Title 犬三次元培養表皮モデルの免疫バリア機能に関する研究( 本文(Fulltext) ) Author(s) 寺本, 圭志 Report No.(Doctoral Degree) 博士(獣医学) 甲第515号 Issue Date 2018-09-21 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/77276 ※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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≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢචࣂࣜᶵ⬟㛵ࡍࡿ◊✲
2018 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉
㸦ᒱ㜧Ꮫ㸧
ᑎᮏ ᆂᚿ
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≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢචࣂࣜᶵ⬟㛵ࡍࡿ◊✲
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┠ḟ
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➨㸯❶ ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿࢧࢺ࢝ࣥࡢ㌿㔞
ゎᯒ
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ᗎㄽ
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➨㸰❶ ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ
ZO-1 ࠾ࡼࡧ claudin-1 ࡢ
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➨㸱❶ ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ
ZO-1 ࠾ࡼࡧ claudin-1
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TNF-alpha ࠾ࡼࡧ IL-13 ࡢᙳ㡪
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3.2.
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AD: atopic dermatitis㸦ࢺࣆ࣮ᛶ⓶⅖㸧 BSA: bovine serum albumin㸦∵⾑Ύࣝࣈ࣑ࣥ㸧 CAD: canine atopic dermatitis㸦≟ࢺࣆ࣮ᛶ⓶⅖㸧 CCL: CC chemokine ligand
CCR: CC chemokine receptor
CPEK: canine progenitor epidermal keratinocyte DPBS: Dulbecco’s phosphate-buffered saline
GAPDH: glyceraldehyde 3- phosphate dehydrogenase GM-CSF: granulocyte macrophage colony-stimulating factor HDM: house dust mite㸦ࣁ࢘ࢫࢲࢫࢺ࣐ࢺ㸧
HPRT1: hypoxanthine phosphoribosyl-transferase 1 IL: Interleukin Ig: immunoglobulin㸦චࢢࣟࣈࣜࣥ㸧 LPS: lipopolysaccharide㸦࣏ࣜከ⢾㸧 PBS: phosphate-buffered saline㸦ࣜࣥ㓟⦆⾪㣗ሷỈ㸧 RPL: ribosomal protein L RPS18: ribosomal protein S18
RT-qPCR: quantitative reverse transcription PCR TARC: thymus and activation regulated chemokine TBS: Tris buffered saline
TER: transepithelial electrical resistance
TEWL: transepidermal water loss㸦⤒⓶ⓗỈศᩓ㔞㸧 TGF: transforming growth factor
TNF: tumor necrosis factor
Th2: 㸰ᆺ࣊ࣝࣃ࣮T ⣽⬊㸦Type 2 helper T㸧 ZO: zonula occluden
⥴
⥴ㄽ
≟ ࢺ ࣆ ࣮ ᛶ ⓶ ⅖ (CAD) ࡣ 㸪 ⎔ ቃ ࣞ ࣝ ࢤ ࣥ ᑐ ࡍ ࡿ ≉ ␗ ⓗ ࡞ Immunogloblin (Ig) E ᢠయࢆࡋࡓ㸪៏ᛶ⅖ᛶᥙᛶ⓶ᝈ࡛࠶ࡾ㸪㑇ఏⓗ ⣲ᅉ࠾ࡼࡧ⎔ቃせᅉࡀ㛵ࡍࡿ[50]ࠋయෆධࡋࡓࣞࣝࢤࣥࢆᤕᤊࡋࡓᢠཎ ᥦ♧⣽⬊ࡣ㸪ࢼ࣮ࣈ࣊ࣝࣃ࣮T ⣽⬊ᢠཎᥦ♧ࢆ⾜࠸㸪Th2 ⣽⬊ඃ࡞ศࢆ ᑟࡃࠋࡲࡓ㸪άᛶࡋࡓB ⣽⬊ࡣ㸪IL-4 ࡲࡓࡣ IL-13 ࡢᏑᅾୗ࠾࠸࡚ IgE ࢆ⏘ ⏕ࡍࡿᙧ㉁⣽⬊ศࡍࡿ[53]ࠋࡇ࠺ࡋ࡚⏘⏕ࡉࢀࡓ IgE ࡣ㸪ࡑࡢᚋ┿⓶ࡢ⫧ ‶⣽⬊⤖ྜࡋ㸪ࣞࣝࢤࣥࡀ IgE ࢆᯫᶫࡍࡿࡇࡼࡾ⫧‶⣽⬊ࡽࡢ⬺㢛 ⢏ࡀច㉳ࡉࢀ㸪CAD ࡢⓎ⮳ࡿ[53]ࠋデ᩿ࡣ㸪ឤᰁࡢ㝖እデ᩿࠾ࡼࡧほ ࡼࡿ⓶⑈ศᕸࡢホ౯ࡀయ࡛࠶ࡾ㸪ᐈほⓗ࡞デ᩿ἲࡣᏑᅾࡋ࡞࠸[66]ࠋࡲࡓ㸪 ⒪࠾࠸࡚ࡣ㸪ࢫࢸࣟࢻ⒪ἲࢆࡣࡌࡵࡍࡿ㠀≉␗ⓗ࡞චᢚไ⒪ἲ㢗ࡗ ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿ[65, 66]ࠋࡇࢀࡽデ᩿⒪࠾ࡅࡿၥ㢟Ⅼࡋ࡚㸪ࣞ ࣝࢤࣥᭀ㟢ࡽឤస⮳ࡿヲ⣽࡞ᶵᗎࡀ༑ศゎ᫂ࡉࢀ࡚࠸࡞࠸ࡇࡀ࠶ࡆࡽ ࢀࡿࠋ CAD Ⓨ⮳ࡿࣞࣝࢤࣥࡢධࡣ㸪྾ჾ㸪ᾘჾࡢ⢓⭷࠾ࡼࡧ⓶ࢆ ࡋ࡚㉳ࡇࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ[53]ࠋࡇࢀࡲ࡛㸪ࣃࢵࢳࢸࢫࢺ࠾ࡼࡧ⓶ෆᛂ ヨ㦂࠾࠸࡚㸪house dust mite㸦HDM㸧ᑐࡋ㝧ᛶᛂࢆ♧ࡍࣅ࣮ࢢࣝ≟㸪HDM ⁐ᾮࢆሬᕸࡍࡿࡇࡼࡾ㸪CAD ࣔࢹࣝ≟ࡀస〇ࡉࢀ࡚࠸ࡿࠋࡑࢀࢆ⏝࠸ࡓ㸪 ⮫ᗋࢫࢥ㸦modified Canine atopic dermatitis extent and severity index, mCADESI㸧 [67]ࡢホ౯ࡽ㸪⤒⓶ⓗ࡞ឤసࡢ㔜せᛶࡀ᫂ࡽ࡞ࡗࡓ[47]ࠋ ⓶ࡣయෆእ⏺ࡢቃ⏺ࢆᡂࡋ㸪ࡑࡢ᭱እᒙࡣ⾲⓶ࡀᏑᅾࡋ࡚࠸ࡿࠋ⾲⓶ ࡣࡉࡽእᒙࡼࡾゅᒙ㸪㢛⢏ᒙ㸪᭷Ჲᒙ࠾ࡼࡧᇶᗏᒙศ㢮ࡉࢀ㸪⾲⓶ࡢ⣙85% ࡣࢣࣛࢳࣀࢧࢺࡼࡗ࡚ᵓᡂࡉࢀ࡚࠸ࡿ[55]ࠋᇶᗏᒙࢣࣛࢳࣀࢧࢺࡣቑṪࡋ㸪 ศࡋ࡞ࡀࡽୖᒙ⛣⾜ࡍࡿࠋゅᒙࡣ㸪㢛⢏ᒙࡢࢣࣛࢳࣀࢧࢺࡀ⬺᰾ࡋࡓᚋ ᙧᡂࡉࢀ㸪࿘㎶ᖏࡤࢀࡿᵓ㐀≀ࡀ⣽⬊⭷ࢆᡴࡕࡋ࡚࠸ࡿࠋ࿘㎶ᖏࡣ㸪᭷Ჲ ᒙ࠾ࡼࡧ㢛⢏ᒙ࡛సࡽࢀࡿࣥ࣎ࣝࢡࣜࣥࡸࣟࣜࢡࣜࣥࢆ࡞ᵓᡂせ⣲ࡋ㸪
ࡇࢀࡽ㢛⢏⣽⬊⏤᮶ࡢࣇࣛࢢࣜࣥࡀᯫᶫ⤖ྜࡋ࡚࠸ࡿ[36]ࠋࣇࣛࢢࣜࣥࡣ ᚋゅᒙୖᒙ࡛ศゎࡉࢀ[29]㸪㢛⢏ᒙࡢᒙᯈ㢛⢏⏤᮶ࡍࡿࢭ࣑ࣛࢻࢆࡣࡌ ࡵࡋࡓゅ㉁⣽⬊㛫⬡㉁ඹ㸪Ỉศಖᣢ࠾ࡼࡧ⓶ࣂࣜᶵ⬟ࡢ⥔ᣢ㔜せ ࡞ᙺࢆᯝࡓࡍ[11, 14, 28, 33, 82, 93]ࠋࣄࢺࡢࢺࣆ࣮ᛶ⓶⅖㸦Atopic dermatitis, AD㸧࠾࠸࡚㸪ࣇࣛࢢࣜࣥࡢኚ␗ࡀሗ࿌ࡉࢀ㸪ඛኳⓗ࡞⓶ࣂࣜᶵ⬟ ࡢḞᦆࡀAD Ⓨࡢ㔜せ࡞ᅉᏊ࡛࠶ࡿࡇࡀ᫂ࡽ࡞ࡗࡓ[71]ࠋࡲࡓ㸪CAD ࣔ ࢹࣝ≟࠾࠸࡚㸪ࣇࣛࢢࣜࣥࡀṇᖖ≟ẚ࡚ῶᑡࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ ࡚࠸ࡿ[52]ࠋࡉࡽ㸪ࣄࢺ AD ࠾ࡼࡧ CAD ࠾࠸࡚㸪ࢭ࣑ࣛࢻࡀṇᖖ⩌ ẚ㍑ࡋ࡚ῶᑡࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[32, 101]ࠋCAD ࠾࠸࡚㸪⤒ ⓶ⓗỈศᩓ㔞㸦Transepidermal water loss, TEWL㸧ࡀቑຍࡋ㸪⓶ࣂࣜᶵ⬟ࡢ పୗࡀ♧ࡉࢀ࡚࠸ࡿ[89]ࡇࡽ㸪ࣇࣛࢢࣜࣥࡸࢭ࣑ࣛࢻ࡞ࡢศᏊࢆ⏘⏕ࡍ ࡿࢣࣛࢳࣀࢧࢺࡢᶵ⬟␗ᖖࡀCAD ែ㛵ࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡿࠋ ࢣࣛࢳࣀࢧࢺࡣᵝࠎ࡞ᾮᛶᅉᏊࢆ⏘⏕ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ[25, 93]ࠋ HDM ࡼࡗ่࡚⃭ࡉࢀࡓ≟ࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪⅖ᛶࢧࢺ࡛࢝ࣥ࠶ ࡾ㸪ᵝࠎ࡞⣽⬊ࡽࡢࢣࣔ࢝ࣥࢆㄏᑟࡍࡿtumor necrosis factor (TNF) -alpha ࡸ㸪 㢛⢏⌫࠾ࡼࡧ༢⌫ࡢάᛶᅉᏊ࡛࠶ࡿ granulocyte macrophage colony-stimulating factor (GM-CSF)㸪ࡲࡓᶞ≧⣽⬊ࡢ㐟㉮࠾ࡼࡧάᛶࡸ㸪ࢼ࣮ࣈ T ⣽⬊ࡽ Th2 ⣽⬊ࡢศࢆಁࡋ㸪ࣞࣝࢠ࣮ᛶ⅖㔜せ࡞ᙺࢆᯝࡓࡍ Thymic stromal lymphopoietin (TSLP) ࡢ㌿㔞ࡢቑຍࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ[41, 46]ࠋࡉࡽ㸪 CAD ࡢ ኚ 㒊 ࠾ ࠸ ࡚ ࡣ 㸪 ⮋ ⓶ ࡢ ཎ ᅉ ⳦ ࡛ ࠶ ࡿ Staphylococcus pseudintermedius ࡢ㐣ቑṪࡀࡋࡤࡋࡤㄆࡵࡽࢀࡿ[19]ࠋࡑࡢ⣽⬊ቨᵓᡂᡂศࡢ ่⃭ࡼࡗ࡚㸪≟ࢣࣛࢳࣀࢧࢺ࠾࠸࡚ࡶ TSLP ࡢ㌿ࡀㄆࡵࡽࢀࡿࡇࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ[81]ࠋࡇࢀࡽࡢ⤖ᯝࡽ㸪ࢣࣛࢳࣀࢧࢺࡣ⾲⓶ࡢ≀⌮ⓗࣂࣜ ࡢࡳ࡞ࡽࡎ㸪ࢧࢺ࢝ࣥ࠾ࡼࡧࢣࣔ࢝ࣥࢆࡋ㸪චᏛⓗࡶCAD ࡢែ 㛵ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋ
