Title 乳酸菌Lactobacillus brevis KB290の抗感染作用とその作用機序に関する研究( 本文(Fulltext) ) Author(s) 岡本, 尚子 Report No.(Doctoral Degree) 博士(獣医学) 乙第172号 Issue Date 2021-03-15 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/81634 ※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。
ங㓟⳦
Lactobacillus brevis
KB290 䛾ᢠឤᰁస⏝䛸
䛭䛾స⏝ᶵᗎ䛻㛵䛩䜛◊✲
2020 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉
ᒸᮏ
ᑦᏊ
ங㓟⳦
Lactobacillus brevis
KB290 䛾ᢠឤᰁస⏝䛸
䛭䛾స⏝ᶵᗎ䛻㛵䛩䜛◊✲
┠ ┠ḟ ᗎㄽ ... 1 ➨1 ❶㻌 䜲䞁䝣䝹䜶䞁䝄䜴䜲䝹䝇ឤᰁ䝬䜴䝇䛻ᑐ䛩䜛Lactobacillus brevis KB290 䛾ឤᰁ 㜵ᚚస⏝䛚䜘䜃䛭䛾స⏝ᶵᗎ ... 5 ➨1 ⠇㻌 ᑠᗎ ... 5 ➨2 ⠇㻌 ᮦᩱ䛸᪉ἲ ... 6 ➨3 ⠇㻌 ⤖ᯝ ... 11 ➨4 ⠇㻌 ⪃ᐹ ... 13 ➨2 ❶㻌 ᑠᏛ⏕䛻䛚䛡䜛䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛻ᑐ䛩䜛Lactobacillus brevis KB290 ᦤ ྲྀ䛾ᙳ㡪䡚㠀┣᳨ᖹ⾜⩌㛫ヨ㦂䛷䛾ホ౯䡚 ... 21 ➨1 ⠇㻌 ᑠᗎ ... 21 ➨2 ⠇㻌 ◊✲ᑐ㇟⪅䠈ᮦᩱ䛚䜘䜃᪉ἲ ... 22 ➨3 ⠇㻌 ⤖ᯝ ... 25 ➨4 ⠇㻌 ⪃ᐹ ... 27
➨3 ❶㻌 Citrobacter rodentiumឤᰁ䝬䜴䝇䛻ᑐ䛩䜛Lactobacillus brevis KB290 䛾ឤ ᰁ㜵ᚚస⏝ ... 35 ➨1 ⠇㻌 ᑠᗎ ... 35 ➨2 ⠇㻌 ᮦᩱ䛸᪉ἲ ... 38 ➨3 ⠇㻌 ⤖ᯝ ... 43 ➨4 ⠇㻌 ⪃ᐹ ... 46 ➨4 ❶㻌 ⥲ྜ⪃ᐹ ... 64 ➨5 ❶㻌 ⥲ᣓ ... 67 ㅰ㎡ ... 70 ཧ⪃ᩥ⊩ ... 71
␎
␎ㄒ䝸䝇䝖
BALF: bronchoalveolar lavage fluid Ẽ⟶ᨭ⫵⬊Ὑίᾮ BLI: bioluminescent imaging ⏕≀Ⓨග䜲䝯䞊䝆䞁䜾 EHEC: enterohemorrhagic Escherichia coli ⭠⟶ฟ⾑ᛶ⭠⳦ ELISA: enzyme-linked immunosorbent assay 㓝⣲ච ᐃἲ EPEC: enteropathogenic Escherichia coli ⭠⟶ཎᛶ⭠⳦ EPS: exopolysaccharide ⳦యእከ⢾
GHS: general health score ᗣ≧ែ䝇䝁䜰 HA ヨ㦂: hemagglutination ヨ㦂 ㉥⾑⌫จ㞟ヨ㦂 IFV: influenza virus 䜲䞁䝣䝹䜶䞁䝄䜴䜲䝹䝇 IFN-α: interferon-alpha 䜲䞁䝍䞊䝣䜵䝻䞁-α IFN-γ: interferon-gamma 䜲䞁䝍䞊䝣䜵䝻䞁-γ IFN-λ: interferon-lamda 䜲䞁䝍䞊䝣䜵䝻䞁-λ IgA: immunoglobulin A ච䜾䝻䝤䝸䞁A IgG: immunoglobulin G ච䜾䝻䝤䝸䞁A IL-1β: interleukin-1 beta 䜲䞁䝍䞊䝻䜲䜻䞁-1 IL-6: interleukin-6 䜲䞁䝍䞊䝻䜲䜻䞁-6 IL-12: interleukin-12 䜲䞁䝍䞊䝻䜲䜻䞁-12 MDCK ⣽⬊: Madin-Darby canine kidney ⣽⬊
MLD50: 50% mouse lethal dose 䝬䜴䝇50%⮴Ṛ㔞
NK ⣽⬊: natural killer ⣽⬊ 䝘䝏䝳䝷䝹䞉䜻䝷䞊⣽⬊ PBS: phosphate-buffered saline 䝸䞁㓟⦆⾪⏕⌮㣗ሷỈ ROI: region of interest 㛵ᚰ㡿ᇦ
TCID50: median tissue culture infectious dose 50%⤌⧊ᇵ㣴ឤᰁ㔞
TNF-α: tumor necrosis factor-alpha ⭘⒆ቯṚᅉᏊ ZO-1: zona occludens protein
1 ᗎ ᗎㄽ ங㓟⳦䛿䠈ⅣỈ≀䜢ᾘ㈝䛧䠈᭱⤊௦ㅰ⏘≀䛸䛧䛶ங㓟䜢⏕ᡂ䛩䜛ᚤ⏕≀䛾⥲⛠䛷䛒 䜚䠈䜾䝷䝮㝧ᛶ䠈㠀⬊Ꮚᙧᡂ䠈䜹䝍䝷䞊䝊㝜ᛶ䠈㏻ᛶ᎘Ẽᛶ⳦䛸䛔䛖ᛶ㉁䜢ᣢ䛴䚹ங㓟⳦䛿䠈 Ⓨ㓝䛻䜘䜛ಖᏑᮇ㛫䛾ᘏ㛗䜔㤶䜚䛾䠈㢼ᨵၿ䛺䛹䠈ே㛫䛾㣗⏕ά䛻ྂ䛟䛛䜙⏝䛥 䜜䛶䛝䛯䚹䛥䜙䛻ங㓟⳦䛾୰䛻䛿䛂䝥䝻䝞䜲䜸䝔䜱䜽䝇䛃䛸䛧䛶䛾ຠᯝ䛜▱䜙䜜䛶䛔䜛䜒䛾䜒䛒 䜛䚹 ᅜ㐃㣗⣊㎰ᴗᶵ㛵䠋ᅜ㝿ಖᶵ㛵䠄FAO/WHO䠅䛾䝽䞊䜻䞁䜾䜾䝹䞊䝥䛜Ⓨ⾲䛧䛯㣗ရ ୰䛾䝥䝻䝞䜲䜸䝔䜱䜽䝇䛾ホ౯䛻㛵䛩䜛䜺䜲䝗䝷䜲䞁䛷䛿䠈䛂䝥䝻䝞䜲䜸䝔䜱䜽䝇䛿㐺ᙜ㔞䜢ᦤ ྲྀ䛧䛯䛸䛝䛻ᐟ䛻ᑐ䛧䛶ᗣୖ䛾᭷┈䛺స⏝䜢ᤵ䛩䜛⏕䛝䛯ᚤ⏕≀䠄Live
