391) '˷Ɛ>R`kVFe<Ȕ ȓ?>øɺŲ̒5$!oe,

(392) '.PMA̖Phorbol 12-myristate 13-acetate̗ÚǸ,Ŀ>ʓȪR`kVFe- GM-CSF Ȕȓ-ˠ AD Ŵɯ )½œɯ,

(393) 'Ȝ+>) in vitro (ȸ?'

(394) >[92]!$' 46.

(395) GM-CSF Ȕȓ|LhZ{BƬ<,>)üƮ,½œȆ) CAD ɩŴȆ-R `kVFe,> GM-CSF Ȕȓˠ5!.Ȕȓ|LhZ{-ǜʾǏʡ+< 0,˗¨įĥȜ-ƶǽ,%

(396) 'ǏʡCAD -ȠŹʞƬ-6+<R`kV FeBǓȨ)! CAD ,Ŀ>ƤʚǡȣǢ-˩Ȧ,ƶȕ( >)űA?! WYcFtcD\B! CPEK -ǥŲçǖŚ)'Protease activated receptor̖PAR̗-˫ɮ<?>PAR .ȅĵ-tcD\, ;$'ȅȜȨ,ǥŲç?>ˠ G ^nPÉť-̟čʁʲˉėöĺ( >Ȏē5(,̝%- PAR rCzüĵ?'=-rCz- %( > PAR-2 .ȓË,ŘÔŐʓȪĚśŇ-R`kVFe,

(397) ' 9ȦȎ'

(398) >)ȸ?'

(399) >[49]PAR-2 -ɚɹʁĦ,˽Ó'

(400) > N ƻɈ¾wt`fˣ.etWet^\5!.ʑǪÐĒɉ VIIaXa Ďį Ɋ-tcD\,;$'Õƣ?>Õƣ?! N ƻɈ. Tethered ligand )' PAR-2 Ôį-ɚɹĦɉ̛t,ɞûWQg¨˒?>PAR-2 .,[tcD\,;$'ǥŲç?>)ɮ<?'!˄Ŗ oe-ɚɹǊ,

(401) 'WYcFtcD\,;$'9ǥŲç?>) Ƭ<)+$!)<[100]Der f 1 ,;> CPEK -ǥŲç, PAR-2 ˫ '

(402) >øɺŲɮ<?! oe-R`kVFe,> GM-CSF -Ȕȓ.ǋËʽÍĎį nuclear factor-kappa B̖NF-κB̗B'ȓ>)ȸ?'

(403) >[54]NF-κB . PAR-2 B'9ǥŲç>)ȸ?'

(404) >)<[56]Der f ÚǸ,; > GM-CSF Ȕȓ,

(405) '9üǒ-|LhZ{˫ˋ'

(406) >øɺŲ >) ɮ<?!5!nȏ,> GM-CSF Ȕȓ,.ʽÍĎį nuclear factor activated T-cell̖NFAT̗-ǋËȾʒ;0 Activator protein-1̖AP-1̗ )-ɞû˞ʘ( >)ěā?'=[51]oeR`kVFe,

(407) ' .NFAT B!WQg¨˒ɝʻİē'

(408) >)9ȸ?'

(409) >[2, 3] 47.

(410) !$'ɉɇ(. GM-CSF -Ȕȓ,>WQg¨˒ɝʻBƬ<, >)BȮȨ)'PAR-2 B! NFAT -ǥŲç) GM-CSF -Ȕȓ, %

(411) 'Ǐʡ>))!. 48.

(412) 2 .5. đ. 49.

(413) đ 2-1 Der f ;0 E-64 -ǭãę̍>ɚɹţŹ̖48 Ʈ˪Ŧ̗ a) DPBS -6b) Der f 1̖10μg/mL̗c) E-64̖10-5 M̗d) Der f 1̖10μg/mL̗ ;0 E-64̖10-5 M̗m. 20μm Bȸ. 50.

(414) .

(415) . . . . .

(416) . .

(417) . .

(418) . .

(419) . .

(420) . . . . đ 2-2 Der f ;0 E-64 ǭãę̍,>ɚɹȓİƞ̖48 Ʈ˪Ŧ̗. 51.

(421) . . . . .

(422) .

(423) . . . . . . . . . . . .

(424) . đ 2-3 Der f ǭãę̍,> CPEK - GM-CSF Ȕȓ ̜č-ȈɆ!Ķ̐-ŕĖ +S.D.Bȸ̘̠ P < 0.01̖Tukey-Kramer’s HSD Test̗. 52.

(425) .

(426) . . . . . . . . . . . . . . . . . . .