୍᪉࡛㸪ࢣࣛࢳࣀࢧࢺࡣ㸪ゅᒙ࠾ࡼࡧࢱࢺࢪࣕࣥࢡࢩࣙࣥࡽ࡞ࡿ⓶ࣂ ࣜࢆᙧᡂࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋCAD ࣔࢹࣝ≟࠾࠸࡚ࡣ㸪ኚ㒊࠾ࡼࡧ 㠀ኚ㒊⓶࠾࠸࡚㸪ゅᒙ⣽⬊㛫⬡㉁ࡢᚤ⣽ᵓ㐀ࡢኚࡀ᫂ࡽ࡞ࡗ࡚࠸ ࡿ[51]ࠋ
ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢⓎ⌧ኚ㛵ࡍࡿሗ࿌ࡋ࡚ࡣ㸪CAD ࣔࢹࣝ≟࠾ ࠸࡚㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢ㔜せ࡞ᵓᡂࢱࣥࣃࢡ࡛࠶ࡿzonula occluden (ZO)-1 ࡸ claudin-(ZO)-1 ࠾ࡅࡿⓎ⌧ࡢపୗࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ[37, 79]ࠋࡲࡓ㸪TNF-alpha ࡸ Interleukin (IL)-1beta ࠶ࡿ࠸ࡣ Th2 ࢧࢺ࡛࢝ࣥ࠶ࡿ IL-4, IL-13 ࠾ࡼ ࡧ IL-31 ࡣ㸪ࢣࣛࢳࣀࢧࢺࡢᇵ㣴⣽⬊࠾࠸࡚㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࢆᵓ ᡂࡍࡿࢱࣥࣃࢡⓎ⌧ኚࢆ㉳ࡇࡍࡇࡀ♧ࡉࢀ࡚࠸ࡿ[2, 18, 26, 40, 98]ࠋࡋࡓ ࡀࡗ࡚㸪CAD ࠾ࡅࡿ⓶ࣂࣜᶵ⬟ࡢపୗࡣ㸪CAD ࡢ⓶࠾ࡅ ࡿ Th2 ᆺ⅖ࢆ≉ᚩࡋࡓ⎔ቃ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᶵ⬟␗ᖖࡀ㛵ࡋ ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿ[48]ࠋࡋࡋ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥⓎ⌧ኚࢆ㉳ࡇࡍせ ᅉ࠾ࡼࡧࡑࢀ㉳ᅉࡍࡿࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᶵ⬟㞀ᐖCAD ែ࠾ࡅࡿ 㛵㐃ࡣ࡛᫂࠶ࡿࠋCAD ែࡢࡉࡽ࡞ࡿゎ᫂ྥࡅ㸪ࢣࣛࢳࣀࢧࢺࡀ㛵ࡍ ࡿ⓶චࣂࣜᶵ⬟ࡢ◊✲ࡣ㸪⏕య࠾ࡅࡿᛂᛅᐇ࡞in vitro ࣔࢹࣝࡀ ᚲせࡉࢀ࡚࠸ࡿࠋ ࡇࢀࡲ࡛㸪≟ࢣࣛࢳࣀࢧࢺ⣽⬊ᰴࢆ⏝࠸ࡓ୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡀస〇 ࡉࢀ㸪⾲⓶㢮ఝࡋࡓᙧែࡢᙧᡂ࠾ࡼࡧྛ✀ࡢศ࣐࣮࣮࢝ࡢⓎ⌧ࡀሗ࿌ࡉࢀ ࡓ[99]ࠋࡑࡇ࡛㸪ᮏ◊✲࡛ࡣ㸪≟ࢣࣛࢳࣀࢧࢺ⣽⬊ᰴࢆ⏝࠸ࡓ୕ḟඖᇵ㣴⾲⓶ ࣔࢹࣝࢆస〇ࡋ㸪ࢧࢺ࢝ࣥࡢ⏘⏕⬟࠾ࡼࡧࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢⓎ⌧ ࡘ࠸࡚ホ౯ࡍࡿࡇ࡛㸪CAD ࡢ in vitro ࣔࢹࣝࡋ࡚ࡢ᭷⏝ᛶࢆ᳨ウࡋࡓࠋࡲ ࡓ㸪⅖ᛶࢧࢺ࢝ࣥ࠾ࡼࡧ Th2 ࢧࢺ࢝ࣥࡀࢱࢺࢪࣕࣥࢡࢩࣙࣥࢱࣥ ࣃࢡཬࡰࡍᙳ㡪ࡘ࠸࡚ホ౯ࢆ⾜ࡗࡓࠋ
➨
➨㸯❶
1.1. ᗎ
ᗎㄽ
CAD ࡣ㸪㑇ఏⓗ࡞⫼ᬒࢆ᭷ࡍࡿᢠཎ≉␗ⓗ࡞ IgE ࢆࡋࡓࣞࣝࢠ࣮ᛶ⅖ ᛶᦋᛶ⓶ᝈ࡛࠶ࡿ[53]ࠋCAD Ⓨ⮳ࡿࣞࣝࢤࣥࡢධࡣ㸪྾ჾ࠾ ࡼࡧᾘ⟶ࡢ⢓⭷ຍ࠼㸪⤒⓶ⓗࡶ㉳ࡇࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ[63]ࠋ HDM ឤసࡋࡓ≟ࢆ⏝࠸ࡓ◊✲࡛ࡣ㸪྾ჾ࠾ࡼࡧᾘჾẚ㸪⓶ࡢࣞࣝࢤࣥ ᭀ㟢ࡀCAD ࡢ⮫ᗋࢫࢥ᭱ࡶ㔜せ࡛࠶ࡿࡇࡀ♧ࡉࢀ[47]㸪CAD ែ࠾ࡅ ࡿ⤒⓶ឤసࡢ㔜せᛶࡀ᫂ࡽ࡞ࡗࡓࠋ ࡇࢀࡲ࡛㸪CAD ࣔࢹࣝ≟࠾࠸࡚⓶ࣂࣜᶵ⬟ࡢపୗࡀ᫂ࡽ࡞ࡗ࡚ ࠸ࡿ[89]ࠋࡲࡓ㸪CAD ࣔࢹࣝ≟࠾࠸࡚㸪ṇᖖ≟ẚ࡚ࣇࣛࢢࣜࣥࡢῶᑡࡀ ሗ࿌ࡉࢀ࡚࠸ࡿ[52]㸪ࣄࢺ AD ࠾ࡼࡧ CAD ࠾࠸࡚㸪ࢭ࣑ࣛࢻࡀṇᖖ ⩌ẚ㍑ࡋ࡚ῶᑡࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[32, 101]ࠋࡋࡓࡀࡗ࡚㸪ࡇࢀࡽ ࡢศᏊࢆ⏘⏕ࡍࡿࢣࣛࢳࣀࢧࢺࡢᶵ⬟␗ᖖࡀCAD ែ㛵ࡋ࡚࠸ࡿࡇࡀ ♧၀ࡉࢀࡿࠋ ࢣࣛࢳࣀࢧࢺࡣ㸪ᵝࠎ࡞ᾮᛶᅉᏊࢆ⏘⏕ࡍࡿࡇࡀ▱ࡽࢀ࡚࠾ࡾ[60]㸪CAD ࠾࠸࡚ࡣ㸪ࣞࣝࢤࣥࡢ่⃭ࡼࡾ㸪ࡑࢀࡽࡢᾮᛶᅉᏊࡀᢠཎᥦ♧⣽⬊ࡢάᛶ ࡸ㸪㸰ᆺ࣊ࣝࣃ࣮T ⣽⬊㸦Th2 ⣽⬊㸧ඃࡢᛂࢆㄏᑟࡍࡿ⪃࠼ࡽࢀ࡚࠸ࡿ [3]ࠋࡘࡲࡾ㸪ࢣࣛࢳࣀࢧࢺࡣ㸪CAD ࡢචែ࠾࠸࡚㔜せ࡞ᙺࢆᯝࡓࡋ ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡋࡋ㸪ࢣࣛࢳࣀࢧࢺ⏤᮶ࡍࡿᾮᛶᅉᏊ⓶Ꮡᅾ ࡍࡿච⣽⬊ࡢࢡࣟࢫࢺ࣮ࢡࡘ࠸࡚ࡣ᫂࡞Ⅼࡀከ࠸[3, 53, 75]ࠋ⌧ᅾ㸪CAD ࡢデ᩿ࡣ㸪㝖እデ᩿ࡀయ࡛࠶ࡾ㸪ᐈほⓗ࡞デ᩿ἲࡀᏑᅾࡋ࡞࠸[68]ࠋࡲࡓ㸪 ⒪࠾࠸࡚ࡣ㸪ࢫࢸࣟࢻ⒪ἲࢆࡣࡌࡵࡍࡿ㠀≉␗ⓗ࡞චᢚไ⒪ἲ 㢗ࡗ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿ[65]ࠋࡋࡓࡀࡗ࡚㸪 CAD ែ࠾ࡅࡿࢣࣛࢳࣀࢧ ࢺࡢචᏛⓗᶵ⬟ࢆ᫂ࡽࡍࢀࡤ㸪CAD ࡢデ᩿࠾ࡼࡧ⒪࠸ᐤࡍࡿ ࡶࡢ⪃࠼ࡽࢀࡿࠋࡇࡢࡼ࠺࡞◊✲ࡣࢣࣛࢳࣀࢧࢺࡢ⏕⌮Ꮫⓗᛂࢆᛅᐇࣔࢹࣝࡍࡿࡇࡀ࡛ࡁࡿin vitro ࢶ࣮ࣝࡀᚲせ࡛࠶ࡿࠋ ୕ḟඖᇵ㣴⓶ࣔࢹࣝࡣ㸪1979 ᖺ Bell ࡽࡼࡗ࡚ึࡵ࡚స〇ࡉࢀ㸪⌧ᅾ㸪 ⤌⧊ᕤᏛࡢ㡿ᇦࢆࡣࡌࡵᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿ[4]ࠋ୕ḟඖᇵ㣴⓶ࣔࢹࣝ࠾࠸ ࡚㸪ࢣࣛࢳࣀࢧࢺࡣ┿⓶ࢆᶍࡋࡓᇶ㉁ୖ✀ࡉࢀ㸪ቑṪ࠾ࡼࡧศࡋࡓᚋ㸪 ゅᒙࢆྵࡴከᒙᵓ㐀ࢆᙧᡂࡍࡿࠋᙧែⓗ࠾ࡼࡧᶵ⬟ⓗศࡍࡿ⾲⓶ࢣࣛࢳࣀ ࢧࢺࡢࣔࢹࣝࡋ࡚ࡣ㸪ᚑ᮶ࡽࡼࡾ୍⯡ⓗ⏝࠸ࡽࢀ࡚࠸ࡿ༢ᒙᇵ㣴ࣔࢹ ࣝẚ㸪୕ḟඖᇵ㣴⓶ࣔࢹࣝࡀᙧែⓗࡼࡾ⏕యࢆᫎࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛ ≟ࡢࢣࣛࢳࣀࢧࢺ⣽⬊ᰴࢆ⏝࠸ࡓ୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡀሗ࿌ࡉࢀ㸪᫂░ ࡞ゅᒙ㸪㢛⢏ᒙ࠾ࡼࡧ᭷Ჲᒙᵝࡢከᒙᵓ㐀ࡀㄆࡵࡽࢀ࡚࠸ࡿࠋࡋࡋ㸪ࡑࡢࣔࢹ ࣝࡢචᶵ⬟ࡣศࡗ࡚࠸࡞࠸[99]ࠋ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢචᶵ⬟ࢆ᫂ࡽ ࡍࡿࡣ㸪ᮏࣔࢹࣝ࠾ࡅࡿࢧࢺ࢝ࣥ⏘⏕⬟ࡢホ౯ࡀ᭷ຠ࡛࠶ࡿࠋ TNF-alpha ࡣɾञ ࡋ๖Ͷଲ͢ͱΠϛφʖεηΝ༢ͤΖΆ͖ɾ༹ʓ͵ࡋ๖͖ ΔγφΩϱൽΝ༢ͤΖ͞ͳͲɾԎൕԢ͕Γ;ࣙષ໖ӺઇΝ୴ ͮͱ͏Ζ[85]ࠋࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚ CCL17 ࡸ㸪CCL20 ࡢ⏘⏕ࢆㄏᑟࡍ ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[1, 91, 97]㸪ࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚ TNF-alpha ࡣTh2 ࢧࢺ࢝ࣥ㸦IL-4, IL-5 ࠾ࡼࡧ IL-13㸧ඹ㸪TSLP ࡢ⏘⏕ࢆቑຍࡉࡏ ࡿ ࡇ ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡿ[9]ɽ ࡉ ࡽ 㸪 ≟ࢣ ࣛ ࢳ ࣀ ࢧ ࢺ ࠾ ࠸ ࡚ࡣ 㸪 lipopolysaccharide㸦࣏ࣜከ⢾㸪LPS㸧ࡸ HDM ࡢせࣞࣝࢤ࡛ࣥ࠶ࡿ Der f1 ࡼࡾ⏘⏕ࡉࢀࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ[31, 46]ɽCAD ྭࡢ⓶ኚ㒊࠾࠸࡚㸪 TNF-alpha ࡢ㌿㔞ࡢቑຍࡀሗ࿌ࡉࢀ࡚࠸ࡿ[61]ɽCAD ࠾࠸࡚ࡣ㸪TSLP ࡢ⏘ ⏕ࢆࡋ࡚ᶞ≧⣽⬊ࢆάᛶࡋ㸪⋓ᚓචࡢ㛤ጞࢆಁࡍ[41]㸪ࣞࣝࢤࣥࡢ ධᑐࡋ㸪⅖㒊ච⣽⬊ࢆ㐟㉮ࡉࡏࡿ୍ḟࢩࢢࢼࣝࡢᙺࢆᯝࡓࡍ ⪃࠼ࡽࢀ࡚࠸ࡿ[91]ɽ TGF-beta ࡣᢚไᛶࢧࢺ࡛࢝ࣥ࠶ࡾ[1]ɾT ⣽⬊࠾ࡼࡧ B ⣽⬊ࡢቑṪࢆ㜼 ᐖࡍࡿ㸪ዲ୰⌫ࡸෆ⓶⣽⬊ࡢάᛶࢆᢚไࡍࡿࡇ࡛㸪චㄪ⠇ࢆᢸࡗ࡚࠸