microorganisms which when administered in adequate amounts confer a health benefit on the host䠅䛃䛸ᐃ⩏䛥䜜䛶䛔䜛䠄35䠅䚹䝥䝻䝞䜲䜸䝔䜱䜽䝇䛜ᐟ䛻䜒䛯䜙䛩᭷┈䛺 స⏝䛸䛧䛶䠈ᢠឤᰁస⏝䠈ᢠ⫧‶స⏝䠈ᢠ⅖స⏝䠈ᢠ䛜䜣స⏝䠈ᢠ䜰䝺䝹䜼䞊స⏝䛺䛹 䛜ሗ࿌䛥䜜䛶䛚䜚䠄31䠅䠈㏆ᖺ䠈䛭䛾◊✲䛿┠ぬ䜎䛧䛔ໃ䛔䛷㐍ᒎ䛧䛶䛔䜛䚹ᮏ◊✲䛷䛿䠈᳜ ≀ᛶ㣗ရ⏤᮶ங㓟⳦䛾୍䛴䛷䛒䜚䠈䝥䝻䝞䜲䜸䝔䜱䜽䝇䛸䛧䛶䛾స⏝䜢ᣢ䛴Lactobacillus brevis KB290䠄KB290䠅䛻╔┠䛧䛯䚹 KB290 䛿䠈ி㒔䛾୕ₕ≀䛾୍䛴䛷䛒䜛䛂䛩䛠䛝䠄Fig. 1䠅䛃䜘䜚ศ㞳䛥䜜䛯᳜≀ᛶ㣗ရ ⏤᮶䛾ங㓟⳦䛷䛒䜛䚹ᮏ⳦ᰴ䛿䠈ᱰ⳦䛸䛔䛖ᙧែᏛⓗ≉ᚩ䜔䠈䛹䛾䜘䛖䛺⢾䜢ᰤ㣴※䛸䛧䛶 ⏝䛷䛝䜛䛛䛸䛔䛖䠈䛔䜟䜖䜛⢾䛾㈨ᛶ䠈䛚䜘䜃 DNA 㓄ิゎᯒ䛺䛹䛻䜘䜚䠈⳦✀䛜 Lactobacillus brevis䛸ྠᐃ䛥䜜䛶䛔䜛䚹䛥䜙䛻䝀䝜䝮䠄㑇ఏᏊሗ䠅ゎᯒ䛻䜘䜚䠈ཎ ᛶ㑇ఏᏊ䜢ಖ᭷䛧䛺䛔䛣䛸䛻ྜ䜟䛫䠈䝇䝖䝺䝇⪏ᛶ㑇ఏᏊ䛺䛹䠈䝥䝻䝞䜲䜸䝔䜱䜽䝇䛸䛧䛶䛾 ᭷⏝ᛶ䛻㛵䜟䜛㑇ఏᏊ䜢ಖ᭷䛩䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛶䛔䜛䠄25䠅䚹䛺䛚 KB290 䛿䠈
2 ⏕≀ᮦᩱ㛤Ⓨᐊ䛻ᐤク䛥䜜䛶䛚䜚䠈Ᏻᛶ䛻䛴䛔䛶䛿䠈๓㏙䛾㑇ఏᏊሗ䛻ຍ䛘䠈䝷䝑䝖 䛻䛚䛔䛶ᛴᛶ䞉ளᛴᛶẘᛶ䜔ദወᙧᛶ䜢♧䛥䛺䛔䛣䛸䠄108䠈109䠅䠈䜎䛯䝠䝖䛻䛚䛔䛶㐣㔞 䜢ᦤྲྀ䛧䛯ሙྜ䛷䜒㔜⠜䛺ᗣ⿕ᐖ䛜ㄆ䜑䜙䜜䛺䛔䛣䛸䛜䠄74䠅䠈䛭䜜䛮䜜ሗ࿌䛥䜜䛶䛔䜛䚹 ᮏ⳦ᰴ䛾ᶵ⬟ᛶ䛻䛴䛔䛶䛿䠈➨୍䛻ᩚ⭠స⏝䛜▱䜙䜜䛶䛔䜛䚹KB290 䛿⳦యእ䛻ከ⢾ 䠄Exopolysaccharide: EPS䠅䜢⏘⏕䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛚䜚䠈EPS 䛷⳦య䜢そ䛖䛣䛸 䛻䜘䜚⫹Ồ㓟䛺䛹䛾ᾘᾮ䛻ᑐ䛧䛶㧗䛔⪏ᛶ䜢ᣢ䛴䛣䛸䛜in vitroヨ㦂䛻䜘䜚♧၀䛥䜜䛶䛔 䜛䠄97䠅䚹䛥䜙䛻䝠䝖䛻䛚䛔䛶䜒䠈KB290 䜢ྵ䜐㣧ᩱ䜢ᦤྲྀ䛧䛯䝠䝖䛾౽䛛䜙⏕䛝䛯 KB290 䛜 ᳨ฟ䛥䜜䛶䛚䜚䠄74䠅䠈KB290 䛜⏕⳦䛾≧ែ䛷⭠䜎䛷฿㐩䛧䠈䝥䝻䝞䜲䜸䝔䜱䜽䝇ຠᯝ䜢Ⓨ 䛩䜛䛣䛸䛜ᮇᚅ䛥䜜䛶䛔䜛䚹䛣䜜䜎䛷䛻䠈KB290 䜢ᦤྲྀ䛩䜛䛣䛸䛻䜘䜛⭠ෆ⳦ྀᨵၿస⏝ 䠄2, 72, 74䠅䜔౽㏻ᨵၿస⏝䠄2, 74䠅䠈㐣ᩄᛶ⭠ೃ⩌䠄72䠅䛻ᑐ䛩䜛ᨵၿస⏝䛜䠈䝠䝖䛷䛾 ヨ㦂䛷᫂䜙䛛䛻䛺䛳䛶䛔䜛䚹 ➨䛾ᶵ⬟ᛶ䛸䛧䛶䠈KB290 䛜ච㈿άస⏝䜢♧䛩䛣䛸䜒」ᩘ䛾◊✲䛻䜘䜚♧䛥䜜䛶䛔 䜛䚹1993 ᖺ䛻䠈KB290 䜢 2 㐌㛫䜎䛯䛿 4 㐌㛫⥅⥆䛧䛶ᦤྲྀ䛧䛯䝠䝖䛻䛚䛔䛶䠈ᮎᲈ⾑䛻䛚 䛡䜛interferon (IFN) -α ⏘⏕⬟䜔䠈ᮎᲈ⾑༢᰾⣽⬊䛾⣽⬊യᐖάᛶ䛜᭷ព䛻ୖ᪼䛩䜛 䛣䛸䛜ึ䜑䛶ሗ࿌䛥䜜䠄51, 52䠅䠈䛭䛾ච㈿άస⏝䛾䝯䜹䝙䝈䝮䛻䛴䛔䛶䛥䜙䛻◊✲䛜⾜䜟 䜜䛯䚹KB290 䛿ศἪᆺ䛾 EPS 䛸⣽⬊⾲ᒙ⤖ྜᆺ䛾 EPS䠄EPS-b䠅䛾䠎✀㢮䛾 EPS 䜢⏕ ⏘䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛚䜚䠈䛣䜜䜙 EPS 䛜ච㈿άస⏝䛻㛵䛩䜛䛣䛸䛜♧၀䛥䜜䛶 䛔䜛䠄97䠅䚹Sasaki 䜙䠄86䠅䛿䠈KB290 䛾ච㈿άస⏝䛻ᐤ䛩䜛⳦యᡂศ䜢≉ᐃ䛩䜛䛯 䜑䠈KB290 䛾⏕⳦య䠈ẅ⳦య䠈EPS-b 䛾⏘⏕䛜ᴟ䜑䛶ᑡ䛺䛔 KB290 䛾ኚ␗ᰴ 䠄KB392䠅䠈䛚䜘䜃 KB290 䜘䜚⢒ᢳฟ䛧䛯 EPS-b 䛻䜘䜛⣽⬊യᐖάᛶୖ᪼స⏝䜢ホ౯䛧 䛯䚹䝬䜴䝇䛻⿕㦂≀㉁䜢ྵ䜐㣫ᩱ䜢䛭䜜䛮䜜䠍᪥㛫⮬⏤ᦤྲྀ䛥䛫䛯ᚋ䠈⭁⣽⬊䛾⣽⬊യᐖ άᛶ䜢ᑐ↷⩌䛸ẚ㍑䛧䛯⤖ᯝ䠈KB290 䛾⏕⳦య䛚䜘䜃ẅ⳦య䛾ᢞ⩌䛿䠈ᑐ↷⩌䛸ẚ㍑
3 䛧䛶䠈⣽⬊യᐖάᛶ䛜᭷ព䛻ୖ᪼䛧䛶䛔䛯䚹୍᪉䠈EPS-b 䛾⏘⏕⬟䛾ప䛔 KB392 ⏕⳦య ᢞ⩌䛾⣽⬊യᐖάᛶ䛿ୖ᪼䛧䛶䛔䛺䛛䛳䛯䚹䛣䛾䛣䛸䛛䜙䠈EPS-b 䛜⣽⬊യᐖάᛶ䛻㛵 䛩䜛ᡂศ䛾୍䛴䛷䛒䜛䛣䛸䛜♧၀䛥䜜䛶䛔䜛䠄86䠅䚹 䛥䜙䛻䠈ච㈿άస⏝䛜Ⓨ䛥䜜䜛㝿䛾ᐟഃ䛾䝯䜹䝙䝈䝮䜒᫂䜙䛛䛻䛺䜚䛴䛴䛒䜛䚹 Fukui 䜙䠄26, 27䠅䛿䠈KB290 䜢ᦤྲྀ䛥䛫䛯䝬䜴䝇䛾⭁⣽⬊䛾⣽⬊യᐖάᛶ䜢 ᐃ䛩䜛䛸䛸 䜒䛻䠈DNA 䝬䜲䜽䝻䜰䝺䜲䜢⏝䛔䛶㑇ఏᏊⓎ⌧䛾⥙⨶ⓗゎᯒ䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䠈KB290 䛾⣽⬊യᐖάᛶୖ᪼స⏝䜢ᢸ䛖䜶䝣䜵䜽䝍䞊⣽⬊䛸䛧䛶䠈natural killer (NK)⣽⬊䜔⣽⬊ യᐖᛶT ⣽⬊䛜㛵䛧䛶䛔䜛ྍ⬟ᛶ䜢ぢฟ䛧䛶䛔䜛䚹 㻌 ୖグ䛾䜘䛖䛻KB290 䛿䠈ච㈿άస⏝䜢ᣢ䛴䛣䛸䜔䛭䛾䝯䜹䝙䝈䝮䛜᫂䜙䛛䛻䛺䜚䛴䛴䛒 䜛䚹䛧䛛䛧䛺䛜䜙䠈䛭䛾ච㈿άస⏝䜢䛧䛶Ⓨ䛥䜜䜛䛣䛸䛜ᮇᚅ䛥䜜䜛ಖຠᯝ䛸䛧䛶䠈 KB290 䛜ᢠឤᰁస⏝䜢Ⓨ䛷䛝䜛䛛䛹䛖䛛䛻䛴䛔䛶䛿᫂䜙䛛䛻䛥䜜䛶䛔䛺䛔䚹䛭䛣䛷䠈ᮏ ◊✲䛷䛿䛣䛾Ⅼ䛻䛴䛔䛶᳨ド䛧䛯䚹➨1 ❶䛷䛿䠈䝬䜴䝇䜢⏝䛔䛶䠈྾ჾឤᰁ䜴䜲䝹䝇䛷䛒 䜛䜲䞁䝣䝹䜶䞁䝄䜴䜲䝹䝇䛻ᑐ䛩䜛KB290 䛾ᢠឤᰁຠᯝ䜔䛭䛾䝯䜹䝙䝈䝮䜢᳨ド䛧䛯䚹䛥䜙 䛻➨2 ❶䛷䛿䠈䝠䝖䛻䛚䛔䛶䜒 KB290 䛾ᢠឤᰁస⏝䜢☜ㄆ䛷䛝䜛䛛ྰ䛛䜢᳨ド䛩䜛䛣䛸䜢 ┠ⓗ䛻䠈ᑠᏛ⏕䜢ᑐ㇟䛸䛧䛶䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛻ᑐ䛩䜛KB290 䛾ຠᯝ䜢㠀┣᳨ᖹ⾜ ⩌㛫ヨ㦂䛻䛶ホ౯䛧䛯䚹䛥䜙䛻➨3 ❶䛷䛿䠈྾ჾឤᰁ௨እ䛾せ䛺ឤᰁ䛷䛒䜛ᾘ ⟶ឤᰁ䜈䛾KB290 䛾ຠᯝ䜢᳨ド䛩䜛䛯䜑䠈ᾘ⟶ឤᰁ⣽⳦䛸䛧䛶 Citrobacter rodentium䜢⏝䛧䠈KB290 䛾⭠⟶ឤᰁ⣽⳦䛻ᑐ䛩䜛ᢠឤᰁస⏝䛻䛴䛔 䛶䝬䜴䝇䜢⏝䛔䛶ホ౯䛧䛯䚹
4
F
Figure 1. Suguki
KB290 were isolated from a Japanese traditional pickle made from a turnip, Suguki, which is cultivated in the Kyoto area.