(427) . ! . đ 2-4 GM-CSF Ȕȓ,>WYcFtcD\ˮĸÞ-ƃÙå Ǆ ̜č-ȈɆ!Ķ̐-ŕĖ + S.D.Bȸ̘̠ P < 0.01̖Der f -6Bǭã !ɬBĿǿ)! Dunnet’s Test̗. 53.

(428) ɉ ̜ɇ. WYcFtcD\B!FiR`kVFe,> GM-CSF-Ȕȓ)NFAT-˫. 54.

(429) 3 .1. Śʫ. R`kVFe.ʓȪ-95̕¤Bí8>Ǒžɚɹ( =ǒ +VFeLF BȔȓ>),;$'Ȫʀ-ǻȡ:Åȝóů,˫'

(430) >[4, 29]R `kVFe,

(431) 'Ȕȓ?>VFeLF-(9IL-1IL-8CCL17; 0TSLP.D OŲȪʀȟŴ-ȠŹ,

(432) '˞ʘ+VFeLF( > LPS ( ǥ Ų ç ? ! o e - R ` k V F e . NF-κB ; 0 extracellular signal-regulated kinase̖ERK̗B'IL-1β;0IL-8BȔȓ>)ȸ ?![70]Fi-ʓȪR`kVFeɚɹǊ( >CPEK,

(433) '.TNF-α-Ú Ǹ,;$'p38˜ç?CCL17 mRNA-ʽÍʧŁ?>[111]LP F5B!R`kVFe<-TSLP-Ȕȓ,.PAR-2)-Ǧ,İē >NF-κB-ǥŲç˫'

(434) >[13]-; ,R`kVFe,

(435) ' .Ħ˙<-ǒ +ÚǸ,ů'?. ?Ȝ+>WQg¨˒ɝʻǥŲç. ?VFeLFȔȓ?> Ûɇ,

(436) 'FiR`kVFe<-ȔȓƬ<)+$!GM-CSF9D O-ÅȝȠŹ,˫>Ôį( >oe-nȏ,

(437) '.PMAB !GM-CSF-Ȕȓ,NFAT-ǥŲç˫'

(438) >)ȸ?'

(439) >[51]˄ ŖoeR`kVFe,

(440) '9NFATİē[2, 3]WPKNWSg \-2-Ȕȓ,˫ˋ'ǥŲç>)Ƭ<,?'

(441) >[34]R` kVFe,>GM-CSF-Ȕȓ)NFAT-˫ˋŲ.Ǐʡ?'

(442) +

(443) (Ƽɇ(.WYcFtcD\(ǥŲç?!Fi-R`kV Fe- GM-CSFȔȓ,

(444) 'NFAT˫'

(445) >BǏʡ!Ûɇ5( HDMBȕ

(446) '!ĩǾƆÓȄ-ÆƂď˻)+$!)<Ƽȡ(.HDM üǒ,WYcFtcD\ǥŲBƶ>nnFBȕ

(447) !nnF.y GY-Th2ɚɹ;0ǔȇɚɹ-ÔçBµˍ>3[118]PAR-2 B'o 55.

(448) e-R`kVFeBȯƐǥŲç>)ȸ?'

(449) >[100](Ƽɇ (.nnFB!FiR`kVFeɚɹǊ̖CPEK̗<-GM-CSFȔȓBELISA,;$'ȷʦŦWrH\Da[FBȕ

(450) 'NFAT-ǋË Ⱦʒ,%

(451) 'ʣ³!<,WPYx̖Ciclosporine, CsA̗Bȕ

(452) ! ˮĸĶ̐,;$'CPEK-GM-CSFȔȓ,>NFAT-˫-ȿŜ,%

(453) 'Ǐ ʡ!. 56.

(454) 3 .2. ƾƢ)ƥǢ. 3.2.1. ƾƢ F i ʓ Ȫ R ` k V F e - ɚ ɹ Ǌ ( > CPEK ) - ę ̍ Ǫ CnT-09 . CellnTEC advanced cell system<ʷÆ!5!ǽʑǮęĕOpti-MEM I ̖Invitrogen̗;0ɠ£ę̍Ʈ-ɚɹÝ˺,etWÑȐÞTrypLE express ̖Invitrogen̗B°ȕ!DPBSWYcFtcD\( >nnF; 0WYcFtcD\ˮĸÞ( > Trans-Epoxysuccinyl- L-Leucylamido̖4-Guanidino̗Butane̖E-64̗.Sigma-Aldrich<ÆƂ!. 3.2.2. ɚɹę̍ ̞č<10čɠ£!CPEKB10 mL-CnT-09,Ǩˏ375% CO2ȑĠ(75 cm2rYT̖NUNC̗Bȕ