ࡿ[1]ࠋࣄࢺ࠾࠸࡚㸪T ⣽⬊ࡼࡿ IL-4 ࠾ࡼࡧ IL-5 ࡢ⏘⏕ࢆᢚไࡍࡿࡇࡀ᫂ ࡽ࡞ࡗ࡚࠸ࡿ[21]㸪ࢼ࣮ࣈ T ⣽⬊ࡽไᚚᛶ T ⣽⬊ࡢศ㔜せ ࡞ᙺࢆᯝࡓࡍࡇࡀ▱ࡽࢀ࡚࠸ࡿ[80]ɽCAD ࡢ⓶࠾࠸࡚㌿㔞ࡢῶ ᑡࡀሗ࿌ࡉࢀ࡚࠸ࡿ[61]ࠋࡋࡓࡀࡗ࡚㸪CAD ࠾࠸࡚ࡣ㸪TGF-beta ࡢⓎ⌧పୗ ࡀ㸪T ࠾ࡼࡧ B ⣽⬊࡞ࡽࡧᶞ≧⣽⬊ࡢᶵ⬟ᑐࡍࡿᢚไᶵᵓࢆపୗࡉࡏࡿࡇ ࡛Ⓨ㛵ࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋ
CCL2㸦monocyte chemotactic protein 1: MCP-1㸧ࡣ㸪༢⌫ࡸ࣐ࢡࣟࣇ࣮ࢪ㸪 ᶞ≧⣽⬊࠾࠸࡚Ⓨ⌧ࡍࡿCC ࢣࣔ࢝ࣥࣞࢭࣉࢱ࣮2㸦CCR2㸧ᑐࡍࡿࣜ࢞ࣥ ࢻ࡛㸪ࡇࢀࡽࡢ⣽⬊ᑐࡋ㉮ᅉᏊࡋ࡚స⏝ࡍࡿ[6]ɽࣛࣥࢤࣝࣁࣥࢫ⣽⬊ࡣ㸪 ᐃᖖ≧ែ࡛ࡣ⾲⓶ෆࡢ๓㥑⣽⬊ࡀ⣽⬊ቑṪࢆ⾜࠸ศࡍࡿ[54]ࠋࡋࡋ㸪⅖ ࡣ⾑ᾮࢆࡋ㸪CCR2 ౫Ꮡᛶ㐟㉮ࡋࡓᚠ⎔⾑ᾮ୰ࡢ༢⌫ࡽศࡍࡿࡇࡀ ࣐࢘ࢫ࠾ࡼࡧࣄࢺ࠾࠸࡚ሗ࿌ࡉࢀ࡚࠸ࡿ[54]ࠋࡲࡓ㸪 CAD ࠾ࡼࡧ CAD ࣔ ࢹࣝ≟ࡢ⓶ኚ㒊࠾࠸࡚㸪CCL2 ࡢ㌿㔞ࡀቑຍࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ[72, 76]ࠋࡋࡓࡀࡗ࡚㸪CAD ࠾࠸࡚ࡣ㸪ᮎᲈ⾑୰ࡢ༢⌫ࢆ⾲⓶㐟㉮ࡉࡏ ࡿࡇ࡛ࣛࣥࢤࣝࣁࣥࢫ⣽⬊ࡢศࢆಁࡋ㸪⋓ᚓචᛂࢆಁ㐍ࡋ࡚࠸ࡿ ⪃࠼ࡽࢀࡿࠋ
CCL20 (liver and activation-regulated chemokine: LARC/macrophage inflammatory protein-3: MIP-3Ș/Exodus-1) ࡣ㸪ࣜࣥࣃ⌫ᑐࡍࡿ㉮ᅉᏊࡋ࡚ሗ࿌ࡉࢀࡓ [27]ࠋCCR6 స⏝ࡍࡿ၏୍ࡢࢣࣔ࢝ࣥࡋ࡚▱ࡽࢀ࡚࠾ࡾ[86]㸪CCR6 ࡣࣄࢺ ࠾ࡼࡧ࣐࢘ࢫࡢᮍ⇍ᶞ≧⣽⬊ࡸࣛࣥࢤࣝࣁࣥࢫ⣽⬊࡛ࡢⓎ⌧ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [16, 44]ࠋࣄࢺࣛࣥࢤࣝࣁࣥࢫ⣽⬊ࡣ㸪⾲⓶ࣔࢹࣝ࠾࠸࡚ CCL20 ࡢస⏝ࡼࡾ 㐟㉮ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ[69]㸪ࡉࡽ㸪CCL20 ࡣࣄࢺࡢⓄ࠾ࡼࡧ AD ࡢ⓶ኚ㒊࠾࠸࡚㸪CCR6 ඹⓎ⌧ࡀቑຍࡋ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ [57]ࠋࡋࡓࡀࡗ࡚ CAD ࠾࠸࡚ࡣ㸪CCL20 ࡀࣛࣥࢤࣝࣁࣥࢫ⣽⬊ࢆኚ㒊㐟 ㉮ࡉࡏࡿࡇ࡛㸪ࡑࡢᚋTSLP ࢆࡋ࡚⏘⏕ࡉࢀࡿ CCL17 ࡞ࡢᾮᛶᅉᏊࡢാ
ࡁࡼࡾ㸪Th2 ᆺᛂࢆㄏᑟࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ ௨ୖࡢࡇࡽ㸪ࡇࢀࡽࡢࢧࢺ࢝ࣥ࠾ࡼࡧࢣࣔ࢝ࣥࡣ㸪CAD ࡢච ែࡢ࡞࡛ࡶ㸪⋓ᚓචᛂࡢಁ㐍࠾࠸࡚ࡑࢀࡒࢀ㔜せ࡞ᙺࢆᯝࡓࡍ⪃ ࠼ࡽࢀࡿࠋࣞࣝࢤࣥࡢ⤒⓶ⓗ࡞ධࡽጞࡲࡿึᮇࡢ⋓ᚓචᛂ࠾࠸࡚ ࡣ㸪ࢣࣛࢳࣀࢧࢺᢠཎᥦ♧⣽⬊ࡢ┦స⏝ࡀ㔜せ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋࡑ ࡇ࡛㸪➨1 ❶࡛ࡣ㸪≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࢆస〇ࡋ㸪ࡇࡢࣔࢹࣝࡢචᶵ⬟ ࡘ࠸࡚㸪⓶ࡢ㢮ఝᛶࢆ༢ᒙᇵ㣴ẚ㍑ࡍࡿࡇࢆ┠ⓗࡋ࡚㸪TNF-Ș, TGF-ș, CCL2 ࠾ࡼࡧ CCL20 ࡢ㑇ఏᏊ㌿㔞ࢆࣜࣝࢱ࣒ RT-PCR ࢆ⏝࠸࡚ホ౯ࡋ ࡓࠋ
1.2. ᮦᩱ᪉ἲ
1.2.1. ᮦᩱ࠾ࡼࡧື≀
ṇᖖ࡞≟ࡢ⾲⓶ࡽ☜❧ࡉࢀࡓࢣࣛࢳࣀࢧࢺ⣽⬊ᰴ࡛࠶ࡿ CPEK (canine progenitor epidermal keratinocyte) ࠾ࡼࡧࡑࡢᇵ㣴ᾮ࡛࠶ࡿ CnT-09 ࡣ CELLnTEC advanced cell systems㸦Bern, Switzerland㸧ࡼࡾධᡭࡋࡓࠋ↓⾑Ύᇵᆅ࡛࠶ࡿ Opti-MEMϨ࠾ࡼࡧ⥅௦ᇵ㣴ࡢ⣽⬊㞳ࡢ㝿⏝࠸ࡓࢺࣜࣉࢩࣥฎ⌮㸦TrypLE Express㸧ࡣ Invitrogen㸦Carlsbad, CA, USA㸧ࡼࡾධᡭࡋࡓࠋDulbecco’s phosphate-buffered saline㸦DPBS㸧࠾ࡼࡧࢣࣛࢳࣀࢧࢺ่⃭ࡋ࡚⏝࠸ࡓࣃࣃࣥࡣ Sigma-Aldrich㸦St. Louis, MO, USA㸧ࡽධᡭࡋࡓࠋ⓶ࡣᗣ࡞ࣅ࣮ࢢࣝ≟㸦ᖺ 㱋5㸫7 ṓ㱋㸪ᖹᆒ 6 ṓ㱋㸪࢜ࢫ 1 㢌㸪ཤໃ῭ࡳ࢜ࢫ 1 㢌࠾ࡼࡧ㑊ዷ῭ࡳ࣓ࢫ 1 㢌㸧ࡢయᖿ⭡ഃ㒊ࡼࡾ᥇ྲྀࡋࡓࠋᐇ㦂ື≀㛵ࢃࡿࡍ࡚ࡢᡭᢏࡣ㸪ᒱ㜧Ꮫື ≀ᐇ㦂ጤဨࡢᢎㄆࢆᚓ࡚⾜ࡗࡓࠋ㸦#15001㸧 1.2.2. ⣽⬊ᇵ㣴 ༢ᒙᇵ㣴 7 ᅇࡽ 9 ᅇ⥅௦ࡋࡓ CPEK ࢆ 10 ml ࡢ CnT-09 ᾋ㐟ࡉࡏ㸪37Υ㸪5% CO2ࡢ
⎔ቃୗ࡛75cm2ࣇࣛࢫࢥ㸦NUNC, Roskilde, Denmark㸧ࢆ⏝࠸࡚ᇵ㣴ࡋࡓࠋ80–90%
ࢥࣥࣇ࢚ࣝࣥࢺ㐩ࡋࡓẁ㝵࡛ᇵ㣴ᾮࢆ྾ᘬ㝖ཤࡋ㸪10 ml ࡢ DPBS ࢆ⏝࠸࡚⣽ ⬊ࢆὙίࡋࡓࠋTrypLE Express ࢆ 3 ml ῧຍࡋࡓᚋ㸪37Υ࡛ 5–10 ศ㛫ࣥ࢟ࣗ࣋ ࣮ࢩࣙࣥࡋ㸪⣽⬊ࢆ㞳ࡋࡓࠋCnT-09 ࢆ 7 ml ຍ࠼㸪㞳ࡋࡓ⣽⬊ᾋ㐟ᾮࢆ 15 ml centrifuge tube㸦NUNC㸧ᅇࡋ㸪180 G㸪5 ศ㛫㐲ᚰࡋࡓࠋ㐲ᚰᚋୖΎࢆ྾ ᘬ㝖ཤࡋ㸪⣽⬊ሢࢆࢱࢵࣆࣥࢢᚋCnT-09 ᠱ⃮ࡋࡓࠋࡧ 75 cm2ࣇࣛࢫࢥ ࡚ 37Υ㸪5% CO2ࡢ⎔ቃୗ࡛ᇵ㣴ࡋ㸪ࡑࡢᚋᇵ㣴ᾮࢆ Opti-MEMϨኚ᭦ࡋ㸪 ↓⾑Ύୗ࡛24 㛫ᇵ㣴ࡋࡓࠋ
୕ ୕ḟඖᇵ㣴㸦ᅗ1-1 ࠾ࡼࡧ 1-2㸧 ୕ḟඖᇵ㣴⏝࠸ࡿ CPEK ࡣ㸪༢ᒙᇵ㣴ྠᵝ 75cm2ࣇࣛࢫࢥࡽ⣽⬊ࢆ 㞳ࡋࡓᚋ㸪ࣞࢳࣀࣥ㓟㸦10−8 M㸧㸪ࣄࢻࣟࢥࣝࢳࢰࣥ㸦0.4 μg/ml㸧㸪ࢥࣞࣛ ࢺ࢟ࢩࣥ㸦0.1 nM㸧㸪ࢺࣛࣥࢫࣇ࢙ࣜࣥ㸦5 μg/ml㸧㸪3,3′,5-ࢺ࣮ࣜࣚࢻ-L-ࢳࣟࢽ ࣥࢼࢺ࣒ࣜ࢘ሷ㸦2 nM㸧㸪⤌࠼ࣄࢺୖ⓶ቑṪᅉᏊ㸦10 ng/ml㸧࠾ࡼࡧ⤌࠼ࣄ ࢺࣥࢫࣜࣥ㸦5 μg/ml㸧㸦Sigma-Aldrich㸧ࢆῧຍࡋࡓ CnT-09 ᠱ⃮ࡋࡓࠋ㸴 ࢙࢘ࣝࢥࣥࣃࢽ࢜ࣥࣉ࣮ࣞࢺ㸦BD Falcon, San Jose, CA, USA㸧╔ࡋࡓࣥࢧ ࣮ࢺࢹࢵࢩࣗ㸦BD Falcon㸧㸪1 ࢙࢘ࣝ࠶ࡓࡾ 2 ml ࡞ࡿࡼ࠺ࢱࣉϨ-A ࢥ࣮ࣛࢤࣥ㸪10 ಸ⃰⦰ MEM ࣁࣥࢡࢫᇵᆅ࠾ࡼࡧᵓᡂ⏝⦆⾪ᾮ㸦ࢡࣛ࣎࢘㸪 㜰㸪᪥ᮏ㸧ࢆ⏝࠸࡚స〇ࡋࡓࢥ࣮ࣛࢤࣥࢤࣝࢆὀධࡋࡓࠋࢥ࣮ࣛࢤࣥࢤࣝୖ㸪 ⣽⬊ᠱ⃮ᾮࢆ0.5×106 cells/ml ࡢᐦᗘ࡛ 1 ml ࡎࡘศὀࡋࡓ㸦ᇵ㣴 0 ᪥┠; ᅗ 1-1㸧ࠋ 37Υ㸪5% CO2ࡢ⎔ቃୗ࡛ᇵ㣴ࡋ㸪ᇵ㣴5 ᪥┠㸪0.4 μg/ml ࡢࣄࢻࣟࢥࣝࢳࢰࣥ ࢆῧຍࡋࡓCnT-09 ᇵᆅࢆኚ᭦ࡋ㸪௨㝆ࡢᐇ㦂ࡣࡇࢀࢆ⏝࠸ࡓࠋᇵ㣴 8 ᪥┠ ࣥࢧ࣮ࢺࢹࢵࢩࣗࡽᇵᆅࢆ㝖ཤࡋ࡚⣽⬊⾲㠃ࢆ✵Ẽᭀ㟢ࡋ㸪ᇵᆅࢆ2 ml ࡎࡘ㸪ᾮ㠃ࡀࣥࢧ࣮ࢺࢹࢵࢩࣗࡢᗏ㒊᥋ࡍࡿࡼ࠺ࢥࣥࣃࢽ࢜ࣥࣉࣞ ࣮ࢺࡢ࢙࢘ࣝෆὀධࡋࡓࠋᇵᆅࡣᇵ㣴 14 ᪥┠ࡲ࡛ẖ᪥㸯ᅇ⾜ࡗࡓ[99]ࠋ ᇵ㣴13 ᪥┠ࡢᇵᆅࡣ Opti-MEMϨࢆ⏝࠸ࡓࠋ 1.2.3 ⓶࠾ࡼࡧ CPEK ࡽࡢࢺ࣮ࢱࣝ RNA ᢳฟ
ࢺ࣮ࢱࣝRNA ࡢᢳฟࡣ RNeasy Mini Kit㸦Qiagen, Valencia, CA, USA㸧ࢆ ⏝ࡋࡓࠋ⓶࠾ࡼࡧ୕ḟඖᇵ㣴ࡋࡓCPEK ࡣ㸪᥇ྲྀᚋ┤ࡕ RNAlater (QIAGEN, Valencia, CA, USA) ᾐₕࡋ㸪ࢺ࣮ࢱࣝ RNA ᢳฟࡲ࡛−30Υ࡛ಖᏑࡋࡓࠋᅇ ࡋࡓࢺ࣮ࢱࣝRNA ࡣ TURBO DNA-Free Kit㸦Applied Biosystems, Foster City, CA, USA㸧ࢆ⏝ࡋ࡚ࢤࣀ࣒ DNA ࡢ㝖ཤࢆ⾜ࡗࡓᚋ㸪−80Υ࡛ಖᏑࡋࡓࠋ
1.2.4 ᐃ㔞ࣜࣝࢱ࣒ RT-PCR㸦RT-qPCR㸧