5
➨
➨1 ❶㻌 䜲䞁䝣䝹䜶䞁䝄䜴䜲䝹䝇ឤᰁ䝬䜴䝇䛻ᑐ䛩䜛Lactobacillus brevis KB290 䛾ឤᰁ 㜵ᚚస⏝䛚䜘䜃䛭䛾స⏝ᶵᗎ
➨1 ⠇㻌 ᑠᗎ
䜲䞁䝣䝹䜶䞁䝄䛿䠈䜲䞁䝣䝹䜶䞁䝄䜴䜲䝹䝇䠄influenza virus; IFV䠅ឤᰁ䛻䜘䜛྾ჾឤ ᰁ䛷䛒䜚䠈㧗⇕䜔ᐮẼ䠈䛾䛹䛾③䜏䠈㢌③䛺䛹䜢≉ᚩ䛸䛩䜛䠄4䠅䚹䛣䜜䜙䛾≧䛿ஙᗂඣ 䜔㧗㱋⪅䛷䛧䜀䛧䜀㔜⠜䛧䠈୰䛻䛿Ṛ䛻⮳䜛ሙྜ䜒䛒䜛䠄29, 30䠅䚹䜲䞁䝣䝹䜶䞁䝄ᑐ⟇䛸 䛧䛶䝽䜽䝏䞁᥋✀䛜ᬑཬ䛧䛶䛔䜛䚹䛧䛛䛧䠈IFV 䛿ᢠཎኚ␗䜢㉳䛣䛧䜔䛩䛔䛯䜑䠈ᚲ䛪䛧䜒䝽 䜽䝏䞁᥋✀䛻䜘䜛ண㜵ຠᯝ䛿༑ศ䛸䛿ゝ䛘䛺䛔䠄21, 78䠅䚹䛧䛯䛜䛳䛶䠈䜲䞁䝣䝹䜶䞁䝄䜢ண 㜵䛩䜛䛯䜑䛻䛿䠈᪥䚻䛾⏕ά䛛䜙චຊ䜢㧗䜑䠈IFV 䛻ᑐ䛩䜛⪏ᛶ䜢㧗䜑䛶䛚䛟䛣䛸䛜㔜 せ䛷䛒䜛䚹 㻌 IFV 䛾䛺ឤᰁ㒊䛿Ẽ㐨䜔⫵⬊➼䠈྾ჾ䛾ୖ⓶⣽⬊䛷䛒䜛䚹IFV 䛜䛣䜜䜙䛾⣽⬊ 䛻ឤᰁ䛩䜛䛸䠈᭱ึ䛻⮬↛චᛂ⟅䛜ㄏᑟ䛥䜜䜛䚹䛩䛺䜟䛱䠈IFV ឤᰁ⣽⬊䛛䜙 IFN-α 䛺 䛹䛾Ϩᆺ䜲䞁䝍䞊䝣䜵䝻䞁䜔䠈㏆ᖺ᪂䛯䛻ぢ䛴䛛䛳䛯IFN-λ 䛸䜘䜀䜜䜛Ϫᆺ䜲䞁䝍䞊䝣䜵䝻 䞁䛜⏘⏕䛥䜜䠄14, 15䠅䠈⣽⬊⭷ୖ䛻䛒䜛 IFN ཷᐜయ䛻⤖ྜ䛩䜛䛣䛸䛷䠈⣽⬊ෆ䜈䛾 IFV 䛾ධ䜔⣽⬊ෆ䛷䛾IFV 䛾ቑṪ䛜ᢚไ䛥䜜䜛䠄22, 49, 68䠅䚹ຍ䛘䛶䠈NK ⣽⬊䜔༢⌫䠈ዲ ୰⌫䠈ᶞ≧⣽⬊䛺䛹䛾㈎㣗⣽⬊䛜ឤᰁ㒊䛻㐟㉮䛧䠈IFV 䛻ឤᰁ䛧䛯⣽⬊䜢㈎㣗䛧㝖 䛩䜛䠄15, 44䠅䚹䛥䜙䛻䛣䜜䜙䛾㈎㣗⣽⬊䛾୍㒊䛜㈎㣗䛧䛯䜴䜲䝹䝇᩿∦䜢䝦䝹䝟䞊T ⣽⬊ 䛻ᥦ♧䛩䜛䛣䛸䛷䠈⋓ᚓචᛂ⟅䛜ㄏᑟ䛥䜜䜛䚹䛩䛺䜟䛱䠈䝦䝹䝟䞊T ⣽⬊䛜 B ⣽⬊䜢ά ᛶ䛥䛫䠈B ⣽⬊䛿ᙧ㉁⣽⬊䜈䛸ศ䛧䠈⤖ᯝⓗ䛻ᙧ㉁⣽⬊䛛䜙 immunoglobulin (Ig) A 䜔 IgG 䛺䛹䛾 IFV ≉␗ⓗᢠయ䛜⏘⏕䛥䜜䠈IFV 䜢୰䛩䜛䚹
KB290 䛾ᦤྲྀ䛿䠈䝠䝖䛻䛚䛔䛶 IFN-α ⏘⏕⬟䜢ஹ㐍䛩䜛䛣䛸䠄51䠅䠈䝠䝖䜔䝬䜴䝇䛻䛚䛔 䛶NK ⣽⬊䛺䛹䛻䜘䜛⣽⬊യᐖάᛶ䜢ୖ᪼䛥䛫䜛䛣䛸䠄26, 52䠅䛜ሗ࿌䛥䜜䛶䛔䜛䚹ୖ㏙䛾
6 ㏻䜚䠈IFN-α 䜔 NK ⣽⬊䛿䜴䜲䝹䝇ឤᰁ䛻ᑐ䛩䜛⮬↛චᛂ⟅䛻䛚䛔䛶㔜せ䛺ᙺ䜢ᢸ䛖 䛣䛸䛛䜙䠈KB290 䛿 IFV ឤᰁ䛻ᑐ䛧䛶㜵ᚚస⏝䜢♧䛩ྍ⬟ᛶ䛜䛒䜛䚹䛭䛣䛷ᮏ◊✲䛷䛿䠈 KB290 䜢ᦤྲྀ䛥䛫䛯䝬䜴䝇䛻 IFV 䜢ឤᰁ䛥䛫䜛䛣䛸䛻䜘䜚䠈KB290 䛜 IFV 䛻ᑐ䛧䛶㜵ᚚ ຠᯝ䜢♧䛩䛛ྰ䛛䜢᳨ド䛧䛯䚹 ➨2 ⠇㻌 ᮦᩱ䛸᪉ἲ 2-䠍䠊⏝ື≀ ᮏヨ㦂䜢ඹྠ䛷ᐇ䛧䛯◊✲ᶵ㛵䛷䛒䜛AgResearch 䛻䛚䛔䛶 IFV ឤᰁヨ㦂䛾ᐇ ⦼䜔▱ぢ䛜㇏ᐩ䛷䛒䛳䛯䠈BALB/c 䝬䜴䝇䛾䝯䝇䜢⏝ື≀䛸䛧䛶㑅ᢥ䛧䛯䚹ලయⓗ䛻 䛿䠈7-8 㐌㱋䛾䝯䝇䛾 BALB/c 䝬䜴䝇䠄AgResearch Ruakura Small Animal Facility, Palmerston North, New Zealand䠅䜢⏝䛔䠈㣫⫱ᮇ㛫୰䛿䠈ᅛᙧ㣫ᩱ䠄Prolab® RMH 1800; LabDiet, Richmond, IN, USA)䠅䛸Ỉ䜢⮬⏤ᦤྲྀ䛷䛘䠈12 㛫䛾᫂ᬯ 䝃䜲䜽䝹䠈 ᗘ21Υ䠈‵ᗘ 50.1±1.4䠂䛾⎔ቃ䛷㣫⫱䛧䛯䚹䛺䛚ᮏヨ㦂䛿 AgResearch Ltd., Hopkirk Research Institute 䠄Palmerston North, New Zealand䠅䛻䛶ᐇ 䛧䠈䛭䛾ィ⏬䛿Agresearch Grasslands Animal Ethics Committee 䠄Palmerston North, New Zealand䠅䛷ᢎㄆ䜢ཷ䛡䛯䚹
2-2䠊ヨ㦂ヨᩱ
ヨ㦂ヨᩱ䛸䛧䛶䠈KB290 䜢 1×1011 cfu/g ྵ᭷䛩䜛㤿㕥⸉䝕䞁䝥䞁䠄䝃䞁䜶䜲⢾ᰴᘧ
♫䠈ឡ▱䠅䜢⏝䛔䛯䚹䜎䛯䠈䝥䝷䝉䝪䛸䛧䛶KB290 䜢ྵ䜎䛺䛔㤿㕥⸉䝕䞁䝥䞁䠄᪥⃦ Ꮫᰴᘧ♫䠈㜰䠅䜢⏝䛔䛯䚹䛭䜜䛮䜜䛾ヨᩱ䛿䝬䜴䝇䜈䛾ᢞ┤๓䛻䝸䞁㓟⦆⾪⏕⌮ 㣗ሷỈ䠄phosphate buffered saline; PBS䠅䛻ᠱ⃮䛧䛯䚹
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2-3䠊ឤᰁ⏝ IFV 䛾ㄪ〇
ឤᰁ⏝䛾IFV 䛻䛿䠈A/PR/8/34 (H1N1)ᆺ䝬䜴䝇㡰ᰴ䠄University of Otago, Dunedin, New Zealand䠅䜢⏝䛧䛯䚹10 ᪥㱋䛾Ꮴ㭜༸䛾ᒀ⭷⭍䛻 IFV 䜢᥋✀䛧䛶 3 ᪥㛫ᇵ㣴䛧䠈IFV 䜢ቑṪ䛥䛫䛯䚹ᒀ⭷⭍ᾮ䜢ᅇ䛧䠈䝇䝖䝑䜽䛸䛧䛶䠉80Υ䛻䛶ಖ⟶䛧 䛯䚹䝇䝖䝑䜽䛾50%⤌⧊ᇵ㣴ឤᰁ㔞䠄median tissue culture infectious dose; TCID50䠅
䛿䠈䜲䝚⭈⮚ᒀ⣽⟶ୖ⓶⏤᮶Madin-Darby Canine Kidney䠄MDCK䠅⣽⬊䜢⏝䛔䛯㉥ ⾑⌫จ㞟䠄Hemagglutination, HA䠅ヨ㦂䠄18䠅䛻䜘䜚 ᐃ䛧䛯䚹䛩䛺䜟䛱䠈୍ᬌᇵ㣴䛧䛯 MDCK ⣽⬊䛻 IFV 䜢ឤᰁ䛥䛫䠈3 ᪥ᚋ䛻 HA ヨ㦂䜢ᐇ䛧䛯䚹䛭䛾⤖ᯝ䠈TCID50 䛿10 6.75 /mL 䛷䛒䜚䠈⤒㰯ᢞ 14 ᪥ᚋ䜎䛷䛾䝬䜴䝇 50%⮴Ṛ㔞䠄50% mouse lethal dose, MLD50䠅䛿2 × TCID50䛷䛒䛳䛯䚹 2-4. IFV ឤᰁᐇ㦂 㻌 ヨ㦂䝇䜿䝆䝳䞊䝹䜢Fig. 2 䛻♧䛧䛯䚹60 ༉䛾䝬䜴䝇䜢 5 ༉/䜿䞊䝆䛻䛶 12 䜿䞊䝆䛷㣫 ⫱䛧䛯䚹䝬䜴䝇䜢㠀ឤᰁ⩌䠈ᑐ↷⩌䠈KB290 ᢞ⩌䛾 3 ⩌䛻ศ䛡䠄4 䜿䞊䝆/⩌䠅䠈㠀ឤᰁ ⩌䛚䜘䜃ᑐ↷⩌䛻䛿㤿㕥⸉䝕䞁䝥䞁䜢䠈KB290 ᢞ⩌䛻䛿 KB290 ྵ᭷㤿㕥⸉䝕䞁䝥 䞁䜢䠈10 mg/100 μL 䛷 PBS 䛻ᠱ⃮䛧䛯䜒䛾䜢 14 ᪥㛫䠄Day 䠉14䡚䠉1䠅↓㯞㓉䛻䛶 ᙉไ⤒ཱྀᢞ䛧䛯䠄KB290 ᢞ⩌䛾 KB290 ᦤྲྀ㔞䠖1×109 cfu/䝬䜴䝇/᪥䠅䚹15 ᪥┠ 䠄Day 0䠅䛻䠈㠀ឤᰁ⩌䜢ู䛾㣫⫱ᐊ䛻㝸㞳䛧䛯ᚋ䠈ᑐ↷⩌䛚䜘䜃 KB290 ᢞ⩌䛻䛿䜿 䝍䝭䞁/䜻䝅䝷䝆䞁 (0.1 mg/g body weight)䜢⭡⭍ෆᢞ䛧䠈PBS 䛷ᕼ㔘䛧䛯 IFV ⁐ᾮ 䜢㯞㓉ୗ䛻䛶⤒㰯ᢞ䛧䛯䠄2㽢MLD50/20 μL/䝬䜴䝇䠅䚹ឤᰁ 3 ᪥ᚋ䠄Day 3䠅䛻ྛ⩌ 10