ࢺ࣮ࢱࣝRNA ࡣ PrimeScript RT Reagent Kit㸦ࢱ࢝ࣛࣂ࢜㸪㈡㸪᪥ᮏ㸧ࢆ ⏝࠸࡚㏫㌿ᛂࢆ⾜࠸㸪cDNA ࢆྜᡂࡋࡓࠋTNF-alpha, TGF-beta, CCL2 ࠾ࡼࡧ CCL20 ࢆ≉␗ⓗቑᖜࡍࡿࣉ࣐࣮ࣛ㸦⾲ 1-1㸧ࢆ⏝࠸࡚㸪RT-PCR 㸦Thermal Cycler Dice Real Time System TP800 ࠾ࡼࡧ SYBR Premix Ex Taq II㸦ࢱ࢝ࣛࣂ ࢜㸧㸧ࢆ⾜ࡗࡓࠋ࡞࠾㸪RT-PCR ࡣ RT ᛂࢆ 42Υ 5 ศ㛫࠾ࡼࡧ 95Υ 10 ⛊㛫㸪 PCR ᛂࢆ 95Υ 5 ⛊㛫࠾ࡼࡧ 60Υ 30 ⛊㛫ࢆ 40 ࢧࢡࣝ㸪ゎ㞳ࡣ 95Υ 15 ⛊ 㛫㸪60Υ 30 ⛊㛫㸪95Υ 15 ⛊㛫ࡢ᮲௳࡛ᐇࡋࡓࠋゎᯒඛ❧ࡕ㸪┦ᑐᐃ㔞 ⏝ ࠸ ࡿ ࣜ ࣇ ࣞ ࣥ ࢫ 㑇 ఏ Ꮚ ࡢ ᳨ ウ ࢆ ⾜ ࡗ ࡓ ࠋHypoxanthine phosphoribosyl-transferase 1㸦HPRT1㸧, Glyceraldehyde 3-phosphate dehydrogenase㸦GAPDH㸧, Ribosomal protein S18㸦RPS18㸧, Ribosomal protein L㸦RPL㸧13A ࠾ࡼࡧ RPL32 ࢆ ೃ⿵[84]ࡋ㸪༢ᒙᇵ㣴࠾ࡼࡧ୕ḟඖᇵ㣴ࡋࡓ CPEK ࢆ⏝࠸࡚ RT-qPCR ࢆ⾜ࡗ ࡓ ࠋ ᚓ ࡽ ࢀ ࡓ ࢹ ࣮ ࢱ ࡽ GeNorm 㸦 https://genorm.cmgg.be 㸧 㸪 NormFinder 㸦https://moma.dk/normfinder-software 㸧 ࠾ ࡼ ࡧ BestKeeper 㸦 http://www.gene-quantification.de/bestkeeper.html㸧ࢆ⏝࠸࡚᭱㐺࡞ࣜࣇࣞࣥࢫ㑇ఏᏊࡢ㡰ࢆỴ ᐃࡋ㸪㸯ࡽ㡰㑇ఏᏊࡈ㓄Ⅼࢆࡋ㸪ࢫࢥࢆẚ㍑ࡋ࡚ࣜࣇࣞࣥࢫ㑇ఏ Ꮚࡢ㑅ฟࢆ⾜ࡗࡓࠋPCR ᛂࡢቑᖜ᭤⥺㜈್ࡀᕪࡍࡿⅬ㸦Ct ್㸧ࡣ㸪Thermal Cycler Dice Real Time System Multiplate RQ software, Version 3.00㸦ࢱ࢝ࣛࣂ࢜㸧 ࢆ⏝࠸࡚⟬ฟࡋࡓࠋTNF-alpha, TGF-beta, CCL2 ࠾ࡼࡧ CCL20 ࡢ㌿ࣞ࣋ࣝࡣ㸪 ẚ㍑Ct ἲ㸦ΔΔCt ἲ㸧ࢆ⏝࠸࡚ࣜࣇࣞࣥࢫ㑇ఏᏊᑐࡍࡿ┦ᑐᐃ㔞್ࡋ࡚⟬ ฟࡋࡓࠋ࡚ࡢࢧࣥࣉࣝࡣ㸰ᅇ㔜」ࡋ࡚ศᯒࡋ㸪ᖹᆒ್ࢆ⟬ฟࡋࡓࠋ࡚ࡢᐇ㦂 ࡣ⊂❧ࡋ࡚㸱ᅇ⾜ࡗࡓࠋ 1.2.5. ⤫ィ ༢ᒙᇵ㣴㸪୕ḟඖᇵ㣴࠾ࡼࡧ⓶࠾ࡅࡿ୕⩌㛫ࡢ᭷ពᕪࢆホ౯ࡍࡿࡓࡵ㸪
Bonferroni/Dunn ᳨ᐃࢆ⏝࠸࡚⤫ィゎᯒࢆ⾜ࡗࡓࠋP < 0.05 ࢆ᭷ពᕪ࠶ࡾࡋࡓࠋ ゎᯒࡣ㸪⤫ィࢯࣇࢺJMP version 13.2㸦SAS Institute, Cary, NC, USA㸧ࢆ⏝ࡋ ࡓࠋ
1.3. ⤖ᯝ
1.3.1 ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ⤌⧊ീࡢኚ ᇵ㣴㸲᪥┠ࡲ࡛ᇵ㣴⣽⬊ࡣ3 ࡽ 4 ᒙࡢᒙᵓ㐀ࢆᙧᡂࡋࡓ㸦ᅗ 1-3 Day 4㸧ࠋᇵ㣴 8 ᪥┠ࡲ࡛ᇵ㣴⣽⬊ࡣ 4 ࡽ 6 ᒙࡢᒙᵓ㐀ࢆᙧᡂࡋࡓ㸦ᅗ 1-3 Day8㸧ࠋᇵ㣴 12 ᪥┠ࡣ 7 ᒙࡽ 8 ᒙࡢᒙᵓ㐀ࡢᙧᡂࡀㄆࡵࡽࢀ㸪ࡉࡽࡑ ࢀࡽࡢୖᒙࡣ㸪⬺᰾ࡼࡿゅᒙᵝᒙࡢᙧᡂࡀㄆࡵࡽࢀࡓࠋࡲࡓ㸪ゅᒙᵝᒙࡢ ୗᒙࡣ㸪⣽⬊ෆ㢛⢏ࢆྵࡴ㢛⢏ᒙᵝᒙࡢᙧᡂࡀㄆࡵࡽࢀࡓ㸦ᅗ1-3 Day 12㸧ࠋᇵ㣴 17 ᪥┠࠾࠸࡚ࡶ 12 ᪥┠ࡰྠᵝࡢᵓ㐀ࡀㄆࡵࡽࢀࡓ㸦ᅗ 1-3 Day 17㸧ࠋ 1.3.2 ࣜࣇࣞࣥࢫ㑇ఏᏊࡢ㑅ฟ 3 ✀㢮ࡢࣝࢦࣜࢬ࣒ࢆ⏝࠸࡚᭱㐺࡞ࣜࣇࣞࣥࢫ㑇ఏᏊࢆホ౯ࡋࡓ⤖ᯝ㸪༢ ᒙᇵ㣴࠾࠸࡚ࡣ㸪RPL32, HPRT1, RPL13A, RPS18, GAPDH ࡢ㡰㸪ࡲࡓ㸪୕ḟ ඖᇵ㣴࠾࠸࡚ࡣ㸪RPL32, HPRT1, RPS18, RPL13A, GAPDH ࡢ㡰᭱㐺࡞ࣜࣇ ࣞࣥࢫ㑇ఏᏊࡀ㑅ฟࡉࢀࡓࠋ༢ᒙ࠾ࡼࡧ୕ḟඖᇵ㣴࠾࠸࡚ࡑࢀࡒࢀࡢ㑇ఏ Ꮚ࡛ᚓࡽࢀࡓࢫࢥࢆẚ㍑ࡋ㸪᭱ࡶ㧗࠸ࢫࢥࡀᚓࡽࢀࡓ RPL32, HPRT1 ࠾ࡼ ࡧRPS18 ࢆࣜࣇࣞࣥࢫ㑇ఏᏊࡋ࡚㑅ฟࡋࡓࠋ 1.3.3 RT-qPCR TNF-alpha ࡣ㸪༢ᒙᇵ㣴࠾࠸࡚ࡣ㌿㔞ࡀ᳨ฟ㝈ᗘ௨ୗ࡞ࡾ㸪୕ḟඖᇵ㣴 ࠾ࡼࡧ⓶࠾࠸࡚ࡢࡳ㸪㌿ࡀㄆࡵࡽࢀࡓ㸦ᅗ1-4 a㸧ࠋTGF-beta, CCL2 ࠾ࡼ ࡧCCL20 ࡣ㸪࡚ࡢࢧࣥࣉࣝ࠾࠸࡚㌿ࡀㄆࡵࡽࢀࡓ㸦ᅗ 1-4 b, c, d㸧ࠋTGF-beta ࡣ㸪ࢧࣥࣉࣝ㛫࠾࠸࡚㌿㔞᭷ពᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦ᅗ 1-4 b㸧ࠋ CCL2 ࠾ࡼࡧ CCL20 ࡣ㸪༢ᒙᇵ㣴࠾ࡼࡧ⓶࠾࠸࡚㌿㔞᭷ពᕪࡀㄆࡵࡽ ࢀࡓࠋ1.4. ⪃ᐹ
ࡇࢀࡲ࡛㸪ࣄࢺAD ࡢ⣽⬊ࣔࢹࣝࡋ࡚㸪ࣄࢺࢣࣛࢳࣀࢧࢺ⣽⬊ᰴ࡛࠶ࡿ HaCaT ⣽⬊ࢆ⏝࠸ࡓሗ࿌ࡀ࠶ࡿ[97]ࠋHaCaT ⣽⬊ࡣ㸪ᇶᗏᒙ࠾ࡼࡧᇶᗏୖᒙㄆ ࡵࡽࢀࡿࢣࣛࢳࣥࢆⓎ⌧ࡋ࡚࠾ࡾ㸪⾲⓶ࡢྛᒙࡢศ⬟ࢆ᭷ࡋ࡚࠸ࡿ[8, 10]ࠋ ༢ᒙᇵ㣴ࡋࡓCPEK ࡣ㸪ࢣࣛࢳࣥࡢⓎ⌧ࣃࢱ࣮ࣥࡸ proliferating cell nuclear antigen (PCNA) ࠾ࡼࡧȘ6ࣥࢸࢢࣜࣥࡢⓎ⌧ࡼࡾ㸪ᇶᗏᒙࢣࣛࢳࣀࢧࢺࡢ⾲⌧ᆺ ࢆ᭷ࡍࡿࡇࡀ᫂ࡽ࡞ࡗ࡚࠾ࡾ[88]㸪ࡉࡽ CPEK ࡣ HaCaT ⣽⬊ྠᵝࡢ ࢧࢺ࢝ࣥ࠾ࡼࡧࢣࣔ࢝ࣥࡢ⏘⏕⬟ࢆ᭷ࡍࡿࡇࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ [46]ࠋࡇࢀࡽࡢᐇࡣ㸪ᾮᛶᅉᏊࡢ⏘⏕⬟࠾࠸࡚㸪CPEK ࡀࣄࢺ AD ࡢ◊✲ ࠾ࡅࡿHaCaT ⣽⬊ྠᵝ㸪CAD ࡢ⣽⬊ࣔࢹࣝࡋ࡚᭷⏝࡛࠶ࡿࡇࢆ♧၀ࡍ ࡿࠋࡋࡋ㸪⾲⓶ࡣᇶᗏᒙࡢࢣࣛࢳࣀࢧࢺࡀᙧែⓗ࠾ࡼࡧᶵ⬟ⓗศࡍࡿࡇ ࡼࡗ࡚ᙧᡂࡉࢀ࡚࠾ࡾ㸪ࡼࡾ⏕యࢆᫎࡋࡓࣔࢹࣝࡋ࡚ࡣ୕ḟඖᇵ㣴ࡢ 㐺⏝ࡀᮃࡲࡋ࠸⪃࠼ࡽࢀࡿࠋࡋࡋ㸪ᮏ◊✲࡛⏝࠸ࡓ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ ࡢචᏛⓗᶵ⬟ࡣ࡛᫂࠶ࡗࡓࠋ ᅇホ౯ࡋࡓࢧࢺ࢝ࣥ࠾ࡼࡧࢣࣔ࢝ࣥࡣ㸪࠸ࡎࢀࡶCAD ࡢែ࠾࠸ ࡚㔜せ࡞ᙺࢆᯝࡓࡍ⪃࠼ࡽࢀࡿࠋ㌿㔞ゎᯒࡢ⤖ᯝ㸪ᮏࣔࢹࣝ࠾࠸࡚ TNF-alpha, CCL2 ࠾ࡼࡧ CCL20 ࡣ࠸ࡎࢀࡶ༢ᒙᇵ㣴ẚ㸪⓶㢮ఝࡋࡓ㌿ࣃࢱ ࣮ࣥࢆ♧ࡋࡓࠋࡋࡓࡀࡗ࡚㸪ࢧࢺ࢝ࣥ㑇ఏᏊࡢ㌿ゎᯒ࠾࠸࡚㸪ᮏࣔࢹࣝ ࡣᚑ᮶ࡢ༢ᒙᇵ㣴ẚ㐺ษ࡞ࣔࢹ࡛ࣝ࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ᮏ◊✲ࡣ㸪ᐃᖖ≧ែ࠾ࡅࡿᮏࣔࢹࣝࡢࢧࢺ࢝ࣥ㑇ఏᏊࡢ㌿ゎᯒ࡛࠶ ࡿࠋࡋࡓࡀࡗ࡚㸪ⓗ⎔ቃ࠾ࡅࡿࢣࣛࢳࣀࢧࢺࡢ⏕⌮Ꮫⓗᶵ⬟ࡢホ౯ࢆ⾜࠺ ࠶ࡓࡗ࡚ࡣ㸪ᚋ㸪่⃭ࡢᮏࣔࢹࣝࡢᣲືࢆᢕᥱࡋ࡚࠾ࡃᚲせࡀ࠶ࡿ⪃࠼ ࡽࢀࡿࠋ ᮏࣔࢹ࡛ࣝࡣࡇࢀࡲ࡛㸪ࣟࣜࢡࣜࣥ㸪Keratinocyte differentiation-associated
protein (Kdap), Ki-67 ࠾ࡼࡧࢣࣛࢳࣥ 14 ࠸ࡗࡓศ࠾ࡼࡧ⣽⬊ቑṪ࣐࣮࣮࢝ࡢ Ⓨ⌧ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ㸪≀⌮Ꮫⓗ≉ᛶࡘ࠸࡚ࡣ᫂࡞Ⅼࡶከ࠸[99]ࠋ⾲⓶ࡀ ഛ࠼ࡿ≀⌮Ꮫⓗ≉ᛶࡣ㸪⣽⬊ࢆᙧᡂࡍࡿྛ✀ࡢᵓᡂᡂศ࠾ࡼࡧ⣽⬊ྠኈࡢ᥋╔ ศᏊࡼࡗ࡚⥔ᣢࡉࢀࡿࠋࡑࢀࡽࡣࢣࣛࢳࣀࢧࢺࡢࢱ࣮࣮ࣥ࢜ࣂ࣮ࡼࡿᜏ ᖖᛶࡢ⥔ᣢࡸ㸪⓶ࣂࣜᶵ⬟ࡢᙧᡂᐤࡋ࡚࠸ࡿ[90]ࠋCAD ࠾࠸࡚ ࡣ⓶ࣂࣜᶵ⬟ࡢపୗࡀㄆࡵࡽࢀ࡚࠾ࡾ㸪ែࡢ㛵㐃ࡀ♧၀ࡉࢀ࡚࠸ࡿ[48]ࠋ ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡣ㸪ゅᒙඹ⓶ࣂࣜᶵ⬟ࢆᵓᡂࡍࡿせ⣲࡛࠶ࡾ㸪㢛 ⢏ᒙࡀᙧᡂࡉࢀࡿ㝿࣋ࣝࢺ≧⣽⬊㛫ࢆࢩ࣮ࣝࡋ࡚㸪⣽⬊ෆእࡽࡢ≀㉁ࡢ ㏱㐣ࢆไ㝈ࡋ࡚࠸ࡿ[34]ࠋCAD ࣔࢹࣝ≟࠾࠸࡚ࢱࢺࢪࣕࣥࢡࢩࣙࣥࢆᵓᡂ ࡍࡿࢱࣥࣃࢡⓎ⌧ኚࡀㄆࡵࡽࢀ࡚࠸ࡿ[37, 79]ࡇࡽ㸪ᮏࣔࢹࣝࡢ in vitro ࢶ࣮ࣝࡋ࡚ࡢ᭷⏝ᛶࢆホ౯ࡍࡿ࠶ࡓࡗ࡚ࡣ㸪ᮏ◊✲࡛᫂ࡽ࡞ࡗࡓච Ꮫⓗ≉ᛶຍ࠼㸪≀⌮Ꮫⓗ≉ᛶ㔜せ࡞ᙺࢆᯝࡓࡍࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢ ホ౯ࡀᚲせ⪃࠼ࡓࠋࡑࡇ࡛㸪➨❶࡛ࡣ㸪ᮏࣔࢹࣝ࠾ࡅࡿࢱࢺࢪࣕࣥࢡࢩ ࣙࣥࡢⓎ⌧ࡘ࠸࡚ホ౯ࡍࡿࡇࡋࡓࠋ
1.5 ᅗ⾲
ᅗ
ᅗ1-1 ୕ḟඖᇵ㣴⏝࠸ࡓᇵ㣴─࠾ࡼࡧᇵ㣴ࡢእほ
㸦A㸧ࣥࢧ࣮ࢺࢹࢵࢩࣗࢆ㸴࢙࢘ࣝࢥࣥࣃࢽ࢜ࣥࣉ࣮ࣞࢺ╔ࡋࡓ≧ែ, 㸦B㸧ࢥ࣮ࣛࢤࣥࢤࣝୖ⣽⬊ᠱ⃮ᾮࡀὀධࡉࢀࡓ≧ែ
ᅗ ᅗ1-2 ୕ḟඖᇵ㣴ࡢ࡞ࡀࢀ࠾ࡼࡧᇵ㣴⣽⬊ࡢእほࡢኚ 㸦A, E㸧ࣥࢧ࣮ࢺࢹࢵࢩࣗࢥ࣮ࣛࢤࣥࢤࣝࢆసᡂࡋࡓ≧ែ,㸦B, F㸧⣽⬊ ᠱ⃮ᾮࢆὀධࡋࡓࡇࢁ (Day 0㸧,㸦C, G㸧ࢣࣛࢳࣀࢧࢺࡣࢥ࣮ࣛࢤࣥࢤࣝୖ ᥋╔ࡍࡿ㸦Day 7 ࡲ࡛㸧,㸦D㸧Day 8 ௨㝆ࡣᇵᆅࢆࣥࢧ࣮ࢺࢹࢵࢩࣗࡽ 㝖ཤࡋ㸪௦ࢃࡾ࢘ࢱ࣮࢙࢘ࣝὀධ,㸦H㸧ᇵ㣴⤊
ᅗ ᅗ1-3 ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ⤌⧊ീࡢኚ (Day 4, Day 8) ᇵ㣴 4 ᪥┠௨㝆ᇵ㣴 8 ᪥┠ࡲ࡛㸪ᇵ㣴⣽⬊ࡣ 4–6 ᒙࡢᒙᵓ㐀 ࢆᙧᡂࡋࡓ, (Day 12, Day 17) ⬺᰾ࡼࡾ㸪ゅᒙᵝᒙࡀᙧᡂࡉࢀࡓࠋࡲࡓ㸪ゅᒙ ᵝᒙࡢୗᒙ࠾࠸࡚㸪⣽⬊ෆ㢛⢏ࢆྵࡴ㢛⢏ᒙᵝᒙࡀᙧᡂࡉࢀࡓࠋࢫࢣ࣮ࣝࣂ࣮ ࡣ20 μm ࢆ♧ࡍࠋ
ᅗ
ᅗ1-4 ༢ᒙᇵ㣴㸪≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡼࡧ≟ṇᖖ⓶࠾ࡅࡿࢧࢺ࢝
ࣥ㑇ఏᏊ㌿
(a) TNF-alpha, (b) TGF-beta, (c) CCL2 ࠾ࡼࡧ (d) CCL20 ࡢ㌿㔞ࠋⓑ㸪⅊㸪㯮 Ⰽࡢࣂ࣮ࡣࡑࢀࡒࢀ༢ᒙᇵ㣴㸪≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ㸪≟ṇᖖ⓶ࢧࣥࣉࣝࢆ ♧ࡍࠋࣜࣇࣞࣥࢫ㑇ఏᏊࡋ࡚RPL32, HPRT1 ࠾ࡼࡧ RPS18 ࢆ⏝࠸ࡓࠋN.D.: ᳨ฟ㝈⏺௨ୗ㸪n=3㸪*P < 0.05㸪Bonferroni/Dunn ᳨ᐃ
(a)