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× g䠈15 ศ㛫䠅䛻䛶⾑Ύ䜢ศ㞳䛧䠈䠉20Υ䛻䛶ಖᏑ䛧䛯䚹⫵䛿䠉80Υ䛻䛶ಖᏑ䛧䛯䚹㻌 ṧ 䛳䛯ྛ⩌10 ༉䛾䝬䜴䝇䛿䠈ឤᰁ᪥䠄Day 0䠅䛛䜙ឤᰁ 7 ᪥ᚋ䠄Day 7䠅䜎䛷ẖ᪥య㔜䜢 ᐃ䛧䠈ྠ䛻㌟ほᐹ䛻䜘䜚ᗣ≧ែ䝇䝁䜰䠄general health score; GHS䠅䜢⟬ฟ䛧䛯䚹 GHS 䛿௨ୗ䛾䜘䛖䛻 5 ẁ㝵䛻䝇䝁䜰䛧䛯䠄48䠅䚹 5 = ᗣ 䠄⮫ᗋ≧䛺䛧䠈ືస䛜ᶵᩄ䠈ẟⰏ䛜Ⰻ䛔䠈࿘ᅖ䛾⎔ቃ䛻⯆䜢♧䛩䠅 4 = ㍍ᗘ 䠄ẟ䛜䜟䛪䛛䛻㏫❧䛴䠅 3 = ୰ᗘ 䠄ẟ䛜୰⛬ᗘ䛻㏫❧䛴䠈ືస䛜άⓎ䠅 2 = 㔜ᗘ 䠄ẟ䛜จ㞟䛩䜛䠈⑭䛫䜛䠅 1 = 㠀ᖖ䛻㔜ᗘ 䠄่⃭䛻ᛂ䛧䛺䛔䠅 ឤᰁ7 ᪥ᚋ䠄Day 7䠅䛻䝬䜴䝇䜢 CO2䜺䝇䛻䛶ᏳᴦṚ䛥䛫䠈⾑ᾮ䛚䜘䜃⭁⮚䜢᥇ྲྀ䛧 䛯䚹⾑ᾮ䛛䜙䛿⾑Ύ䜢ศ㞳䛧䠉20Υ䛻䛶ಖᏑ䛧䛯䚹⭁⮚䛿⣽⬊യᐖάᛶ䛾 ᐃ䛻౪䛧䛯 䠄2-8, 3-5 䛾㡯ཧ↷䠅䚹䜎䛯䠈ษ㛤䛻䛶㟢ฟ䛥䛫䛯Ẽ⟶䛾ෆ⭍䛻䜹䝙䝳䞊䝺䜢ᤄධ䛧䠈⦭ྜ 䛧䛶ᅛᐃ䛧䛯䚹ḟ䛻䜹䝙䝳䞊䝺䛻䝅䝸䞁䝆䜢᥋⥆䛧䛶1 mL 䛾 PBS 䜢Ẽ⟶䛻ὀධ䛧䠈3 ᅇ ⛬ᗘPBS 䜢ฟ䛧ධ䜜䛩䜛䛣䛸䛻䜘䜚䠈Ẽ⟶ᨭ⫵⬊Ὑίᾮ䠄bronchoalveolar lavage fluid; BALF䠅䜢᥇ྲྀ䛧䛯䚹BALF 䛿㐲ᚰศ㞳䠄150 × g䠈15 ศ㛫䠅ᚋ䛻ୖΎ䜢ᅇ䛧䠈䠉 80Υ䛻䛶ಖᏑ䛧䛯䚹 2-5. ⫵䛻䛚䛡䜛 IFV ຊ౯䛾 ᐃ 㻌 䠉80Υ䛻䛶ಖ⟶䛧䛯⫵䜢⼥ゎ䛥䛫䠈2.0 mM 䛾䜾䝹䝍䝭䞁䠄Invitrogen, California䠈 USA䠅䠈100 U/mL 䛾䝨䝙䝅䝸䞁䠄Invitrogen䠅䠈䛚䜘䜃 100 μg/mL 䛾䝇䝖䝺䝥䝖䝬䜲䝅䞁 䠄Invitrogen䠅䜢ῧຍ䛧䛯䝎䝹䝧䝑䝁ᨵኚ䜲䞊䜾䝹ᇵᆅ䠄Invitrogen䠅䜢ຍ䛘䛶䝩䝰䝆䝘䜲 䝈䛧䠈10% (w/v)䛾⫵⤌⧊ᠱ⃮ᾮ䜢స〇䛧䛯䚹䛣䛾ᠱ⃮ᾮ䛛䜙㐲ᚰ䠄7,000 × g䠈20 ศ㛫䠅
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䛻䜘䜚ୖΎ䜢ᅇ䛧䠈ୖΎ୰䛾IFV ຊ౯䜢䠈MDCK ⣽⬊䜢⏝䛔䛯 HA ヨ㦂䛻䜘䜚 ᐃ䛧 䛯䚹
2-6. ⾑Ύ୰䛻䛚䛡䜛 IFN-α 㔞䛾 ᐃ
㻌 ⾑Ύ୰䛾IFN-α 㔞䛿䠈Mouse IFN-alpha Platinum ELISA䠄eBioscience, Bender MedSystems Gmbh, Vienna, Austria䠅䜢⏝䛔䠈ῧ䛾䝥䝻䝖䝁䞊䝹䛻ᚑ䛔 ᐃ䛧䛯䚹 䛺䛚䠈 ᐃ䛻䛿4 ಸᕼ㔘䛧䛯⾑Ύ䜢⏝䛧䠈䛭䜜䛮䜜 duplicate 䛷 ᐃ䛧䛯䚹᳨ฟ㝈⏺ 䛿7.48 pg/mL 䛷䛒䛳䛯䚹
2-7. BALF ୰䛻䛚䛡䜛 IFV ≉␗ⓗ IgA 㔞䛾 ᐃ
㻌 BALF ୰䛻䛚䛡䜛 IFV ≉␗ⓗ IgA 㔞䛿 ELISA ἲ䛻䛶 ᐃ䛧䛯䚹䛩䛺䜟䛱䠈2-3 䛾㡯 䛻䛶ㄪ〇䛧䛯IFV 䛛䜙ᐦᗘ໙㓄㉸㐲ᚰἲ䛻䜘䜚⢭〇䛧䛯 A/PR/8/34 䜴䜲䝹䝇ᢠཎ䜢䠈 0.05 M 䛾Ⅳ㓟Ỉ⣲ሷ䝞䝑䝣䜯䞊䠄pH 9.6, Sigma, St. Louis, MO, USA䠅䛻 5 μg/mL 䛾⃰ᗘ䛷⁐ゎ䛧䠈96 well 䝥䝺䞊䝖䛻 50 μL/well 䛪䛴ຍ䛘 4Υ䛷୍ᬌᨺ⨨䛧䛯䚹0.5 (v/v) Tween20 䜢ྵ䜐 PBS 䛷䝥䝺䞊䝖䜢Ὑί䛧䠈1% (v/v)䛾∵⾑Ύ䜰䝹䝤䝭䞁䠈0.5% (v/v)䛾 Tween 20 䜢ྵ䜐 PBS 䜢 100 μL/well ຍ䛘䠈ᐊ 䛷 60 ศ㛫ᨺ⨨䛧䛯䚹䜃 0.5 (v/v)Tween20 䜢ྵ䜐 PBS 䛷䝥䝺䞊䝖䜢Ὑί䛧䠈BALF 䜢 50 μL/well ຍ䛘䠈ᐊ 䛷 60 ศ㛫ᨺ⨨䛧䛯䚹䛭䛾ᚋ䠈ྠᵝ䛻䝥䝺䞊䝖䜢Ὑί䛧䠈5000 ಸ䛻ᕼ㔘䛧䛯 Goat-anti-Mouse-IgA-HRP䠄Bethyl, Montgomery, TX, USA䠅䜢 50 μL/well ຍ䛘䠈ᐊ 䛷 60 ศ㛫ᨺ⨨䛧䛯䚹䛥䜙䛻䠈䝥䝺䞊䝖䜢Ὑίᚋ䠈0.15 M 䛾䜽䜶䞁㓟䝸䞁㓟䝞䝑䝣䜯䞊䠄pH 5.0, Sigma䠅䛻 0.5 mg/mL 䛾⃰ᗘ䛷⁐ゎ䛧䛯 o-䝣䜵䝙䝺䞁䝆䜰䝭䞁䠄Sigma䠅䜢 50 μL/well ຍ䛘䛯䚹᭱ᚋ䛻䠈1 M 䛾 H2SO4䜢50 μL/well ຍ䛘䜛䛣䛸䛷ᛂ䜢Ṇ䛥䛫䠈䝥䝺䞊䝖
10
䝸䞊䝎䞊䠄VERSAmax; Molecular Devices, Sunnyvale, CA, USA䠅䛻䛶 490 nm 䛚䜘 䜃ᑐ↷Ἴ㛗650 nm 䛾྾ගᗘ䜢 ᐃ䛧䛯䚹䛺䛚䠈ᑐ↷Ἴ㛗䛿㠀≉␗ⓗ䛺྾ග䛻㉳ᅉ䛩 䜛䝞䝑䜽䜾䝷䜴䞁䝗䜢 ᐃ䛩䜛䛯䜑䛾䜒䛾䛷䠈ᑐ↷Ἴ㛗䛷ᚓ䜙䜜䛯྾ග್䜢┠ⓗἼ㛗䛷ᚓ 䜙䜜䛯྾ග್䜘䜚ᘬ䛟䛣䛸䛷䠈㠀≉␗ⓗ䛺྾ග䜢㝖እ䛩䜛䛯䜑䛻⏝䛔䛯䚹
2-8. ⭁⣽⬊䛾⣽⬊യᐖάᛶ ᐃ
㻌 40 μm 䝉䝹䝇䝖䝺䞊䝘䞊䠄Product Number 431750, Corning, NY, USA䠅䛻⭁⮚䜢 䛾䛫䠈䝝䝃䝭䛷⣽᩿䛧䛯ᚋ䠈10%䛾䜴䝅⫾ඣ⾑Ύ䠄Invitrogen䠅䜢ྵ䜐 RPMI 1640 ᇵᆅ 䜢ຍ䛘䛺䛜䜙䝣䜱䝹䝍䞊䜢㏻䛩䛣䛸䛷⭁⣽⬊ᠱ⃮ᾮ䜢ᚓ䛯䚹䛣䛾ᠱ⃮ᾮ䜢120 × g䛷5 ศ 㛫㐲ᚰ䛧䛯ᚋ䠈⁐⾑ᾮ䠄17 mM Tris–HCl, 140 mM NH4Cl䠅䜢ຍ䛘ΰ䛧䠈ᐊ 䛷⣙ 3 ศ㛫㟼⨨䛥䛫䜛䛣䛸䛷㉥⾑⌫䜢⁐ゎ䛥䛫䛯䚹ᗘ120 × g䛷5 ศ㛫㐲ᚰ䛧䛯ᚋ䠈20% (v/v) 䝆䝯䝏䝹䝇䝹䝩䜻䝅䝗䠄Sigma䠅䜢ྵ䜐䜴䝅⫾ඣ⾑Ύ䠄Invitrogen䠅䛻⭁⣽⬊䜢ᠱ⃮ 䛧䠈䠉80Υ䛻䛶ಖᏑ䛧䛯䚹⣽⬊യᐖάᛶ䛿䠈᪤ሗ䛻ᚑ䛔䠈⭁⣽⬊䜢䜶䝣䜵䜽䝍䞊⣽⬊䠈 YAC-1 ⣽⬊䠄䝬䜴䝇䝸䞁䝟⭘⏤᮶䠅䜢ᶆⓗ⣽⬊䛸䛧䛯䠈ᨺᑕᛶ䜽䝻䝮䠄51Cr䠅ᨺฟἲ䛻䛶 ᐃ䛧䛯䠄111䠅䚹䛣䛾᪉ἲ䛿䠈⭁⣽⬊䛸䠈䛒䜙䛛䛨䜑51Cr 䛷ᶆ㆑䛧䛯 YAC-1 ⣽⬊䛸䜢ඹᇵ 㣴䛧䠈⭁⣽⬊䛜യᐖ䛧䛯YAC-1 ⣽⬊䛾ྜ䠄䠙⣽⬊യᐖάᛶ䠅䜢䠈ඹᇵ㣴ᚋ䛾ᇵ㣴ୖ Ύ୰䛻⁐ฟ䛧䛯51Cr 㔞䜢 ᐃ䛩䜛䛣䛸䛷ホ౯䛩䜛᪉ἲ䛷䛒䜛䚹䛘䜀䠈䛶䛾 YAC-䠍⣽ ⬊䛜യᐖ䛥䜜䛯ሙྜ䛿⣽⬊യᐖάᛶ䛜100%䛸䛺䜚䠈䛔䛪䜜䛾 YAC-1 ⣽⬊䜒യᐖ䛥䜜䛺 䛛䛳䛯ሙྜ䛻䛿0%䛸䛺䜛䚹䛺䛚ᮏヨ㦂䛷䛿䠈ඹᇵ㣴䛾䜶䝣䜵䜽䝍䞊⣽⬊䛸䝍䞊䝀䝑䝖⣽ ⬊䛸䛾ẚ䛿20:1 䜎䛯䛿 40:1 䛸䛧䛯䚹 2-9. ⤫ィゎᯒ
11
್䛿䛩䜉䛶ᖹᆒ್ ± ᶆ‽ㄗᕪ䛷♧䛧䛯䚹⩌୰䛻᳨ฟ㝈⏺௨ୗ䛾ಶయ䛜Ꮡᅾ䛧䛯ሙ ྜ䛻䛿䠈䛭䛾ಶయ䜢㝖䛔䛯ᖹᆒ್ ± ᶆ‽ㄗᕪ䜢⟬ฟ䛧䛯䚹య㔜ኚ㔞䛿⥺ᙧΰྜຠᯝ 䝰䝕䝹䜢⏝䛔䛶ẚ㍑䛧䠈false discovery rate䠄FDR䠅ἲ䛻䛶ከ㔜ᛶ䜢ㄪᩚ䛧䛯ୖ䛷P್ 䜢⟬ฟ䛧䛯䠄6䠅䚹GHS 䛿 Kruskal–Wallis test 䜢⏝䛔䛶ゎᯒ䛧䛯䚹⾑Ύ୰䛾 IFN-α 㔞䠈 BALF ୰䛾 IFV ≉␗ⓗ IgA 㔞䠈⭁⣽⬊䛾⣽⬊യᐖάᛶ䛿୍ඖ㓄⨨ศᩓศᯒ䜢⏝䛔䛶 ゎᯒ䛧䛯䚹P್䛜0.05 ᮍ‶䛾䛻⤫ィᏛⓗ䛻᭷ពᕪ䛜䛒䜛䛸ุ᩿䛧䛯䚹䛺䛚䠈య㔜ኚ 㔞䛾ゎᯒ䛻⏝䛧䛯⥺ᙧΰྜຠᯝ䝰䝕䝹䛸䛿䠈䛻」ᩘ䛾䝃䞁䝥䝹䛻ᑐ䛧䛶⧞䜚㏉䛧 ᐃ䛥䜜䛯⤒䝕䞊䝍䛻ᑐ䛧䛶⏝䛥䜜䜛ゎᯒ᪉ἲ䛷䛒䜛䚹t ᳨ᐃ䜔ศᩓศᯒ䛺䛹䛸␗䛺 䜚䠈ಶయᕪ䜔㛫ⓗᙳ㡪䛺䛹䠈䝕䞊䝍䛸䛧䛶ྲྀᚓ䛷䛝䛺䛔せᅉ䛾ᙳ㡪䠄ኚ㔞ຠᯝ䠅䜢⪃៖ 䛧䛯ୖ䛷䠈┠ⓗ䛾ຠᯝ䠄ᅛᐃຠᯝ䠈ᮏヨ㦂䛷䛿KB290 䛾ධ䜢ᣦ䛩䠅䜢ホ౯䛩䜛䛣䛸䛜 䛷䛝䜛ゎᯒ᪉ἲ䛷䛒䜛䠄28䠅䚹 ➨ ➨3 ⠇㻌 ⤖ᯝ 3-1. IFV ឤᰁᚋ䛾య㔜䛚䜘䜃 GHS 䛾ኚ
IFV ឤᰁᚋ䛾య㔜ኚ䠄A䠅䛚䜘䜃 GHS䠄B䠅䜢 Fig. 3 䛻♧䛧䛯䚹㠀ឤᰁ⩌䠄Normal ⩌䠅䛷䛿㢧ⴭ䛺య㔜䛾ኚ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䛾䛻ᑐ䛧䠈IFV 䜢ឤᰁ䛥䛫䛯ᑐ↷⩌ 䠄Control ⩌䠅䛚䜘䜃 KB290 ᢞ⩌䛷䛿䠈IFV ឤᰁᚋ䛻㢧ⴭ䛺య㔜ῶᑡ䛜ㄆ䜑䜙䜜䛯䚹 䛧䛛䛧䛺䛜䜙䠈KB290 ᢞ⩌䛷䛿ᑐ↷⩌䛸ẚ䜉䠈ឤᰁ 3 ᪥ᚋ௨㝆䛷య㔜ῶᑡ⋡䛜᭷ព 䛻ప್䜢♧䛧䛯䚹GHS 䛻䛴䛔䛶䜒ྠᵝ䛻䠈ᑐ↷⩌䛚䜘䜃 KB290 ᢞ⩌䛷䛿 IFV ឤᰁ ᚋ䛻ᚎ䚻䛻ప್䜢♧䛧䛯䚹䛧䛛䛧䠈䛭䛾್䛿䠈ឤᰁ4 ᪥ᚋ௨㝆䛻䛚䛔䛶䠈ᑐ↷⩌䛸ẚ䜉 KB290 ᢞ⩌䛾᪉䛜᭷ព䛻㧗䛔್䜢♧䛧䛯䚹
12 3-2. ⫵⤌⧊୰䛾 IFV ຊ౯ 㻌 㠀ឤᰁ⩌䛷䛿䛩䜉䛶䛾䝬䜴䝇䛷⫵⤌⧊୰䛾IFV ຊ౯䛜᳨ฟ㝈⏺௨ୗ䛰䛳䛯䚹୍᪉䠈 ឤᰁ3 ᪥ᚋ䛻䛚䛡䜛ᑐ↷⩌䛚䜘䜃 KB290 ᢞ⩌䛾ᖹᆒ್䛿䛭䜜䛮䜜䠈104.80±0.62 TCID50/mL䠈104.65±0.57 TCID50/mL 䛷䛒䜚䠈୧⩌䛾㛫䛻᭷ពᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹 3-3䠊⾑Ύ୰䛾 IFN-α 㔞
㻌 IFV ឤᰁ 3 ᪥ᚋ䛚䜘䜃 7 ᪥ᚋ䛾⾑Ύ୰ IFN-α 㔞䜢 Fig. 4 䛻♧䛧䛯䚹3 ᪥ᚋ䛷䛿䠈㠀 ឤᰁ⩌䛚䜘䜃ᑐ↷⩌䛾䛩䜉䛶䛾䝬䜴䝇䛷᳨ฟ㝈⏺௨ୗ䛷䛒䛳䛯䛾䛻ᑐ䛧䠈KB290 ᢞ ⩌䛻䛚䛔䛶䛿䛩䜉䛶䛾䝬䜴䝇䛷᳨ฟ䛥䜜䛯䚹7 ᪥ᚋ䛷䛿䠈㠀ឤᰁ⩌䛻䛚䛔䛶䛿 3 ᪥ᚋ䛸 ྠᵝ䛩䜉䛶䛾䝬䜴䝇䛷᳨ฟ㝈⏺䛷䛒䜚䠈ᑐ↷⩌䛻䛚䛔䛶䛿10 ༉୰ 3 ༉䛾䝬䜴䝇䛷᳨ฟ 䛥䜜䛯䚹୍᪉䠈KB290 ᢞ⩌䛷䛿 3 ᪥ᚋ䛸ྠᵝ䛩䜉䛶䛾䝬䜴䝇䛷᳨ฟ䛥䜜䠈䛭䛾ᖹᆒ್ 䛿ᑐ↷⩌䛸ẚ㍑䛧䛶᭷ព䛻㧗䛛䛳䛯䚹
3-4. BALF ୰䛾 IFV ≉␗ⓗ IgA 㔞
㻌 IFV ឤᰁ 7 ᪥ᚋ䛾 BALF ୰䛾 IFV ≉␗ⓗ IgA 㔞䜢 Fig. 5 䛻♧䛧䛯䚹KB290 ᢞ ⩌䛾BALF ୰ IFV ≉␗ⓗ IgA 㔞䛿ᑐ↷⩌䛸ẚ䜉䛶᭷ព䛻㧗䛛䛳䛯䚹㠀ឤᰁ⩌䛷䛿䠈 䛩䜉䛶䛾䝬䜴䝇䛷᳨ฟ㝈⏺௨ୗ䛰䛳䛯䚹
3-5䠊⭁⣽⬊䛾⣽⬊യᐖάᛶ
IFV ឤᰁ 3 ᪥ᚋ䛚䜘䜃 7 ᪥ᚋ䛻䛚䛡䜛䠈䜶䝣䜵䜽䝍䞊⣽⬊䠖䝍䞊䝀䝑䝖⣽⬊䠙20:1 䛾 ᮲௳ୗ䛷䛾⭁⣽⬊䛾⣽⬊യᐖάᛶ䜢Fig. 6 䛻♧䛧䛯䚹䛔䛪䜜䛾 ᐃ᪥䛻䛚䛔䛶䜒䠈㠀 ឤᰁ⩌䛻ᑐ䛧ᑐ↷⩌䛚䜘䜃KB290 ᢞ⩌䛷᭷ព䛻㧗䛟䠈䛥䜙䛻䠈KB290 ᢞ⩌䛾್
13 䛿䠈ᑐ↷⩌䛻ᑐ䛧᭷ព䛻㧗䛛䛳䛯䚹䜎䛯䠈䜶䝣䜵䜽䝍䞊⣽⬊䠖䝍䞊䝀䝑䝖⣽⬊䠙40:1 䛾᮲௳ ୗ䛷䜒ྠᵝ䛻䠈䛔䛪䜜䛾 ᐃ᪥䛻䛚䛔䛶䜒ᑐ↷⩌䛸ẚ㍑䛧KB290 ᢞ⩌䛾್䛜᭷ព䛻 㧗್䜢♧䛧䛯䠄3 ᪥ᚋ䠖ᑐ↷⩌ 7.8 ± 0.15% vs KB290 ᢞ⩌ 13.3 ± 0.25% , 7 ᪥ᚋ䠖 ᑐ↷⩌8.4 ± 0.17% vs KB290 ᢞ⩌ 16.3 ± 0.32%, 䛔䛪䜜䜒p < 0.05䠅䚹 ➨ ➨4 ⠇㻌 ⪃ᐹ ᮏ◊✲䛷䛿䠈IFV ឤᰁ๓䛻 KB290 䜢 14 ᪥㛫⤒ཱྀᢞ䛧䛯⤖ᯝ䠈IFV ឤᰁ 3 ᪥ᚋ௨ 㝆䛷య㔜ῶᑡ䛜䠈4 ᪥ᚋ௨㝆䛷 GHS 䛾పୗ䛜䠈ᑐ↷⩌䛸ẚ㍑䛧䛶᭷ព䛻ᢚไ䛥䜜䛯䚹య 㔜ῶᑡ䛾⛬ᗘ䛿䠈IFV ឤᰁ䛻䜘䜛≧䛾㔜⠜ᗘ䛾ᣦᶆ䛸䜏䛺䛥䜜䛶䛔䜛䠄64䠅䚹䜎䛯䠈GHS 䛾䜘䛖䛻㌟ほᐹ䛻䜘䜚ᗣ≧ែ䜢䝇䝁䜰䛧䛯䜒䛾䜒䠈㔜⠜ᗘ䛾ᣦᶆ䛸䛧䛶ᩘከ䛟ሗ࿌䛥䜜 䛶䛔䜛䠄61, 95, 106䠅䚹䛧䛯䛜䛳䛶ᮏ◊✲䛛䜙䠈IFV ឤᰁ๓䛻 KB290 䜢ᦤྲྀ䛩䜛䛣䛸䛻䜘䜚 䜲䞁䝣䝹䜶䞁䝄≧䜢⦆䛷䛝䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹 IFN-α 䜢䛿䛨䜑䛸䛩䜛Ϩᆺ䜲䞁䝍䞊䝣䜵䝻䞁䛿䠈ṇᖖ䛺⏕యෆ䛷䛿䜋䛸䜣䛹⏘⏕䛥䜜䛪䠈 䜴䜲䝹䝇䜔⣽⳦ឤᰁ䛺䛹䛾እⓗ่⃭䛻䜘䜚⏘⏕䛥䜜䜛䠄8䠅䚹ᐇ㝿䛻ᮏヨ㦂䛷䜒䠈IFV 䜢ឤᰁ 䛥䛫䛶䛔䛺䛔㠀ឤᰁ⩌䛷䛿䛔䛪䜜䛾䝬䜴䝇䛻䛚䛔䛶䜒IFN-α 䛜᳨ฟ䛥䜜䛺䛛䛳䛯䚹䛥䜙䛻䠈 IFV 䜢ឤᰁ䛥䛫䛯ᑐ↷⩌䛻䛴䛔䛶䜒ឤᰁ 3 ᪥ᚋ䛷䛿 IFN-α 䛜᳨ฟ䛥䜜䛺䛛䛳䛯䛾䛻ᑐ 䛧䠈KB290 ᢞ⩌䛷䛿䛩䜉䛶䛾䝬䜴䝇䛻䛚䛔䛶᳨ฟ䛥䜜䛯䚹ຍ䛘䛶 7 ᪥ᚋ䛻䛚䛔䛶䛿䠈ᑐ ↷⩌䛻䛚䛔䛶䛿10 ༉୰ 3 ༉䛾䝬䜴䝇䛷䛾䜏᳨ฟ䛥䜜䛯䛾䛻ᑐ䛧䠈KB290 ᢞ⩌䛷䛿䛩 䜉䛶䛾䝬䜴䝇䛷᳨ฟ䛥䜜䠈䛭䛾ᖹᆒ್䛿ᑐ↷⩌䛸ẚ㍑䛧䛶᭷ព䛻㧗䛛䛳䛯䚹ங㓟⳦䛾ᢞ 䛻䜘䛳䛶IFV ឤᰁᚋ䛾 IFN-α ⏘⏕䛜ஹ㐍䛩䜛䛣䛸䛿䛣䜜䜎䛷䜒ሗ࿌䛥䜜䛶䛔䛯䛜䠄59, 99䠅䠈䛣䜜䜙䛾ሗ࿌䛿䛩䜉䛶䠈IFV ឤᰁᚋ䜒ங㓟⳦ᢞ䜢⥅⥆䛧䛶䛔䛯䚹୍᪉䠈ᮏヨ㦂䛷 䛿䠈ᙉไ⤒ཱྀᢞ䛾ฎ⨨䛻䜘䜚IFV ឤᰁᚋ䛾䝬䜴䝇䛻䛥䜙䛻䝎䝯䞊䝆䜢䛘䜛䛣䛸䜢㑊䛡䜛