(b)
➨
➨㸰❶
≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ
ZO-1 ࠾ࡼࡧ claudin-1 ࡢ
2.1. ᗎㄽ
⓶ࡣࣂࣜࢆᙧᡂࡍࡿࡇࡼࡗ࡚㸪ᚤ⏕≀㸪Ꮫ≀㉁࠾ࡼࡧ≀⌮ⓗ࡞่⃭ ࡽ⏕యࢆಖㆤࡍࡿඹ㸪యෆࡽࡢỈศࡸ㟁ゎ㉁࠾ࡼࡧࢱࣥࣃࢡ㉁ࡢ₃ฟ ࢆ㜵࠸࡛࠸ࡿ[43]ࠋ⓶ࣂࣜࡣゅᒙ࠾ࡼࡧࢱࢺࢪࣕࣥࢡࢩࣙࣥࡽᵓᡂࡉࢀ ࡿࡇࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿ[18, 38]ࠋゅᒙࣂࣜࡣ㸪ゅᒙ⣽⬊ࡑࡢ⣽⬊⭷ࢆ ᙉ㠌ᡴࡕࡍࡿ࿘㎶ᖏ࠾ࡼࡧ⣽⬊㛫⬡㉁ࡼࡾᙧᡂࡉࢀࡿ[59]ࠋࡉࡽ㸪㢛⢏ ᒙࡢ⣽⬊㛫᥋ྜ㒊ࡣࢱࢺࢪࣕࣥࢡࢩࣙࣥࡤࢀࡿࢱࣥࣃࢡࡀᏑᅾࡍࡿࠋ ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡣ㸪」ᩘࡢࢱࣥࣃࢡࡢ㞟ྜయ࡛࠶ࡾ㸪claudin ࡸ occludin ࡞ ࡢ⣽⬊⭷㈏㏻ᆺࢱࣥࣃࢡ㸪ZO ࡞ࡢࢡࢳࣥࣇ࣓ࣛࣥࢺࡢ㊊ሙ࡞ࡿࢱࣥ ࣃࢡࡲࡓࡣZO-1-associated nucleic acid-binding protein (ZONAB) ࡞ࡢ⣽⬊㉁ෆ ࠾࠸࡚ࢩࢢࢼࣝศᏊࡸ㌿ᅉᏊࡋ࡚ᶵ⬟ࡍࡿࢱࣥࣃࢡศ㢮ࡉࢀࡿ[5, 70]ࠋ ZO-1 ࡣ㸪1986 ᖺྠᐃࡉࢀ[92]㸪⣽⬊ෆ࠾࠸࡚ࡣ claudin ࡸ occludin ඹ 」ྜయࢆᙧᡂࡋ࡚࠸ࡿ[58]ࠋZO-1 ࠾ࡼࡧ ZO-2 ࢆࣀࢵࢡ࢘ࢺࡋࡓ࣐࢘ࢫࡢࢣࣛ ࢳࣀࢧࢺ࠾࠸࡚ࡣ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᙧᡂࡀⴭࡋࡃ㜼ᐖࡉࢀࡿඹ ⤒ୖ⓶㟁Ẽᢠ (Transepithelial electrical resistance; TER) ࡢῶᑡ࠾ࡼࡧ⣽⬊㛫 ࠾ࡅࡿ≀㉁㏱㐣ᛶࡀஹ㐍ࡍࡿ[95]ࠋࡇࡢࡇࡽ㸪ZO-1 ࡣࢱࢺࢪࣕࣥࢡࢩ ࣙࣥࡢᙧᡂ࡞ࡽࡧࣂࣜᶵ⬟ࡢ⥔ᣢ㔜せ࡞ᙺࢆᢸࡗ࡚࠸ࡿࡇࡀ⪃࠼ࡽ ࢀࡓࠋ claudin ࡣ㸪1998 ᖺྠᐃࡉࢀ[23]㸪⌧ᅾࣄࢺ࠾ࡼࡧ࣐࢘ࢫ࠾࠸࡚ 27 ࡢ ࢯࣇ࢛࣮࣒ࡀ▱ࡽࢀ࡚࠸ࡿ[56]ࠋ༢୍ࡢ⣽⬊ࡣᑡ࡞ࡃࡶ㸰✀㢮ࡢ claudin ࡀ Ⓨ⌧ࡋ࡚࠾ࡾ㸪ඹ㔜ྜࡋ࡚㸯ᮏࡢࢱࢺࢪࣕࣥࢡࢩࣙࣥ㙐ࢆᙧᡂࡋ㸪ࡉࡽྥ ࠸ྜ࠺⣽⬊⭷ୖࡢࢱࢺࢪࣕࣥࢡࢩࣙࣥ㙐ࡀ⤖ྜࡍࡿ[94]ࠋclaudin-1 ࢆࣀࢵࢡࢲ ࢘ࣥࡉࡏࡓࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚ࡣ㸪TER ࡢపୗ࠾ࡼࡧ⣽⬊㛫࠾ࡅࡿ ≀㉁㏱㐣ᛶࡢஹ㐍ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[18, 100]ࠋࡉࡽ㸪claudin-1 ࢆࣀࢵࢡ࢘ࢺࡋࡓ࣐࢘ࢫ࠾࠸࡚ࡣ㸪⤒⓶ⓗỈศᩓ㔞 (TEWL) ࡢቑຍࡀㄆࡵࡽࢀ㸪⣽⬊㛫 ࡢ≀㉁㏱㐣ᛶࡢஹ㐍ࡶ࣐࢘ࢫࡣ⬺Ỉࡼࡾ⏕ᚋ1 ᪥࡛Ṛஸࡍࡿ[24]ࠋࡇࢀ ࡽࡢ⤖ᯝࡽ㸪claudin-1 ࡣ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢࣂࣜᶵ⬟㔜せ࡞ᙺ ࢆᯝࡓࡋ࡚࠸ࡿᛮࢃࢀࡿࠋ ࡇࢀࡲ࡛㸪claudin-1 Ḟᦆ࣐࢘ࢫ࠾ࡅࡿࣥ࣎ࣝࢡࣜࣥ࠾ࡼࡧࣇࣛࢢࣜ ࣥࡢⓎ⌧పୗࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸪ࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚ claudin-1 ࡢ ࣀࢵࢡࢲ࢘ࣥࡼࡾ㸪ࢣࣛࢳࣀࢧࢺࡢቑṪࡀㄆࡵࡽࢀࡿࡇࡀ♧ࡉࢀ࡚࠸ࡿ [18, 39]ࠋࡲࡓ㸪ࣛࣥࢤࣝࣁࣥࢫ⣽⬊ࡣᶞ≧✺㉳ claudin-1 ࢆⓎ⌧ࡋ࡚࠾ࡾ㸪ά ᛶࡋ࡚ᢠཎᥦ♧ࢆ⾜࠺㝿ࡣ㸪ᶞ≧✺㉳ࢆ⓶⾲ᒙྥࡗ࡚ఙ㛗ࡋ㸪㢛⢏ᒙ ࡢࢣࣛࢳࣀࢧࢺࡢ㛫ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᵓ⠏ࡼࡿࣂࣜࢆᙧᡂ ࡍࡿࡇࡀ࣐࢘ࢫ࠾࠸࡚᫂ࡽ࡞ࡗ࡚࠸ࡿ[42]ࠋࡘࡲࡾ㸪ࢱࢺࢪࣕࣥࢡࢩ ࣙࣥࡣ≀⌮ⓗ࡞ࣂࣜࡢᙧᡂຍ࠼㸪ࡉࡽࢣࣛࢳࣀࢧࢺࡢศࡢ㛵࠾ ࡼࡧචᏛⓗࡶࣂࣜᶵ⬟ࡢ⥔ᣢ㛵ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋ ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢ␗ᖖࡣ㸪ᵝࠎ࡞⓶ᝈࡢᅉ⪃࠼ࡽࢀ࡚࠾ࡾ㸪ࣄ ࢺ࡛ࡣࡇࢀࡲ࡛ᑜᖖᛶⓄ㸪NISCH ೃ⩌ (neonatal sclerosing cholangitis associated with ichthyosis)㸪ᡥᖹⱏⓄ㸪AD ࡢ㛵㐃ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[12]ࠋࡇࢀ ࡽࡢᝈ࠾࠸࡚ࡣ㸪ZO-1, claudin-1㸪claudin-4㸪occludin ࡞ࡢࢱࢺࢪࣕࣥࢡ ࢩࣙࣥࢱࣥࣃࢡࡢⓎ⌧ኚࡀㄆࡵࡽࢀ࡚࠸ࡿ[18, 22, 40, 77]ࠋ୍᪉㸪≟࠾࠸࡚ ࡣ㸪 CAD ࣔࢹࣝ≟ࡢ㠀ኚ㒊⓶࠾࠸࡚㸪ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧పୗ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[37, 79]ࠋࣄࢺ AD ྠᵝ㸪CAD ࠾࠸࡚ࡶࢱࢺࢪࣕࣥࢡ ࢩࣙࣥࡢᶵ⬟㞀ᐖ㉳ᅉࡍࡿ⓶ࣂࣜᶵ⬟ࡢపୗࡀែ㛵ࡋ࡚࠸ࡿࡇ ࡀ♧၀ࡉࢀࡿࡀ㸪ヲ⣽ࡣࢃࡗ࡚࠸࡞࠸ࠋᚋ㸪ಶࠎࡢࢱࢺࢪࣕࣥࢡࢩࣙࣥࢱ ࣥࣃࢡࡢᙺࢆ᫂ࡽࡋ࡚࠸ࡃ࠶ࡓࡾ㸪≟ࢣࣛࢳࣀࢧࢺࢆ⏝࠸ࡓ in vitro ࣔࢹࣝࡀồࡵࡽࢀ࡚࠸ࡿࠋ ๓❶࠾࠸࡚㸪CAD ࡢ⣽⬊ࣔࢹࣝࡋ࡚㸪༢ᒙᇵ㣴ẚ㍑ࡋࡓ≟୕ḟඖᇵ㣴
⾲⓶ࣔࢹࣝࡢඃᛶࡀࢧࢺ࢝ࣥ࠾ࡼࡧࢣࣔ࢝ࣥࡢ㌿㔞ゎᯒࡽ᫂ࡽ ࡞ࡗࡓࠋࡋࡋ㸪ᮏࣔࢹࣝ࠾ࡅࡿࢱࢺࢪࣕࣥࢡࢩࣙࣥࢱࣥࣃࢡࡢⓎ⌧ࡣ ࡛᫂࠶ࡿࠋࡑࡇ࡛㸪ᮏ❶࡛ࡣCAD ࠾࠸࡚Ⓨ⌧ኚࡀሗ࿌ࡉࢀ࡚࠸ࡿࡶࡢࡢ࠺ ࡕ㸪≉ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢࣂࣜᶵ⬟୰ᚰⓗ࡞ᙺࢆᯝࡓࡍZO-1 ࠾ࡼ ࡧclaudin-1 ࢆᑐ㇟㸪≟ࡢ⾲⓶ᮏࣔࢹࣝ࠾ࡅࡿⓎ⌧ࢆẚ㍑ࡋࡓࠋ
2.2. ᮦᩱ᪉ἲ
2.2.1 ᮦᩱ࠾ࡼࡧື≀
≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢస〇ࡣ㸪CPEK (CELLnTEC advanced cell systems, Bern, Switzerland㸧࠾ࡼࡧࡑࡢᇵ㣴ᾮ࡛࠶ࡿ CnT-09 (CELLnTEC advanced cell systems) ࢆ⏝࠸ࡓࠋ⓶ࡣ㸰㢌ࡢᗣ࡞ࣅ࣮ࢢࣝ≟㸦ᖺ㱋 4 ṓ㱋㸪࢜ࢫ࠾ࡼࡧ 5 ṓ㱋㸪࣓ࢫ㸧ࡢయᖿ⭡ഃ㒊ࡼࡾ㸪8 mm ⏕᳨ࣃࣥࢳࢆ⏝࠸࡚᥇ྲྀࡋ㸪10%୰ᛶ⦆ ⾪࣐࣍ࣝࣜࣥᾮ࡛ᅛᐃࡋࡓࠋᐇ㦂ື≀㛵ࢃࡿࡍ࡚ࡢᡭᢏࡣ㸪ᒱ㜧Ꮫື≀ᐇ 㦂ጤဨࡢᢎㄆࢆᚓ࡚⾜ࡗࡓࠋ㸦#15001㸧 2.2.2 ୕ḟඖᇵ㣴 ୕ḟඖᇵ㣴ࡣ 1 ❶࡛㏙ࡓ᪉ἲྠᵝ⾜ࡗࡓࠋᇵ㣴 5 ᪥┠ࡽᇵ㣴⤊ࡲ ࡛㸪ᇵᆅ0.4 μg/ml ࡢࣄࢻࣟࢥࣝࢳࢰࣥ㸦Sigma-Aldrich㸧ࢆῧຍࡋࡓ CnT-09 ࢆ ⏝ࡋࡓࠋᇵ㣴14 ᪥┠㸪ᇵᆅࡢධࡗࡓእഃࡢᇵ㣴─ࡽᇵ㣴⣽⬊ࡢධࡗࡓෆ ഃࡢᇵ㣴─ࢆྲྀࡾฟࡋ㸪᭰ล࣓ࢫ㸦No.11; ࢝ࣥࢲࢫࢺ࣮ࣜࢬ㸪ᒱ㜧㸪᪥ᮏ㸧 ࢆ⏝࠸࡚㸪ࢥ࣮ࣛࢤࣥࢤࣝ࠾ࡼࡧෆഃࡢᇵ㣴─ࡢᗏ㒊ࡢࣇࣝࢱ࣮ࡈᇵ㣴⣽ ⬊ࢆ᥇ྲྀࡋࡓࠋ᥇ྲྀࡋࡓࢧࣥࣉࣝࡣ㸪10%୰ᛶ⦆⾪࣐࣍ࣝࣜࣥᾮ࡛ᅛᐃࡋࡓࠋ࣍ ࣐ࣝࣜࣥᅛᐃᚋ㸪ࣃࣛࣇࣥໟᇙࢆ⾜ࡗࡓ⓶࠾ࡼࡧᇵ㣴⣽⬊ࢆ3 μm ࡛ⷧษ ࡋ㸪࣐࣊ࢺ࢟ࢩ࢚ࣜࣥ࢜ࢪࣥᰁⰍ࠾ࡼࡧච⤌⧊Ꮫ౪ࡋࡓࠋ 2.2.3 ච⤌⧊Ꮫ ษ∦ࡣ㸪࢟ࢩࣞࣥࢆ⏝࠸࡚2 ᗘ⬺⬡ࡋ㸪↓Ỉ࢚ࢱࣀ࣮ࣝࡼࡿỈࢆ 10 ศ 㛫ࡎࡘ 2 ᗘ⾜ࡗࡓᚋ㸪␃Ỉ࡚Ὑίࡋࡓࠋ95–100Υண⇕ࡋࡓ࢚ࢳࣞࣥࢪ ࣑ࣥᅄ㓑㓟㸦Tris-EDTA㸧⦆⾪ᾮ㸦pH 9.0; Dako, Santa Clara, CA, USA㸧ᾐₕࡋ ࡓษ∦ࢆ㸪ᐙᗞ⏝㟁Ꮚࣞࣥࢪࢆ⏝࠸࡚20 ศ㛫ຍ ࡋ㸪ᢠཎ㈿άฎ⌮ࢆ⾜ࡗࡓࠋ