14
䛯䜑䠈IFV ឤᰁᚋ䛿 KB290 ᢞ䜢⾜䜟䛺䛛䛳䛯䚹䛭䜜䛻䜒㛵䜟䜙䛪䠈ឤᰁ 3 ᪥ᚋ䛚䜘䜃 7 ᪥ᚋ䛻 KB290 ᢞ⩌䛻䛚䛔䛶 IFN-α ⏘⏕䛾ஹ㐍䛜ㄆ䜑䜙䜜䛯䚹䛧䛯䛜䛳䛶䠈KB290 䜢IFV ឤᰁ๓䛻ᦤྲྀ䛧䛶䛚䛟䛣䛸䛻䜘䜚䠈IFV ឤᰁ䛻䜘䜛 IFN-α ⏘⏕䛜䛩䜀䜔䛟ㄏᑟ䛥䜜 䜛䛣䛸䠈䛥䜙䛻䛭䛾ຠᯝ䛿ᦤྲྀ䜢Ṇ䛧䛯ᚋ䜒⥅⥆䛩䜛䛣䛸䛜♧၀䛥䜜䛯䚹Ϩᆺ䜲䞁䝍䞊䝣䜵 䝻䞁䛿䠈IFV ឤᰁ⣽⬊䛻స⏝䛧⣽⬊ෆ䛾䜴䜲䝹䝇」〇䜢ᢚไ䛩䜛䛣䛸䛻ຍ䛘䠄68, 91䠅䠈ឤᰁ ึᮇ䛾ᢠయ⏘⏕ᛂ䜔⣽⬊യᐖάᛶ䜢ቑᙉ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛䠄7, 55䠅䚹䛧䛯䛜䛳 䛶䠈ᮏヨ㦂䛷䛿䠈ឤᰁ7 ᪥ᚋ䛻䛚䛔䛶 BALF ୰䛾 IFV ≉␗ⓗ IgA 㔞䛜䠈ឤᰁ 3 ᪥ᚋ䛚 䜘䜃7 ᪥ᚋ䛻䛚䛔䛶⭁⣽⬊䛾⣽⬊യᐖάᛶ䛜䠈ᑐ↷⩌䛸ẚ㍑䛧 KB290 ᢞ⩌䛷㧗್䜢 ♧䛧䛯䛜䠈䛣䜜䜙䛿IFN-α 䛾⏘⏕ஹ㐍䜢䛧䛶ㄏᑟ䛥䜜䛯䜒䛾䛷䛒䜛ྍ⬟ᛶ䛜䛒䜛䚹IFV ≉␗ⓗIgA 䛿䠈IFV ឤᰁึᮇ䛻 IFV 䛻≉␗ⓗ䛻⤖ྜ䛧䛶ឤᰁᛶ䜢ኻ䜟䛫䜛䛸䛔䛖㔜せ䛺 ᙺ䜢ᯝ䛯䛧䛶䛔䜛䠄13, 19, 43䠅䚹䜎䛯䠈NK ⣽⬊䛿䜴䜲䝹䝇ឤᰁึᮇ䛾⮬↛චᛂ⟅䛻䛚 䛔䛶㔜せ䛺ᙺ䜢ᯝ䛯䛧䛶䛔䜛䠄44䠅䚹௨ୖ䜘䜚䠈IFV ឤᰁ๓䛻 KB290 䜢ᦤྲྀ䛥䛫䜛䛣䛸䛻 䜘䜚IFV ឤᰁ䛻䜘䜛 IFN-α 䛾⏘⏕䛜ஹ㐍䛧䠈䛭䜜䛻䜘䜚䠈IFV ≉␗ⓗ IgA ⏘⏕䜔 NK ⣽ ⬊䛻䜘䜛⣽⬊യᐖάᛶ䛾ቑᙉ䛜ㄏᑟ䛥䜜䠈⤖ᯝⓗ䛻䛣䜜䜙䛜IFV ឤᰁ䛻䜘䜛య㔜ῶᑡ䜔 GHS ᝏ䜢ᢚไ䛧䛯䜒䛾䛸⪃䛘䜙䜜䜛䚹 ୍᪉䠈ᮏヨ㦂䛷䛿䠈ឤᰁ3 ᪥ᚋ䛻䛚䛡䜛⫵⤌⧊୰䛾 IFV ຊ౯䛻䛿ᑐ↷⩌䛸 KB290 ᢞ ⩌䛸䛾㛫䛻ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹IFV 䜢⤒㰯ឤᰁ䛧䛯ᚋ䛾⫵䛾䜴䜲䝹䝇ຊ౯䛿ឤᰁ 3 ᪥ᚋ䛻䝢䞊䜽䜢㏄䛘䛭䛾ᚋῶ⾶䛧䛶䛔䛟䛸䛾ሗ࿌䛜䛒䜛䛣䛸䛛䜙䠄59䠅䠈ᮏヨ㦂䛷䛿 IFV ຊ ౯䜢ឤᰁ3 ᪥ᚋ䛻ホ౯䛧䛯䚹୍᪉䛷䠈ங㓟⳦ᦤྲྀ䛻䜘䜛⫵⤌⧊୰䛾 IFV ຊ౯పῶస⏝ 䛿䠈⮫ᗋ≧䛾ᢚไ䛸ྠ䛨䝍䜲䝭䞁䜾䛷ㄆ䜑䜙䜜䛶䛔䜛᪤ሗ䛜ᩘከ䛟Ꮡᅾ䛩䜛䠄34, 39, 48䠅䚹 ᮏヨ㦂䛷䛿IFV ឤᰁᚋ䛾 GHS 䛿䠈ឤᰁ 4 ᪥ᚋ௨㝆䛻䛚䛔䛶ᑐ↷⩌䛸 KB290 ᢞ⩌䛸 䛾㛫䛻ᕪ䛜ㄆ䜑䜙䜜䛯䛣䛸䛛䜙䠈ឤᰁ3 ᪥ᚋ䛷䛾 IFV ຊ౯䛾ホ౯䛿 KB290 䛾స⏝䜢☜
15 ㄆ䛩䜛䛻䛿᪩㐣䛞䛯ྍ⬟ᛶ䛜䛒䜚䠈 GHS ᨵၿస⏝䛜ㄆ䜑䜙䜜䛯ឤᰁᚋᮇ䛷䛒䜜䜀䠈ᑐ↷ ⩌䛸KB290 ᢞ⩌䛸䛾㛫䛷 IFV ຊ౯䛻ᕪ䛜ㄆ䜑䜙䜜䛯䛛䜒䛧䜜䛺䛔䚹KB290 䛾 IFV 䛻 ᑐ䛩䜛㜵ᚚຠᯝ䛾ಙ㢗ᛶ䜢㧗䜑䜛ୖ䛷䠈ឤᰁᚋᮇ䛻䛚䛡䜛IFV ຊ౯ホ౯䛿ᚋ䛾ㄢ㢟䛷 䛒䜛䚹 䜎䛯ᮏヨ㦂䛷䛿䠈IFV 䛸䛧䛶 H1N1 ᆺ䛸䛔䛖୍✀㢮䛾䜴䜲䝹䝇ᆺ䛧䛛ホ౯䛧䛶䛔䛺䛔䛣䛸 䜒ㄢ㢟䛸䛧䛶ᣲ䛢䜙䜜䜛䚹IFV 䛻ᑐ䛩䜛 KB290 䛾ᢠឤᰁస⏝䛾ಙ㢗ᛶ䜢䜘䜚ᙉᅛ䛺䜒䛾䛻 䛩䜛䛯䜑䛻䛿䠈H3N2 ளᆺ䛺䛹」ᩘ䛾䜴䜲䝹䝇ᰴ䛻ᑐ䛩䜛 KB290 䛾ᢠឤᰁస⏝䛾᳨ド䜒 ồ䜑䜙䜜䜛䚹 ௨ୖ䛾⤖ᯝ䜘䜚䠈KB290 䛿䜲䞁䝣䝹䜶䞁䝄≧⦆స⏝䜢ᣢ䛴䛣䛸䠈䛥䜙䛻ᮏస⏝䛻䛿䠈 IFN-α ⏘⏕ஹ㐍䜔 IFV ≉␗ⓗ IgA ⏘⏕ஹ㐍䠈⣽⬊യᐖάᛶୖ᪼స⏝䛺䛹䠈KB290 䛻䜘 䜛ᵝ䚻䛺ච㈿άస⏝䛜㛵䛧䛶䛔䜛䛣䛸䛜♧၀䛥䜜䛯䚹ᚋ䛿䠈ឤᰁᚋᮇ䛾IFV ຊ౯䛾 ホ౯䜔䠈␗䛺䜛䜴䜲䝹䝇ᆺ䛾IFV 䛻ᑐ䛩䜛᭷ຠᛶ䛾᳨ド䛺䛹䛜ᮇᚅ䛥䜜䜛䚹
16
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Figure 2. Schedule for animal experiments. Mice were assigned to three groups, Normal, Control, and KB290. Mice in the Normal and Control groups were orally administered potato starch daily from day −14 to day −1, while mice in the KB290 group were orally administered KB290 daily at the same period (solid lines). Control and KB290 group mice were intranasally infected with IFV on day 0, while mice in the Normal groups were not infected. Ten mice in each group were evaluated for IFN-α level in serum, IFV titres in the lungs, and cell-mediated cytotoxic activity of splenocytes on day 3. The remaining 10 mice in each group were weighed daily and their general health status was scored from Day 0 to 7. Levels of IFN-α in serum, cell-mediated cytotoxic activity of splenocytes, and IFV-specific IgA in BALF were evaluated on Day 7 in those mice.
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Figure 3. Body weight (A) and general health score (B) of mice in the
Normal ({, n = 10), Control ( , n = 10), and KB290 (U, n = 10) groups. The comparative body weight loss between mice in the KB290 group and the Control group was analysed using a mixed effects model, and P-values for pairwise comparison were adjusted by ‘FDR’ method (*P < 0.05, **P < 0.01). The comparative general health scores between mice in the KB290 group and the Control group were analysed using the Kruskal-Wallis test (*P < 0.05).
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Figure 4. Levels of IFN-α in sera collected on both days 3 and 7 from mice in Normal, Control, and KB290 groups (n = 10 in each group on days 3 and 7). Data were analysed using one-way ANOVA (*P < 0.05).
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Figure 5. IFV-specific IgA levels in BALF collected from mice in Normal, Control, and KB290 groups (n = 10 in each group) on day 7 after IFV infection. OD490 readings were log10 transformed and analysed using
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Figure 6. The cell-mediated cytotoxic activity of splenocytes using
51Cr release assay. Splenocytes were collected from mice on days 3 and 7
after IFV infection (mice in the Normal group were not challenged with IFV). The splenic NK cell cytotoxicity was assayed at effector (splenocytes) : target (51Cr-labelled YAC-1) cell ratios as 20:1. Means of the specific lysis
were pairwise compared using one-way ANOVA. Means that do not share a letter are significantly different (P < 0.05).
21 ➨ ➨2 ❶㻌 ᑠᏛ⏕䛻䛚䛡䜛䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛻ᑐ䛩䜛Lactobacillus brevis KB290 ᦤ ྲྀ䛾ᙳ㡪䡚㠀┣᳨ᖹ⾜⩌㛫ヨ㦂䛷䛾ホ౯䡚 ➨1 ⠇㻌 ᑠᗎ ➨1 ❶䛷䛿䠈KB290 䛜䜲䞁䝣䝹䜶䞁䝄≧䛾⦆స⏝䜢ᣢ䛴䛣䛸䠈䛥䜙䛻ᮏస⏝䛻䛿 IFN-α ⏘⏕ஹ㐍䜔 IFV ≉␗ⓗ IgA ⏘⏕ஹ㐍䠈⣽⬊യᐖάᛶୖ᪼స⏝䛺䛹䠈KB290 䛻䜘 䜛ᵝ䚻䛺ච㈿άస⏝䛜㛵䛧䛶䛔䜛䛣䛸䛜♧၀䛥䜜䛯䚹䛭䛣䛷ᮏ❶䛷䛿䠈ື≀ヨ㦂䛻䛶 ㄆ䜑䜙䜜䛯KB290 䛾䜲䞁䝣䝹䜶䞁䝄䛻ᑐ䛩䜛㜵ᚚస⏝䛜䝠䝖䛻䛚䛔䛶䜒Ⓨ䛥䜜䜛䛛ྰ䛛 䜢᳨ド䛩䜛䛣䛸䜢┠ⓗ䛸䛧䛯䚹 䛣䜜䜎䛷䛻䠈ங㓟⳦䛾ᦤྲྀ䛜Ⓨ⇕䜔㰯₃䠈Ⓨတ䛺䛹䛾྾ჾ≧䜢⦆䛩䜛䛣䛸䜔䠈 ྾ჾឤᰁ䛚䜘䜃ᾘ⟶ឤᰁ䛾⨯ᝈᮇ㛫䜢▷⦰䛥䛫䜛䛣䛸䛺䛹䛜ሗ࿌䛥䜜䛶䛔䜛䠄60, 69䠅䚹䛧䛛䛧䛺䛜䜙䠈䜲䞁䝣䝹䜶䞁䝄䛻㛵䛧䛶䛿䠈䛭䛾⨯ᝈ⋡䛻ᑐ䛩䜛㣗ရᡂศ䛾ຠᯝ䜢ホ ౯䛧䛯䛿䜋䛸䜣䛹䛺䛔䚹䛣䜜䛿䠈䜲䞁䝣䝹䜶䞁䝄䛾⨯ᝈ⋡䛻ᑐ䛩䜛ຠᯝ䜢ホ౯䛩䜛䛻䛿ከ ᩘ䛾◊✲ᑐ㇟⪅䛜ᚲせ䛺䛣䛸䜔䠈䜲䞁䝣䝹䜶䞁䝄䛻⨯ᝈ䛧䛯䛸ุ᩿䛩䜛䛾䛻་ᖌ䛾デ᩿䛜 ᚲせ䛻䛺䜛䛣䛸䛺䛹䛜ཎᅉ䛰䛸⪃䛘䜙䜜䜛䚹ᐇ㝿䠈྾ჾឤᰁ䛻ᑐ䛩䜛䝥䝻䝞䜲䜸䝔䜱䜽䝇 䛾ຠᯝ䛻䛴䛔䛶グ㏙䛧䛯⥲ㄝ䛷䜒䠈྾ჾឤᰁ䛾≧䛾⦆䜔⨯ᝈᮇ㛫䛾▷⦰䛻ᑐ䛧 䛶䝥䝻䝞䜲䜸䝔䜱䜽䝇䛿᭷⏝䛷䛒䜛䜒䛾䛾䠈⨯ᝈ⋡䛾పῶ䛻ຠᯝ䛜䛒䜛䛸䛿䛔䛘䛺䛔䠈䛸⤖ㄽ 䛡䛶䛔䜛䠄105䠅䚹 ୍⯡ⓗ䛻䠈㣗ရᡂศ䛾ᶵ⬟ᛶ䛻䛴䛔䛶䝠䝖䜢ᑐ㇟䛻ホ౯䛩䜛᪉ἲ䛸䛧䛶䛿䠈↓సⅭ 㔜┣᳨䝥䝷䝉䝪ᑐ↷ẚ㍑ヨ㦂䛜⏝䛔䜙䜜䜛䚹↓సⅭ䛸䛿ᩘ⾲䛺䛹䜢⏝䛔䛶䝷䞁䝎䝮䛻 ⿕㦂⪅䜢䜚䛡䜛᪉ἲ䛷䠈䛔䛪䜜䛛䛾ヨ㦂⩌䛻≉ᐃ䛾≉ᛶ䜢ᣢ䛳䛯⿕㦂⪅䛜೫䜛䛣䛸䜢 㑊䛡䜛䛣䛸䛜┠ⓗ䛷䛒䜛䚹䜎䛯㔜┣᳨䝥䝷䝉䝪ᑐ↷ẚ㍑䛸䛿䠈⿕㦂≀㉁䜢ᦤྲྀ䛩䜛⩌䠄⿕ 㦂⩌䠅䛸䠈㤶䜔እほ䛺䛹䛷⿕㦂≀㉁䛸༊ู䛷䛝䛺䛔䝥䝷䝉䝪≀㉁䜢ᦤྲྀ䛩䜛⩌䠄䝥䝷䝉䝪