20 ศ㛫ὶỈ࡛෭༷ࡋࡓᚋ㸪␃Ỉ࡛ 10 ศ㛫Ὑίࡋࡓࠋ0.1%∵⾑Ύࣝࣈ࣑ࣥ 㸦BSA㸧࠾ࡼࡧ 0.1%Tween20㸦Tween20; ග⣧⸆ᕤᴗ, 㜰, ᪥ᮏ㸧ࢆῧຍࡋࡓ ࢺࣜࢫሷ㓟⦆⾪ᾮ㸦TBS㸧 10 ศ㛫ᾐₕࡋࡓᚋ㸪ෆᅾᛶ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮࡢ άࡢࡓࡵ 0.3%㐣㓟Ỉ⣲Ỉࢆྵࡴ࣓ࢱࣀ࣮ࣝᾮ୰࡛ 10 ศ㛫ᛂࡉࡏ㸪 0.1%BSA ຍ TBS 10 ศ㛫ᾐₕࡋ㸪ࡇࢀࢆ㸱ᅇ⧞ࡾ㏉ࡋ࡚Ὑίࡋࡓࠋ10%ṇᖖ ࣖࢠ⾑Ύࢆῧຍࡋࡓࣜࣥ㓟⦆⾪㣗ሷỈ㸦PBS㸧ࢆ⏝࠸࡚㸪ᐊ ࡚ 30 ศ㛫㠀≉ ␗ⓗᛂࡢᢚไฎ⌮ࢆ⾜ࡗࡓࠋࢿ࢞ࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚㸪୍ḟᢠయࡢ௦ࢃ ࡾṇᖖ࢘ࢧࢠ⾑Ύ⏤᮶࣒ࣀࢢࣟࣈࣜࣥ (X0903; Dako) ࢆྠࡌࢱࣥࣃࢡ⃰ᗘ ᕼ㔘ࡋ࡚⏝ࡋࡓࠋࡲࡓ㸪≟ṇᖖ⓶ࢆ࣏ࢪࢸࣈࢥࣥࢺ࣮ࣟࣝࡋࡓ[78]ࠋ ZO-1 ࡲࡓࡣ claudin-1 ࡢⓎ⌧ゎᯒࡣ㸪≟ࡢ ZO-1 ࠾ࡼࡧ claudin-1 ࢆㄆ㆑ࡍࡿ࢘ ࢧࢠ࣏ࣜࢡ࣮ࣟࢼࣝᢠࣄࢺZO-1 ᢠయ㸦#61-7300; Thermo Fisher Scientific, Waltham, MA, USA㸧ࡲࡓࡣ࢘ࢧࢠ࣏ࣜࢡ࣮ࣟࢼࣝᢠࣄࢺ claudin-1 ᢠయ㸦#ab15098; Abcam, Cambridge, UK㸧ࢆࡑࢀࡒࢀ⏝ࡋࡓ[78]ࠋ୍ḟᢠయ࠾ࡼࡧࢿ࢞ࢹࣈࢥࣥࢺ࣮ࣟ ࣝࡢᕼ㔘ࡣ1.5%ṇᖖࣖࢠ⾑Ύຍ PBS ࢆ⏝࠸ࡓࠋ୍ḟᢠయࡢᕼ㔘ಸ⋡ࡣࡑࢀࡒ ࢀࡢᢠయࡘ࠸࡚㸪ࡇࢀࡲ࡛ࡢሗ࿌[78]ࢆཧ⪃㸪㸱ẁ㝵ࡢᕼ㔘ಸ⋡ (1 μg/ml, 5 μg/ml, 25 μg/ml) ࡛⾜ࡗࡓࠋ⫼ᬒࡢ㠀≉␗ⓗᛂࡀ᭱ࡶᑡ࡞ࡃ㸪᭱ࡶᰁⰍᙉᗘࡀ 㧗ࡃᚓࡽࢀࡓࡶࡢࢆ⮳㐺⃰ᗘࡋ࡚㑅ᢥࡋࡓࠋ≟ṇᖖ⓶࠾ࡼࡧ≟୕ḟඖᇵ㣴 ⾲⓶ࣔࢹࣝ࠾࠸࡚㸪⮳㐺࡞୍ḟᢠయ⃰ᗘࡣZO-1 ࠾ࡼࡧ claudin-1 ඹ㸪1 μg/ml ࡛࠶ࡗࡓ㸦ᅗ2-1 ࠾ࡼࡧ 2-2㸧ࠋ୍ḟᢠయࢆ㸯㛫ᐊ ࡚ᛂࡉࡏ㸪0.1%BSA ຍTBS 10 ศ㛫ᾐₕࡋ㸪ࡇࢀࢆ㸱ᅇ⧞ࡾ㏉ࡋ࡚Ὑίࡋࡓࠋḟḟᢠయࡋ࡚ ࣮࣍ࢫࣛࢹࢵࢩࣗ࣌ࣝ࢜࢟ࢩࢲ࣮ࢮᶆ㆑ࣖࢠ⏤᮶ᢠ࢘ࢧࢠ࣒ࣀࢢࣟࣈࣜࣥ (EnVision+ system- HRP; Dako) ࢆ⏝࠸࡚㸪ᐊ ࡛ 30 ศ㛫ᛂࡉࡏࡓᚋ㸪0.1%BSA ຍTBS 10 ศ㛫ᾐₕࡋ㸪ࡇࢀࢆ㸱ᅇ⧞ࡾ㏉ࡋ࡚Ὑίࡋࡓࠋࢪ࣑ࣀ࣋ࣥࢪࢪࣥ ᾮ࡛ⓎⰍࡉࡏࡓษ∦ࡣ㸪࣐࣊ࢺ࢟ࢩ࡛ࣜࣥ᰾ᰁⰍࢆ⾜ࡗࡓᚋ㸪࢝ࣂ࣮࢞ࣛࢫࢆ⏝ ࠸࡚ᑒධࡋ㸪ගᏛ㢧ᚤ㙾 (DM2500, Leica, Wetzlar, Germany) ࡚ほᐹࡋࡓࠋᐇ㦂
2.3 ⤖ᯝ
2.3.1 ≟ṇᖖ⓶ ZO-1 ࠾ࡼࡧ claudin-1 ࡣ㸪ゅᒙࢆ㝖ࡃࡍ࡚ࡢᒙ࠾࠸࡚Ⓨ⌧ࡀㄆࡵࡽࢀࡓ 㸦ᅗ2-3a, b㸧ࠋclaudin-1 ࠾ࡅࡿᰁⰍᙉᗘࡣ㸪㢛⢏ᒙ࠾࠸࡚᭱ࡶᙉࡃ㸪⻏ࡢᕢ ≧ࡢᰁⰍࣃࢱ࣮ࣥࢆ♧ࡋࡓࠋࢿ࢞ࢸࣈࢥࣥࢺ࣮ࣟࣝ࠾࠸࡚㸪㝧ᛶᰁⰍࡣㄆࡵ ࡽࢀ࡞ࡗࡓ㸦ᅗ2-1a, 2-2a㸧 2.3.2 ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ ZO-1 ࡣゅᒙᵝࡢᒙࢆ㝖ࡃࡍ࡚ࡢᒙ࡛㸪⣽⬊㉁࠾࠸࡚Ⓨ⌧ࡀㄆࡵࡽࢀ ࡓ㸦ᅗ2-3c㸧ࠋclaudin-1 ࡣゅᒙᵝࡢᒙࢆ㝖ࡃ㸪㢛⢏ᒙᵝᒙࡽ᭷Ჲᒙᵝᒙࡢ ୖᒙ࠾࠸࡚Ⓨ⌧ࡀㄆࡵࡽࢀࡓ㸦ᅗ2-3d㸧ࠋclaudin-1 ࡢᰁⰍᙉᗘࡣ㸪㢛⢏ᒙᵝࡢ ᒙ࠾࠸࡚᭱ࡶ㧗ࡃ㸪≟ṇᖖ⓶㢮ఝࡋࡓ⻏ࡢᕢ≧ࡢᰁⰍࣃࢱ࣮ࣥࢆ♧ࡋࡓ 㸦ᅗ 2-3d㸧ࠋࢿ࢞ࢸࣈࢥࣥࢺ࣮ࣟࣝ࠾࠸࡚㸪㝧ᛶᰁⰍࡣㄆࡵࡽࢀ࡞ࡗࡓ 㸦ᅗ2-1e, 2-2e㸧2.4. ⪃ᐹ
ᮏ❶࡛ࡣ㸪ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧ࢆホ౯ࡍࡿࡇࡼࡾ㸪ࢱࢺࢪࣕࣥ ࢡࢩࣙࣥࡢࣂࣜᶵ⬟◊✲ࢶ࣮ࣝࡋ࡚ࡢ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢ᭷⏝ᛶࢆ ᳨ウࡋࡓࠋ ᮏࣔࢹࣝ࠾࠸࡚㸪ZO-1 ࡣ㸪ゅᒙᵝᒙࢆ㝖ࡃ࡚ࡢᒙࡢ⣽⬊㉁ࢆ୰ᚰⓎ⌧ ࡀㄆࡵࡽࢀࡓࠋZO-1 ࠾ࡅࡿࡇࡢᰁⰍࣃࢱ࣮ࣥ࠾ࡼࡧᒁᅾࡣ㸪ᮏ◊✲࠾ࡼࡧࡇ ࢀࡲ࡛ሗ࿌ࡉࢀ࡚࠸ࡿ≟ࡢṇᖖ⓶࠾ࡅࡿⓎ⌧⤖ᯝ㢮ఝࡋ࡚࠸ࡓ[78, 79]ࠋ CAD ࡛ࡣ㸪㠀ኚ㒊⓶࠾࠸࡚㸪ච⤌⧊Ꮫࡼࡿ ZO-1 ࡢᰁⰍᙉᗘ ࡀṇᖖ⓶ẚ㍑ࡋ࡚పୗࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ[37]㸪ࣄࢺ AD ࠾࠸ ࡚ࡶኚ㒊࠾ࡼࡧ㠀ኚ㒊࠾ࡅࡿⓎ⌧㔞ࡢపୗࡀ♧ࡉࢀ࡚࠸ࡿ[102]ࠋࡲࡓ㸪 ࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪IL-1beta ࡶࡋࡃࡣ IL-17 ࡼࡾ ZO-1 ࡢⓎ⌧㔞ࡀ పୗࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾ[40, 102]㸪ᮏࣔࢹࣝࡣ≟ࢣࣛࢳࣀࢧࢺ࠾ࡅࡿ ZO-1 ࡢⓎ⌧ࡘ࠸࡚㸪ࢧࢺ࢝ࣥࡢᙳ㡪ࢆ᳨ドࡍࡿࡢ᭷⏝࡛࠶ࡿᛮࢃࢀ ࡓࠋ ᮏࣔࢹࣝ࠾࠸࡚㸪claudin-1 ࡣ⻏ࡢᕢ≧ࡢᰁⰍࣃࢱ࣮ࣥࢆ♧ࡋࡓࠋࡲࡓ㸪ᰁⰍ ᙉᗘࡣᇶᗏᒙഃྥࡗ࡚ῶᙅࡋࡓࠋࡇࢀࡣᮏ◊✲࠾ࡼࡧࡇࢀࡲ࡛ሗ࿌ࡉࢀ ࡚࠸ࡿ≟ࡢṇᖖ⓶࠾ࡅࡿⓎ⌧⤖ᯝྠᵝ࡛࠶ࡗࡓ[7, 37, 64, 78, 79]ࠋᮏࣔࢹ ࣝ࠾ࡅࡿclaudin-1 ࡢⓎ⌧ࡣ㸪㢛⢏ᒙᵝᒙࡽ᭷Ჲᒙᵝᒙࡢୖᒙࢆ୰ᚰ ㄆࡵࡽࢀࡓࠋࡋࡋ㸪≟ࡢ⾲⓶࠾࠸࡚ࡣ㸪ゅᒙࢆ㝖ࡃ࡚ࡢᒙ࡛ claudin-1 ࡢ Ⓨ⌧ࡀㄆࡵࡽࢀ࡚࠸ࡿ[7, 78]ࠋࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪claudin-1 ࡢⓎ⌧ ࡣᇵᆅ୰㧗࠸Ca2+⃰ᗘࡀᚲせ࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[18]ࠋYagihara ࡽࡣ㸪 ᮏࣔࢹࣝ࠾࠸࡚ࢣࣛࢳࣥ 10 ࡢⓎ⌧ࡀㄆࡵࡽࢀ࡞࠸ࡇࢆሗ࿌ࡋ࡚࠸ࡿ[99]ࠋ ࢣࣛࢳࣥ10 ࡣ Ca2+ࡼࡗ࡚ࢣࣛࢳࣀࢧࢺࡀศࡍࡿ㝿Ⓨ⌧ࡍࡿ࣐࣮࣮࡛࢝ ࠶ࡾ[20]㸪ᮏࣔࢹࣝ࠾࠸࡚ claudin-1 ࡢⓎ⌧ࡀ㸪᭷Ჲᒙࡢ୰㛫ࡼࡾୗᒙࡢ㡿ᇦ࠾࠸࡚ㄆࡵࡽࢀ࡞ࡗࡓཎᅉࡣ㸪ᇵᆅ୰ࡢ Ca2+⃰ᗘࡢ㊊㉳ᅉࡍࡿ CPEK ࡢ ༑ศ࡞ศ࡛࠶ࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ ๓❶࠾࠸࡚㸪ࢣࣛࢳࣀࢧࢺࡢචᏛⓗᶵ⬟◊✲࠾ࡅࡿᮏࣔࢹࣝࡢ᭷⏝ ᛶࡀ♧၀ࡉࢀࡓࠋࡋࡋ㸪ᮏࣔࢹࣝࡣࢣࣛࢳࣀࢧࢺࡢࡳࡽᵓᡂࡉࢀ࡚࠾ࡾ㸪 ࣜࣥࣃ⌫ࡸᶞ≧⣽⬊࡞ࡢ⾲⓶ࢆᵓᡂࡍࡿࡑࡢࡢ⣽⬊ࢆྵࡲ࡞࠸ࠋࡋࡓࡀࡗ ࡚㸪ࡼࡾ㐺ษ࡞ࣔࢹࣝⓎᒎࡉࡏࡿࡓࡵࡣ㸪ᚋࢣࣛࢳࣀࢧࢺ௨እࡢ⾲⓶ᵓ ᡂ⣽⬊ࡢඹᇵ㣴࡞ࡀᚲせ⪃࠼ࡽࢀࡿࠋ ᮏࣔࢹࣝ࠾࠸࡚㸪ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧ࡀ᫂ࡽ࡞ࡗࡓࡇࡽ㸪 ᮏࣔࢹࣝࡀ㢛⢏ᒙ࠾ࡅࡿࢱࢺࢪࣕࣥࢡࢩࣙࣥ◊✲࠾࠸࡚᭷⏝࡛࠶ࡿྍ⬟ ᛶࡀ♧ࡉࢀࡓࠋCAD ࠾࠸࡚ࡣ㸪⓶ࣂࣜᶵ⬟ࡢపୗࡀㄆࡵࡽࢀ࡚࠾ࡾ [89]㸪ࡲࡓ㸪CAD ࣔࢹࣝ≟࠾࠸࡚ ZO-1 ࡸ claudin-1 ࡢⓎ⌧ኚࡀሗ࿌ࡉࢀ࡚ ࠸ࡿ[37, 79]ࠋࡋࡓࡀࡗ࡚㸪CAD ࠾࠸࡚ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᶵ⬟␗ᖖࡀ ែ㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡑࡇ࡛㸪➨㸱❶࡛ࡣ㸪ZO-1 ࠾ࡼࡧ claudin-1 ᑐࡋ㸪Ⓨ⌧ኚࢆㄏᑟࡍࡿᅉᏊࡘ࠸᳨࡚ウࡋࡓࠋ
2.5 ᅗ
ᅗ2-1 ྛ୍ḟᢠయ⃰ᗘ࠾ࡅࡿ ZO-1 ࡢᰁⰍᛶ
(a, b, c, d) ≟ṇᖖ⓶㸪(e, f, g, h) ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ㸪(a, e) 0 μg/ml㸪(b, f) 1 μg/ml㸪(c, g) 5 μg/ml㸪(d, h) 25 μg/mlࠋ⃰ᗘࡣᢠ ZO-1 ᢠయࡢ⃰ᗘࢆ♧ࡍࠋࢫ ࢣ࣮ࣝࣂ࣮ࡣ20 μm ࢆ♧ࡍࠋ
ᅗ
ᅗ2-2 ྛ୍ḟᢠయ⃰ᗘ࠾ࡅࡿ claudin-1 ࡢᰁⰍᛶ
(a, b, c, d) ≟ṇᖖ⓶㸪(e, f, g, h) ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ㸪(a, e) 0 μg/ml㸪(b, f) 1 μg/ml㸪(c, g) 5 μg/ml㸪(d, h) 25 μg/mlࠋ⃰ᗘࡣᢠ claudin-1 ᢠయࡢ⃰ᗘࢆ♧ࡍࠋ ࢫࢣ࣮ࣝࣂ࣮ࡣ20 μm ࢆ♧ࡍࠋ
ᅗ
ᅗ2-3 ≟≟ṇᖖ⓶࠾ࡼࡧ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿච⤌⧊Ꮫ (a, b) ≟ṇᖖ⓶㸪(c, d) ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ㸪(a, c) ZO-1㸪(b, d) claudin-1ࠋᙅᣑࡣ 400 ಸ㸪ᙉᣑࡣ 800 ಸࢆ♧ࡍࠋࢫࢣ࣮ࣝࣂ࣮ࡣ 50 μm ࢆ♧ࡍࠋ
➨
➨㸱❶
≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝ࠾ࡅࡿ
ZO-1 ࠾ࡼࡧ claudin-1 ᑐࡍࡿ
TNF-alpha ࠾ࡼࡧ IL-13 ࡢᙳ㡪
3.1. ᗎㄽ
CAD ࡢᅉࡣ㸪⎔ቃせᅉ࠾ࡼࡧ㑇ఏⓗ⣲ᅉࡼࡿ」ྜⓗ࡞ࡶࡢ࡛࠶ࡾ㸪ࡇࢀ ࡽࡀ⓶ࣂࣜᶵ⬟࠾ࡼࡧᝈ⪅ࡢචᛂᙳ㡪ࢆཬࡰࡍ⪃࠼ࡽࢀ࡚࠸ࡿ [53]ࠋゅᒙࡢỈศಖᣢ㔜せ࡞ᙺࢆᯝࡓࡍࣇࣛࢢࣜࣥࡢ㑇ఏᏊኚ␗࠾ࡼࡧࡑ ࢀ㉳ᅉࡍࡿ⓶ࣂࣜᶵ⬟ࡢ㞀ᐖࡣ㸪AD ࡢ᭱ࡶ㔜せ࡞ᅉᏊ࡛࠶ࡿ⪃࠼ࡽࢀ ࡚࠸ࡿ[17, 50]ࠋࡋࡋ࡞ࡀࡽ㸪AD ᝈ⪅࠾ࡅࡿࣇࣛࢢࣜࣥ㑇ఏᏊኚ␗ࡢྜ ࡣ㸪㞟ᅋࡼࡗ࡚᫂ࡽ࡞ᕪࡀㄆࡵࡽࢀ࡚࠸ࡿ[13, 15]ࠋࡲࡓ㸪㝈ࡽࢀࡓᩘࡢሗ ࿌࡞ࡀࡽ㸪CAD ࠾ࡅࡿࣇࣛࢢࣜࣥࡢⓎ⌧㛵ࡍࡿ◊✲࡛ࡣ㸪CAD ࣇࣛ ࢢࣜࣥ㑇ఏᏊࡢኚ␗ࡢ㛫᭷ព࡞㛵㐃ᛶࡣㄆࡵࡽࢀ࡚࠸࡞࠸[74]ࠋࡋࡓࡀࡗ࡚㸪 AD ࠾ࡼࡧ CAD ࠾ࡅࡿ⓶ࣂࣜᶵ⬟㞀ᐖࡣ㸪ࣇࣛࢢࣜࣥ㑇ఏᏊࡢኚ␗ ௨እࡢせᅉࡀᏑᅾࡍࡿࡇࡀ♧၀ࡉࢀࡿࠋ ࡇࢀࡲ࡛㸪AD ᝈ⪅ࡢ⓶ኚ㒊࠾ࡼࡧ㠀ኚ㒊࠾ࡅࡿ claudin-1 ࡢⓎ⌧ పୗࡢ㸪claudin-1 ࡢ㌿㔞ࡣ㸪⾑Ύ⥲ IgE ࠾ࡼࡧ⥲ዲ㓟⌫ᩘ㈇ࡢ┦㛵ࢆ ♧ࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[18]ࠋࡉࡽ㸪AD ᝈ⪅ࡢ⓶ኚ㒊࠾ࡅࡿ ZO-1 ࡢ Ⓨ⌧㸪㠀ኚ㒊࠾ࡅࡿ ZO-1 ࡞ࡽࡧ claudin-4 ࡢⓎ⌧ࡣṇᖖ⓶ࡼࡾపୗࡋ ࡚࠸ࡿࡇࡶ᫂ࡽ࡞ࡗ࡚࠸ࡿ[102]ࠋࡲࡓ㸪≟࠾࠸࡚ࡣ㸪CAD ࣔࢹࣝ≟ࡢ 㠀ኚ㒊⓶࠾ࡅࡿclaudin-1 ࡢⓎ⌧ࡣᖖ≟ࡢ⓶ẚ࡚పୗࡋ࡚࠾ࡾ㸪 ZO-1 ࠾ࡼࡧ occludin ࡢⓎ⌧ࡣኚࡀㄆࡵࡽࢀ࡞ࡗࡓࡇࡀ♧ࡉࢀ࡚࠸ࡿ [79]ࠋCAD ࣔࢹࣝ≟ࢆ⏝࠸ࡓูࡢሗ࿌࡛ࡣ㸪㠀ኚ㒊⓶࠾ࡅࡿ ZO-1 ࡢⓎ⌧ ࡣᖖ≟ࡢ⓶ẚ㍑ࡋ࡚పୗࡋ࡚࠾ࡾ㸪claudin-1 ࠾ࡼࡧ occludin ࡢⓎ⌧ࡣ ᕪࡀㄆࡵࡽࢀ࡞ࡗࡓࡇࡀ♧ࡉࢀ࡚࠸ࡿ[37]ࠋࡇࢀࡽࡢ⤖ᯝࡣ㸪ࢱࢺࢪࣕࣥ ࢡࢩࣙࣥࢱࣥࣃࢡࡀAD ࠾ࡼࡧ CAD ࡢែ㛵ࡋ࡚࠸ࡿࡇࢆ♧၀ࡍࡿࡶࡢ ࡛࠶ࡿࠋ ࡇࢀࡲ࡛㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࢱࣥࣃࢡࡢⓎ⌧ኚ㛵㐃ࡍࡿᅉᏊࡀᗄࡘሗ࿌ࡉࢀ࡚࠸ࡿࠋᇵ㣴ࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪IL-1beta ࡼࡾ claudin-1 ࡢࢱࣥࣃࢡⓎ⌧㔞ࡢపୗࡀㄆࡵࡽࢀࡓ[98]ࠋࡲࡓ㸪⤌⧊ᇵ㣴ࡋࡓࣄࢺ࠾ࡼࡧࣈ ࢱࡢ⓶࠾࠸࡚㸪IL-1beta ࡢ⓶ෆᢞࡼࡾ㸪ච⺯ගᰁⰍࡼࡿ ZO-1 ࡢᒁ ᅾࡢኚ࡞ࡽࡧ㸪ࢱࣥࣃࢡⓎ⌧㔞ࡢపୗࡀㄆࡵࡽࢀ࡚࠸ࡿ[40]ࠋࡉࡽ㸪ࣄࢺ ⭠ୖ⓶⣽⬊ᰴ࡛࠶ࡿHT-29/B6 ࠾࠸࡚㸪TNF-alpha ࡼࡿ claudin-1 ࡢࢱࣥࣃࢡ Ⓨ⌧㔞ࡢቑຍࡀㄆࡵࡽࢀ࡚࠸ࡿ[2]ࠋࡲࡓ㸪IL-4 ࡸ IL-13 ࡼࡾ㸪ᇵ㣴ࣄࢺࢣࣛࢳ ࣀࢧࢺ࠾࠸࡚㸪claudin-1 ࡢࢱࣥࣃࢡⓎ⌧㔞ࡀቑຍࡋࡓ[18]ࠋࡉࡽูࡢሗ࿌ ࡛ࡣ㸪ࣄࢺࢣࣛࢳࣀࢧࢺࢆ⏝࠸ࡓ୕ḟඖᇵ㣴࠾࠸࡚㸪4, 13 ࠾ࡼࡧ IL-31 ࡼࡾ claudin-1 ࡢ⣽⬊㎶⦕࠾ࡅࡿච⺯ගᰁⰍᙉᗘࡢపୗࡀ♧ࡉࢀ࡚࠸ࡿ [26]ࠋࡇࢀࡽࡢ⤖ᯝࡽ㸪⅖ᛶࢧࢺ࢝ࣥ࠾ࡼࡧ Th2 ࢧࢺ࢝ࣥࡀࢱࢺ ࢪࣕࣥࢡࢩࣙࣥࢱࣥࣃࢡࡢⓎ⌧ኚࢆ㉳ࡇࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ CAD ࡢኚ㒊࠾ࡼࡧ㠀ኚ㒊⓶࠾࠸࡚ࡣ㸪⅖ᛶࢧࢺ࡛࢝ࣥ࠶ࡿ IL-1beta, TNF-alpha ࡢ㸪 IL-4, IL-5, IL-13 ࢆࡣࡌࡵࡋࡓ Th2 ࢧࢺ࢝ࣥࡢ ㌿㔞ࡢቑຍࡀሗ࿌ࡉࢀ࡚࠸ࡿ[45, 61, 62, 83]ࠋࡋࡓࡀࡗ࡚㸪CAD ࠾࠸࡚⅖ ᛶࢧࢺ࢝ࣥࡶࡋࡃࡣ Th2 ࢧࢺ࢝ࣥࡀࢱࢺࢪࣕࣥࢡࢩࣙࣥࢱࣥࣃࢡࡢ Ⓨ⌧ᙳ㡪ࢆ࠼㸪⓶ࣂࣜᶵ⬟ࡢపୗࢆᣍ᮶ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ ࡞࡛ࡶ㸪TNF-alpha ࡣ㸪ࣄࢺࢣࣛࢳࣀࢧࢺ࠾ࡅࡿ Thymus and activation regulated chemokine (TARC)/CCL17 ࡢ⏘⏕ࢆቑຍࡉࡏࡿࡇࡀ♧ࡉࢀ࡚࠾ࡾ[96]㸪 ≟ࢣࣛࢳࣀࢧࢺ࠾࠸࡚ࡣ㸪TNF-alpha ࡼࡾ TARC/CCL17 ࡢ㌿㔞ࡀቑຍ ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[87]ࠋ୍᪉㸪IL-1beta ࡣ TARC/CCL17 ࡢ㌿㔞ኚ ࢆཬࡰࡉ࡞ࡗࡓ[87]ࠋࡘࡲࡾ㸪TNF-alpha ࡣ㸪CAD ࠾࠸࡚⓶ኚ㒊ࡢ Th2 ⣽⬊ࡢ㐟㉮ࢆಁ㐍ࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡿࠋࡲࡓ㸪IL-13 ࡣ㸪⤒⓶ⓗ࡞ HDM ่⃭ ࢆ⾜ࡗࡓ≟ࡢ⓶࠾࠸࡚㸪IL-4, IL-5, IL-10 ࡼࡾࡶ᪩ᮇ㸪ࡼࡾ㧗࠸㌿άᛶ ࢆ♧ࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[49]ࠋࡋࡓࡀࡗ࡚㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡣ㸪CAD ࠾ࡅࡿ Th2 ᆺࡢᛂࡢಁ㐍㔜せ࡞ᙺࢆᯝࡓࡍ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛㸪ᮏ
◊✲࡛ࡣ⅖ᛶࢧࢺ࢝ࣥ࠾ࡼࡧTh2 ࢧࢺ࢝ࣥࡋ࡚ࡑࢀࡒࢀ TNF-alpha ࠾ࡼࡧIL-13 ╔┠ࡋࡓࠋ ࡇࢀࡽࡢࢧࢺ࢝ࣥࡀ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥཬࡰࡍᙳ㡪ࡀ᫂ࡽ࡞ ࢀࡤ㸪CAD ࠾ࡅࡿ⓶ࣂࣜᶵ⬟ࡢ␗ᖖࡘ࠸࡚㸪ࡉࡽ࡞ࡿែࡢゎ᫂ࡀᮇ ᚅ࡛ࡁࡿᛮࢃࢀࡿࡀ㸪⌧ᅾヲ⣽ࡣ࡛᫂࠶ࡿࠋ ๓❶ࡲ࡛࠾࠸࡚㸪ᚑ᮶ࡢ༢ᒙᇵ㣴ẚ㍑ࡋࡓ≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢ᭷ ⏝ᛶࡀ᫂ࡽ࡞ࡗࡓࠋࡑࡇ࡛㸪ᮏ❶࡛ࡣࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᙧᡂ࠾ࡼࡧࣂ ࣜᶵ⬟ࡑࢀࡒࢀ㔜せ࡞ᙺࢆᯝࡓࡍZO-1 ࠾ࡼࡧ claudin-1 ࡘ࠸࡚㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡀཬࡰࡍᙳ㡪ࢆホ౯ࡋࡓࠋ
3.2. ᮦᩱ᪉ἲ
3.2.1 ᮦᩱ࠾ࡼࡧື≀
≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࡢస〇ࡣ㸪CPEK (CELLnTEC advanced cell systems, Bern, Switzerland㸧࠾ࡼࡧࡑࡢᇵ㣴ᾮ࡛࠶ࡿ CnT-09 (CELLnTEC advanced cell systems) ࢆ⏝࠸ࡓࠋ⤌࠼≟ TNF-alpha ࠾ࡼࡧ≟ IL-13 ࡣ㸪ࡑࢀࡒࢀ Sino Biological㸦ி㸪୰ᅜ㸧࠾ࡼࡧ Kingfisher Biotech㸦Saint Paul, MN, USA㸧ࡼࡾධ ᡭࡋࡓࠋ⓶ࡣ㸰㢌ࡢᗣ࡞ࣅ࣮ࢢࣝ≟㸦ᖺ㱋 2 ṓ㱋㸪࢜ࢫ࠾ࡼࡧ 4 ṓ㱋㸪࣓ ࢫ㸧ࡢయᖿ⭡ഃ㒊ࡼࡾ㸪8 mm ⏕᳨ࣃࣥࢳࢆ⏝࠸࡚᥇ྲྀࡋ㸪10%୰ᛶ⦆⾪࣐࣍ࣝ ࣜࣥᾮ࡛ᅛᐃࡋࡓࠋ
3.2.2 TNF-alpha ࠾ࡼࡧ IL-13 ࡢㄪ〇
⤌࠼≟ TNF-alpha ࠾ࡼࡧ⤌࠼≟ IL-13 ࡣ㸪ࡑࢀࡒࢀ RNase ࣇ࣮ࣜỈ࠾ࡼ ࡧ0.1%BSA ࢆῧຍࡋࡓ PBS ࢆ⏝࠸࡚⁐ゎ࠾ࡼࡧᕼ㔘ࢆ⾜ࡗࡓࠋ 3.2.3 ୕ḟඖᇵ㣴 ୕ḟඖᇵ㣴ࡣ1 ❶࡛㏙ࡓ᪉ἲྠᵝ⾜࠸㸪ᇵ㣴 5 ᪥┠ࡽᇵ㣴⤊ࡲ࡛㸪 ᇵ㣴ᾮ0.4 μg/ml ࡢࣄࢻࣟࢥࣝࢳࢰࣥ㸦Sigma-Aldrich㸧ࢆῧຍࡋࡓࠋᇵ㣴 8 ᪥ ┠ࡽࡣࣄࢻࣟࢥࣝࢳࢰࣥຍ࠼㸪ᇵ㣴ᾮᑐࡋ5 ng/ml ࡲࡓࡣ 50 ng/ml ࡞ࡿ ࡼ࠺ㄪ〇ࡋࡓ⤌࠼≟TNF-alpha ࠾ࡼࡧ⤌࠼≟ IL-13 ࢆῧຍࡋࡓࠋࡲࡓ㸪ᑐ ↷ࢧࣥࣉࣝࡢᇵ㣴ᾮࡣCnT-09 ࡶࡋࡃࡣ 0.1%ࡢ BSA ࢆῧຍࡋࡓࠋ ᇵ㣴 14 ᪥┠㸪ᇵᆅࡢධࡗࡓእഃࡢᇵ㣴─ࡽᇵ㣴⣽⬊ࡢධࡗࡓෆഃࡢᇵ㣴 ─ࢆྲྀࡾฟࡋ㸪᭰ล࣓ࢫ㸦No.11; ࢝ࣥࢲࢫࢺ࣮ࣜࢬ㸧ࢆ⏝࠸࡚㸪ࢥ࣮ࣛࢤ ࣥࢤࣝ࠾ࡼࡧෆഃࡢᇵ㣴─ࡢᗏ㒊ࡢࣇࣝࢱ࣮ࡈᇵ㣴⣽⬊ࢆ᥇ྲྀࡋࡓࠋ᥇ྲྀ ࡋࡓࢧࣥࣉࣝࡣ㸪10%୰ᛶ⦆⾪࣐࣍ࣝࣜࣥᾮ࡛ᅛᐃࡋࡓࠋ࣐࣍ࣝࣜࣥᅛᐃᚋ㸪
ࣃࣛࣇࣥໟᇙࢆ⾜ࡗࡓ⓶࠾ࡼࡧᇵ㣴⣽⬊ࢆ3 μm ࡛ⷧษࡋ㸪࣐࣊ࢺ࢟ࢩࣜࣥ ࢚࢜ࢪࣥᰁⰍ࠾ࡼࡧච⤌⧊Ꮫ౪ࡋࡓࠋ 3.2.4 ච⤌⧊Ꮫ ච⤌⧊Ꮫࡣ㸰❶࡛㏙ࡓ᪉ἲྠᵝ⾜ࡗࡓࠋ⏝࠸ࡓ୍ḟᢠయࡣ㸪㸰❶ࡢ ⤖ᯝᇶ࡙ࡁ㸪ZO-1 ࠾ࡼࡧ claudin-1 ඹ 1 μg/ml ࡛⏝ࡋࡓࠋᐇ㦂ࡣ␗࡞ࡿࢧ ࣥࣉࣝࢆ⏝࠸࡚3 ᅇ⾜ࡗࡓࠋ
3.3 ⤖ᯝ
3.3.1 ᇵ㣴⣽⬊ࡢᙧែ࠾ࡅࡿ TNF-alpha ࠾ࡼࡧ IL-13 ࡢᙳ㡪 TNF-alpha ࡲࡓࡣ IL-13 ࡢᏑᅾୗ࠾࠸࡚ࡶ㸪ᇵ㣴⣽⬊ࡣゅᒙᵝࡢᒙࢆᙧᡂࡋ ࡓࠋ᭷Ჲᒙᵝࡢᒙࡽᇶᗏᒙᵝࡢᒙ࠾࠸࡚ᾏ⥥≧ኚ࠾ࡼࡧ␗ᖖゅࢆㄆࡵ ࡓ㸦ᅗ3-1B, C, E, F㸧ࠋ 3.3.2 ࢥࣥࢺ࣮ࣟࣝ࠾ࡅࡿ ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧ ࢿ࢞ࢸࣈࢥࣥࢺ࣮ࣟࣝࡋ࡚⏝࠸ࡓṇᖖ≟⓶࠾ࡼࡧᇵ㣴⣽⬊࠾࠸࡚ࡣ㸪 ࡕࡽࡶ㝧ᛶᡤぢࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ3.3.2 ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧࠾ࡅࡿ TNF-alpha ࡢᙳ㡪
ZO-1 ࡢⓎ⌧ࢥࣥࢺ࣮ࣟࣝࡢᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦ᅗ 3-2B, C㸧ࠋ୍᪉㸪 claudin-1 ࡢⓎ⌧ࡣ㸪5 ng/ml ࡢ TNF-alpha ῧຍࡼࡾ㸪ᰁⰍᛶࡢ㒊ศⓗ࡞ᾘኻࡀ ㄆࡵࡽࢀࡓ㸦ᅗ3-2E㸧ࠋࡲࡓ㸪ࡑࢀࡣ 50 ng/ml ࡢῧຍ࠾࠸࡚ࡼࡾ㢧ⴭ࡛࠶ࡗ ࡓ㸦ᅗ3-3F㸧ࠋ
3.3.3 ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧࠾ࡅࡿ IL-13 ࡢᙳ㡪
ZO-1 ࡢⓎ⌧ࢥࣥࢺ࣮ࣟࣝࡢᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦ᅗ 3-3B, C㸧ࠋ୍᪉㸪 claudin-1 ࡢⓎ⌧ࡣ㸪5 ng/ml ࡢ IL-13 ῧຍࡼࡾ㸪ᰁⰍᛶࡢ㒊ศⓗ࡞ᾘኻࡀㄆࡵ ࡽࢀࡓ㸦ᅗ3-3E㸧ࠋIL-13 ࡢ⃰ᗘࡼࡿᕪࡣㄆࡵࡽࢀ࡞ࡗࡓ㸦ᅗ 3-4E, F㸧ࠋ
3.4. ⪃ᐹ
ᮏ❶࡛ࡣ㸪ZO-1 ࠾ࡼࡧ claudin-1 ࠾ࡅࡿ TNF-alpha ࠾ࡼࡧ IL-13 ࡢཬࡰࡍ ᙳ㡪ࡘ࠸࡚㸪≟୕ḟඖᇵ㣴⾲⓶ࣔࢹࣝࢆ⏝࠸࡚ホ౯ࡋࡓࠋTNF-alpha ࡣ㸪ZO-1 ࡢⓎ⌧ᙳ㡪ࢆ࠼࡞ࡗࡓࠋࡲࡓ㸪claudin-ࡣ㸪ZO-1 ࠾࠸࡚ࡣ㸪⏝㔞౫ᏑᛶᰁⰍ ᛶࡢ㒊ศⓗ࡞ᾘኻࡀㄆࡵࡽࢀࡓࠋ ࡇࢀࡲ࡛㸪ࣄࢺ⭠⟶⒴⣽⬊ᰴ㸦T84 ࠾ࡼࡧ Caco-2㸧࠾࠸࡚㸪TNF-alpha ࡣ㸪 ZO-1 ࡢᒁᅾኚࢆ⏕ࡌࡉࡏ࡞ࡗࡓࡇࡀ♧ࡉࢀ࡚࠾ࡾ㸪୍᪉࡛ claudin-1 ࠾࠸࡚ࡣ TNF-alpha ࡼࡾⓎ⌧ศᕸኚࡀㄆࡵࡽࢀ࡚࠸ࡿࠋࡉࡽࡑࢀࡽࡢ ኚకࡗ࡚㸪⣽⬊㛫࠾ࡅࡿ≀㉁㏱㐣ᛶࡢቑຍࡀㄆࡵࡽࢀ࡚࠸ࡿ[35]ࠋࡲࡓ㸪 ≟⭈⮚ᒀ⣽⟶ୖ⓶⏤᮶⣽⬊ᰴ (MDCK ⣽⬊) ࠾࠸࡚㸪TNF-alpha ࡣ ZO-1 ࡢⓎ ⌧㔞ᙳ㡪ࢆ࠼ࡎ㸪claudin-1 ࡢⓎ⌧㔞ࢆపୗࡉࡏࡓ[73]ࠋࡉࡽච⺯ගᰁⰍ ീ࠾࠸࡚ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᙧែࡣ TNF-alpha ࡢῧຍࡼࡾ◚ቯࡉࢀ࡚ ࠾ࡾ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᙧᡂࡀ㞀ᐖࡉࢀࡓࡇࡀ♧၀ࡉࢀࡓ[73]ࠋࡇࢀࡽ ࡢ⤖ᯝࡣ㸪ᮏ◊✲࠾ࡅࡿ⤖ᯝࢆᨭᣢࡍࡿࡶࡢ࡛࠶ࡿࠋ AD ᝈ⪅ࡢኚ㒊⓶࠾࠸࡚㸪ZO-1 ࡢⓎ⌧㔞ࡀపୗࡋ࡚࠾ࡾ[102]㸪ࡲࡓ㸪 Ⓞᝈ⪅ࡢ⓶࠾࠸࡚ ZO-1 ࡢᒁᅾࡢኚ࠾ࡼࡧ㌿㔞ࡢቑຍࡀㄆࡵࡽࢀࡿ ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[40]ࡇࡽ㸪ZO-1 ࡢⓎ⌧ㄪ⠇ࡣ㸪TNF-alpha ௨እࡢᅉ Ꮚࡀ㛵ࡋ࡚࠸ࡿᛮࢃࢀࡿࠋ ୍᪉㸪AD ᝈ⪅ࡢኚ㒊⓶࠾࠸࡚ claudin-1 ࡢⓎ⌧㔞ࡀపୗࡋ࡚࠾ࡾ[102]㸪 ࡲࡓ㸪Ⓞᝈ⪅ࡢ⓶࠾࠸࡚㸪claudin-1 ࠾ࡅࡿ㌿㔞࠾ࡼࡧච⤌⧊Ꮫ ࡼࡿᰁⰍᙉᗘࡀపୗࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[40]ࠋࡋࡓࡀࡗ࡚㸪ᮏ◊✲ ࡢ⤖ᯝࡼࡾ㸪CAD ࡢࡼ࠺࡞⅖ᛶ⓶ᝈࡢኚ㒊࠾࠸࡚㸪TNF-alpha ࡣ claudin-1 ࡢⓎ⌧ㄪ⠇┤᥋㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ
Ⰽᛶࡢ㒊ศⓗ࡞ᾘኻࡀㄆࡵࡽࢀࡓࠋ ࡇࢀࡲ࡛㸪࢝ࣝࢩ࣒࢘ࡼࡾศㄏᑟࡋࡓࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪 IL-13 ࡢ่⃭ࡼࡾ㸪ZO-1 ࡢⓎ⌧㔞ࡣኚࡋ࡞ࡗࡓࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ[18]ࠋ ࡲࡓ㸪CAD ࣔࢹࣝ≟ࡢ㠀ኚ㒊⓶࠾࠸࡚㸪ZO-1 ࡢⓎ⌧ࡣᖖ≟ẚ㍑ࡋ࡚ ኚࡀㄆࡵࡽࢀ࡞ࡗࡓ[79]ࠋࡉࡽ㸪AD ᝈ⪅࠾ࡼࡧ CAD ࣔࢹࣝ≟ࡢ㠀ኚ 㒊⓶࠾࠸࡚㸪ᖖ⓶ẚ㍑ࡋ㸪ච⤌⧊Ꮫࡼࡿ claudin-1 ࡢᰁⰍᙉᗘ ࡢపୗࡀㄆࡵࡽࢀ࡚࠸ࡿ[18, 79]ࠋࡇࢀࡽࡢᐇࢆ㚷ࡳࡿ㸪ᮏ◊✲࡛ᚓࡽࢀࡓ ⤖ᯝࡼࡾ㸪IL-13 ࡣ㸪ZO-1 ࡢⓎ⌧ㄪ⠇┤᥋ⓗ࡞㛵ࢆࡋ࡚࠸࡞࠸୍᪉㸪claudin-1 ࡢⓎ⌧ㄪ⠇㛵ࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ ࡋࡋ㸪࢝ࣝࢩ࣒࢘ࡼࡾศㄏᑟࡋࡓࣄࢺࢣࣛࢳࣀࢧࢺ࠾࠸࡚㸪IL-13 ࡢῧຍࡼࡾ㸪 claudin-1 ࡢⓎ⌧㔞ࡀቑຍࡍࡿࡇࡀ♧ࡉࢀ࡚࠸ࡿ[18]ࠋࡲࡓ㸪 ࣄࢺࢣࣛࢳࣀࢧࢺࢆ⏝࠸ࡓ୕ḟඖᇵ㣴⓶ࣔࢹࣝ࠾࠸࡚㸪IL-13 ࡼࡿ่⃭ ᑐࡋ㸪claudin-1 ࡢ㌿㔞࠾ࡼࡧⓎ⌧㔞ࡣኚࢆ♧ࡉ࡞ࡗࡓ[30]ࠋࡇࢀࡽࡢ▩ ┪ࡍࡿᐇࡣ㸪⏝࠸ࡓ⣽⬊ࡸᇵ㣴᮲௳㸪࡞ࡽࡧ่⃭᮲௳㸦⃰ᗘ࠾ࡼࡧ่⃭㛫㸧 ࡞㉳ᅉࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀ㸪ゎ㔘ࡣὀពࢆせࡍࡿᛮࢃࢀࡿࠋࡲࡓ㸪 TNF-alpha ࡲࡓࡣ IL-13 ࡢῧຍࡼࡾ㸪ᮏࣔࢹ࡛ࣝࡣ୍㒊ࡢᇵ㣴⣽⬊ኚᛶࡀㄆ ࡵࡽࢀࡓࠋࡋࡓࡀࡗ࡚㸪ᮏ◊✲࡛⏝࠸ࡓᐇ㦂᮲௳ࡘ࠸࡚ࡶ㸪ᚋࢱࣥࣃࢡ㔞ࡢ ᐃ㔞ࡸ่⃭᮲௳ࡢ᳨ウࡀᚲせ⪃࠼ࡽࢀࡓࠋ ᮏ◊✲࡛ࡣ㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡑࢀࡒࢀࡘ࠸࡚㸪ࢱࢺࢪࣕࣥࢡࢩࣙ ࣥ࠾ࡅࡿᙳ㡪ࢆホ౯ࡋࡓࠋ⤌⧊ᇵ㣴ࡋࡓࣄࢺࡢ⓶࠾࠸࡚㸪Th2 ࢧࢺ࢝ ࣥ (IL-4, IL-5, IL-13) ࠾ࡼࡧ TNF-alpha ࡼࡿ TSLP ࡢ⏘⏕ࡣ㸪Th2 ࢧࢺ࢝ ࣥ (IL-4 ࡶࡋࡃࡣ IL-13 ࡲࡓࡣ㸪IL-4 ࠾ࡼࡧ IL-13) ࠾ࡼࡧ TNF-alpha ࢆΰྜࡋ ่࡚⃭ࡋࡓሙྜ࠾࠸࡚ࡢࡳㄆࡵࡽࢀࡓ[9]ࠋࡘࡲࡾ㸪Th2 ࢧࢺ࢝ࣥ࠾ࡼࡧ TNF-alpha ࡣ CAD ࠾ࡅࡿ⓶චែ㸪┦ຍⓗ࡞స⏝ࢆ᭷ࡍࡿྍ⬟ᛶࡀ⪃ ࠼ࡽࢀࡿࠋࡋࡓࡀࡗ࡚㸪ᚋࡢin vitro ࠾ࡅࡿ⓶ࣂࣜᶵ⬟◊✲ࡣ㸪่⃭
᮲௳ࡢࡉࡽ࡞ࡿ᳨ウࡀᚲせᛮࢃࢀࡿࠋ
ᮏ◊✲ࡢ⤖ᯝࡽ㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡣ claudin-1 ࡢⓎ⌧ㄪ⠇㛵ࡋ㸪 CAD ࠾ࡅࡿࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢࣂࣜᶵ⬟ᙳ㡪ࢆ࠼࡚࠸ࡿྍ⬟ᛶࡀ 㧗ࡲࡗࡓࠋZO-1 ࠾ࡼࡧ claudin-1 CAD ࡢែࡢ㛵ಀࢆ᫂ࡽࡍࡿࡣ㸪 ࡼࡾ⏕య࠾ࡅࡿኚࢆ࡛ᫎࡁࡿᵝ࡞in vitro ࣔࢹࣝࡢ㛤Ⓨࢆྵࡵ㸪ࡉࡽ࡞ࡿ
3.5 ᅗ
ᅗ3-1 TNF-alpha࠾ࡼࡧIL-13ࡼࡿᇵ㣴⣽⬊ࡢᙧែኚ
(A) ᇵᆅࡢࡳ㸪(B) TNF-alpha 5 ng/ml㸪(C) TNF-alpha 50 ng/ml㸪(D) ᇵᆅ +0.1%BSA㸪(E) IL-13 5 ng/ml㸪(F) IL-13 50 ng/mlࠋ࣐࣊ࢺ࢟ࢩ࢚ࣜࣥ࢜ࢪࣥᰁⰍࠋ ࢫࢣ࣮ࣝࣂ࣮ࡣ50 μmࢆ♧ࡍࠋ
ᅗ
ᅗ3-2 ᇵ㣴⣽⬊ࡢ ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧࠾ࡅࡿ TNF-alpha ࡢᙳ㡪
(A, B, C) ZO-1㸪(D, E, F) claudin-1㸪(B, E) TNF-alpha 5 ng/ml㸪(C, F) TNF-alpha 50 ng/mlࠋࢫࢣ࣮ࣝࣂ࣮ࡣ 50 μm ࢆ♧ࡍࠋ
ᅗ
ᅗ3-3 ᇵ㣴⣽⬊ࡢ ZO-1 ࠾ࡼࡧ claudin-1 ࡢⓎ⌧࠾ࡅࡿ IL-13 ࡢᙳ㡪
(A, B, C) ZO-1㸪(D, E, F) claudin-1㸪(B, E) IL-13 5 ng/ml㸪(C, F) IL-13 50 ng/mlࠋ ࢫࢣ࣮ࣝࣂ࣮ࡣ50 μm ࢆ♧ࡍࠋ
⥲
⥲ᣓ
claudin-1 ࡢⓎ⌧ᙉᗘࡢపୗࡀㄆࡵࡽࢀࡓࠋ୍᪉㸪ZO-1 ࡢⓎ⌧ኚࡣㄆࡵࡽࢀ ࡞ࡗࡓࠋࡋࡓࡀࡗ࡚㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡣ㸪claudin-1 ࡢⓎ⌧ㄪ⠇㛵 ࡋ㸪ࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢࣂࣜᶵ⬟ᙳ㡪ࢆ࠼࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ᮏ◊✲ࡢ⤖ᯝࡽ㸪≟ࢣࣛࢳࣀࢧࢺ⣽⬊ᰴࢆ⏝࠸࡚స〇ࡋࡓ୕ḟඖᇵ㣴⾲ ⓶ࣔࢹࣝࡢᣢࡘචࣂࣜᶵ⬟ࡣ㸪⓶㢮ఝࡋࡓᛶ㉁ࢆ᭷ࡋ࡚࠸ࡿࡇࡀ᫂ ࡽ࡞ࡗࡓࠋࡋࡓࡀࡗ࡚㸪ᮏࣔࢹࣝࡣ≟ࢣࣛࢳࣀࢧࢺ࠾ࡅࡿ㸪⿕㦂ᑐ㇟ࡢ ⏕⌮Ꮫⓗᶵ⬟◊✲ࡸࢱࢺࢪࣕࣥࢡࢩࣙࣥࡢᙳ㡪ホ౯㸪᭷⏝࡞ࢶ࣮ࣝ࡞ ࡿྍ⬟ᛶࢆᣢࡘ⪃࠼ࡽࢀࡿࠋࡲࡓ㸪CAD ࠾ࡅࡿ⓶ࣂࣜᶵ⬟㞀ᐖࡘ࡞ ࡀࡿཎᅉࡋ࡚㸪TNF-alpha ࠾ࡼࡧ IL-13 ࡼࡿ claudin-1 ࡢⓎ⌧ㄪ⠇ࡀ㛵ࡋ࡚ ࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋᚋࡣ㸪㛵㐃ศᏊࡘ࠸࡚ࡉࡽᗈ⠊࡞ከゅⓗ◊✲ࡀᚲ せᛮࢃࢀࡿࠋࡲࡓ㸪⋓ᚓචᛂࡢึᮇ࠾࠸࡚ࡣ㸪ࢣࣛࢳࣀࢧࢺᢠཎᥦ ♧⣽⬊ࡢ┦స⏝ࡀ㔜せ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋࡋࡓࡀࡗ࡚㸪ᵝࠎ࡞ᾮᛶᅉᏊࢆ ࡋ࡚」㞧ែ㛵ࡋ࡚࠸ࡿࢣࣛࢳࣀࢧࢺᢠཎᥦ♧⣽⬊ࡢࢡࣟࢫࢺ ࣮ࢡࢆ┤᥋ホ౯࡛ࡁࡿࣔࢹࣝࡋ࡚㸪ᮏࣔࢹࣝࡣᑗ᮶ⓗ㸪ᢠཎᥦ♧⣽⬊ࡢඹ ᇵ㣴ࡼࡿ⾲⓶ࣔࢹࣝࡢⓎᒎࡀᮇᚅࡉࢀࡿࠋ
ㅰ
ㅰ㎡
ཧ
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