22 ⩌䠅䜢タᐃ䛧䠈◊✲⪅䛚䜘䜃⿕㦂⪅䛾୧⪅䛜䠈䛹䛾⩌䛻䜚䛡䜙䜜䛶䛔䜛䛛䜢▱䜙䛪䛻ヨ 㦂䜢ᐇ䛩䜛᪉ἲ䛷䛒䜚䠈䝥䝷䝉䝪ຠᯝ䜢㝖እ䛷䛝䜛䛯䜑⤖ᯝ䛾ᐈほᛶ䜢ಖ䛴䛣䛸䛜䛷䛝䜛 䛸䛔䛖䝯䝸䝑䝖䛜䛒䜛䚹୍᪉䛷ᮏヨ㦂䝕䝄䜲䞁䛿䠈䝥䝷䝉䝪䛾タィ䜔䝷䞁䝎䝮䛾ྍྰ䛺䛹 ᵝ䚻䛺᳨ウせ௳䛜䛒䜚䠈ヨ㦂タィ䜔ᐇ䛻ከ㢠䛾䝁䝇䝖䛜䛛䛛䜛䚹䜎䛯䠈ຠᯝ䛜↓䛔䝥䝷䝉 䝪䜢ᦤྲྀ䛩䜛ᚲせ䛜䛒䜛䛯䜑䠈◊✲ᑐ㇟⪅䛻䜒㈇Ⲵ䛜䛛䛛䜛䚹䛭䛣䛷ᮏヨ㦂䛷䛿䠈䜲䞁䝣䝹 䜶䞁䝄䛾⨯ᝈ⋡䛻ᑐ䛩䜛KB290 䛾ຠᯝ䜢䜘䜚⡆౽䛻ホ౯䛩䜛䛯䜑䠈↓సⅭ䜢⾜䜟 䛪䠈䛥䜙䛻䝥䝷䝉䝪⩌䛷䛿䛺䛟㠀ᦤྲྀ⩌䜢ᑐ↷䛸䛧䛯䠈㠀↓సⅭ㠀┣᳨୪⾜⩌㛫ヨ㦂䜢 ᐇ䛧䠈KB290 䛾䜲䞁䝣䝹䜶䞁䝄䛻ᑐ䛩䜛㜵ᚚస⏝䛻䛴䛔䛶᳨ウ䛧䛯䚹 ➨ ➨2 ⠇㻌 ◊✲ᑐ㇟⪅䠈ᮦᩱ䛚䜘䜃᪉ἲ 2-1. ヨ㦂䝕䝄䜲䞁 㻌 ヨ㦂䝕䝄䜲䞁䛿㠀↓సⅭ㠀┣᳨୪⾜⩌㛫ヨ㦂䛸䛧䛯䚹ヨ㦂䛿2 ᮇ㛫䛻ศ䛡䛶⾜䛔䠈 ๓༙ᮇ㛫䛿2013 ᖺ 10 ᭶ 21 ᪥䡚12 ᭶ 13 ᪥䠈ᚋ༙ᮇ㛫䛿 2014 ᖺ 1 ᭶ 14 ᪥䡚3 ᭶ 7 ᪥䛸䛧䛯䚹ᮏヨ㦂䛾ヨ㦂ィ⏬䛿䠈2013 ᖺ 9 ᭶ 12 ᪥䛻ཷ␒ྕ 2013-R04 䛸䛧䛶䜹䝂 䝯ᰴᘧ♫◊✲⌮ᑂᰝጤဨ䛾ᢎㄆ䜢ᚓ䛯䚹 2-2. ヨ㦂㣗ရ ヨ㦂㣗ရ䛻䛿䠈䜹䝂䝯ᰴᘧ♫䛻䛶〇㐀䞉㈍䛥䜜䛶䛔䜛ங㓟⳦㣧ᩱ䜢⏝䛔䛯䚹㣧⏝ ᮇ㛫୰䠈ྛᑠᏛᰯ䛻ᚲせᮏᩘ䜢ẖ᪥෭ⶶ䛻䛶㓄㏦䛧䛯䚹㓄㏦ᚋ䛾ヨ㦂㣗ရ䛿⿕㦂⪅䛻 㓄ᕸ䛩䜛䜎䛷ྛᏛᰯ䛾෭ⶶᗜ䛷ಖᏑ䛧䛯䚹ヨ㦂㣗ရ䛾ᴫせ䜢௨ୗ䛻♧䛩䚹 䞉ཎᮦᩱྡ䠖 䜚䜣䛤ᯝỒ䠈ங〇ရ䠈㇋㣧ᩱ䠈䛻䜣䛨䜣䜶䜻䝇䠈䝷䜲䝮ᯝỒ䠈 ㇋䝨䝥䝏䝗䠈Ⲕ䜶䜻䝇䠈Ᏻᐃ䠄䝨䜽䝏䞁䠅䠈㤶ᩱ
23 䞉㛵ᡂศ䠖 KB290㻌 6.0×109 cfu/ᮏ௨ୖ 䠄〇㐀䠅 䞉ᰤ㣴ᡂศ䠄1 ᮏᙜ䜚䠅䠖䜶䝛䝹䜼䞊䠄35 kcal䠅䠈䛯䜣䜁䛟㉁䠄0.6 g䠅䠈⬡㉁䠄0䡚0.3 g䠅䠈 ⢾㉁䠄7.6 g䠅䠈㣗≀⧄⥔䠄0䡚0.5 g䠅䠈䝘䝖䝸䜴䝮䠄8 mg䠅䠈䝅䝵⢾䠄1.0 g䠅䠈 䜹䝹䝅䜴䝮䠄14 mg䠅䠈䜹䝸䜴䝮䠄65 mg䠅䠈䝸䞁䠄14 mg䠅 2-3. ◊✲ᑐ㇟⪅ 㻌 ᰣᮌ┴㑣㡲ሷཎᕷෆ䛾ᑠᏛᰯ25 ᰯ䛾䛖䛱䠈ྛᏛᰯ䛾௦⾲ᩍㅍ䠄Ꮫᰯ㛗䠅䛛䜙ヨ㦂ཧ ຍ䛾チྍ䜢ᚓ䛯15 ᰯ䜢ཧຍᰯ䛸䛧䛯䚹15 ᰯ䜢ᆅᇦ䛸ேᩘ䛻೫䜚䛜䛺䛔䜘䛖䛻 2 䛴䛾⩌䛻 ศ䛡䠈୍᪉䜢๓༙㣧⏝ᰯ䠈䜒䛖୍᪉䜢ᚋ༙㣧⏝ᰯ䛸䛧䛯䚹ཧຍᰯ䛾ඣ❺䛚䜘䜃䛭䛾ಖ ㆤ⪅䛻䛿䠈᭩㠃䛻䛶ヨ㦂䛾㊃᪨䛚䜘䜃ෆᐜ䜢ㄝ᫂䛧䠈ヨ㦂ཧຍ䜢ᕼᮃ䛩䜛ඣ❺䛾ಖㆤ ⪅䛛䜙䠈䜲䞁䝣䜷䞊䝮䝗䝁䞁䝉䞁䝖䜢᭩㠃䛻䜘䜚ᚓ䛯䚹ಖㆤ⪅䛾䜲䞁䝣䜷䞊䝮䝗䝁䞁䝉䞁䝖䛜ᚓ 䜙䜜䠈௨ୗ䛾ձ䡚յ䛾㝖እᇶ‽䛻ヱᙜ䛧䛺䛔ඣ❺䜢◊✲ᑐ㇟⪅䛸䛧䛶㑅ᐃ䛧䛯䚹 䐟 㐣ཤ䛻㔜ᗘ䛾Ṕ䛜䛒䜛䠈䜎䛯䛿⌧ᅾ⒪୰䛾ᣢ䛜䛒䜚䠈ヨ㦂ཧຍ䛻ၥ㢟䛜䛒䜛 䛸ุ᩿䛥䜜䛯ඣ❺ 䐠 ヨ㦂䛻㛵㐃䛧䛶䜰䝺䝹䜼䞊Ⓨ䛾ᜍ䜜䛜䛒䜛ඣ❺䠄≉䛻㇋䠈ங䠈䝸䞁䝂䠅 䐡 ᾘ⟶䛾ษ㝖䜢క䛖ᡭ⾡䜢ཷ䛡䛯䛣䛸䛾䛒䜛ඣ❺䠄ᆶษ㝖䜢㝖䛟䠅 䐢 ྠᒃ䛩䜛ᐙ᪘䛻䠈䜹䝂䝯ᰴᘧ♫䛾ᚑᴗဨ䛜ྵ䜎䜜䜛ඣ❺ 䐣 䛭䛾䠈ヨ㦂㈐௵་ᖌ䛜㐺ᙜ䛸ุ᩿䛧䛯ඣ❺ 2-4. ヨ㦂䝇䜿䝆䝳䞊䝹
㻌 ヨ㦂䝇䜿䝆䝳䞊䝹䜢Fig. 7A 䛻♧䛧䛯䚹๓༙㣧⏝ᰯ䠄Group A䠅䛚䜘䜃ᚋ༙㣧⏝ᰯ 䠄Group B䠅䛾◊✲ᑐ㇟⪅䠄௨ୗ䠈䛭䜜䛮䜜䠈๓༙㣧⏝⩌䠈ᚋ༙㣧⏝⩌䛸䛩䜛䠅䛿䠈䛭䜜䛮
24 䜜䠈๓༙ᮇ㛫䜎䛯䛿ᚋ༙ᮇ㛫䛾Ⓩᰯ᪥䠄5 ᪥/㐌䠅䛻䠈ヨ㦂㣗ရ䜢 1 ᮏ䠈1 ᪥䛾䛖䛱௵ព䛾 㛫䛻㣧⏝䛧䛯䚹䛺䛚䠈◊✲ᑐ㇟⪅䛜ᑠᏛ⏕䛷䛒䜛䛣䛸䜢⪃៖䛧䠈㣧⏝ᮇ㛫䛷䛒䛳䛶䜒䠈 యㄪ䛜ᝏ䛔᪥䛺䛹䛿ヨ㦂㣗ရ䜢ᚲ䛪䛧䜒㣧⏝䛧䛺䛟䛶䜒䜘䛔᪨䜢๓䛻ఏ䛘䛯䚹䜎䛯䠈㣧 ⏝ᮇ㛫௨እ䛾ヨ㦂㣗ရ䛾㣧⏝䜒ไ㝈䛧䛺䛛䛳䛯䚹 2-5. 䝕䞊䝍䛾ྲྀᚓ 2-5-1. ಖㆤ⪅䜎䛯䛿◊✲ᑐ㇟⪅䜈䛾䜰䞁䜿䞊䝖 ྛᮇ㛫⤊䛻䠈ྛᮇ㛫୰䛾ヨ㦂㣗ရ䛾㣧⏝㢖ᗘ䠈䜲䞁䝣䝹䜶䞁䝄䛾ண㜵᥋✀䛾 ᭷↓䛚䜘䜃᥋✀᪥䠈་ᖌ䛻䜲䞁䝣䝹䜶䞁䝄䛸デ᩿䛥䜜䛯䛛ྰ䛛䠈་ᖌ䛻㢼㑧䜎䛯䛿ᾘ ⟶ឤᰁ䛸デ᩿䛥䜜䛯䛛ྰ䛛䜢䠈ಖㆤ⪅䜒䛧䛟䛿ಖㆤ⪅䛾┘╩ୗ䛷◊✲ᑐ㇟⪅䠄ඣ ❺䠅䛻䠈ㄪᰝ᭩䛾グධ䜢౫㢗䛧䛯䚹䛺䛚䠈◊✲ᑐ㇟⪅䛾䝥䝷䜲䝞䝅䞊䜢Ᏺ䜛䛯䜑䠈ᖺ㱋 䜔ᛶู䠈㌟㛗䠈య㔜䛺䛹䛾䠈ಶே䛾≉ᐃ䛻䛴䛺䛜䜛㡯┠䛿㞟䛧䛺䛛䛳䛯䚹 2-5-2. ྛᏛᰯ䜈䛾ㄪᰝ 㻌 ◊✲ᑐ㇟⪅௨እ䛾ඣ❺䜒ྵ䜑䠈ヨ㦂䛻ཧຍ䛧䛯Ꮫᰯ䛻ᡤᒓ䛩䜛ඣ❺୰䛾䠈ྛヨ㦂 ᮇ㛫୰䛻䛚䛡䜛䜲䞁䝣䝹䜶䞁䝄⨯ᝈேᩘ䛻䛴䛔䛶䠈ྛᮇ㛫⤊ᚋ䛻ㄪᰝ⚊䜈䛾グධ 䜢౫㢗䛧䛯䚹 2-6. ⤫ィゎᯒ ⤖ᯝ䛿䛩䜉䛶x2᳨ᐃ䜢⏝䛔䛶ゎᯒ䛧䠈ゎᯒ䛻䛿EZR 䝋䝣䝖䜴䜵䜰䠄Version 1.35䠅䜢 ⏝䛔䛯䚹P್䛜0.05 ᮍ‶䛾䛻⤫ィᏛⓗ䛻᭷ពᕪ䛜䛒䜛䛸ุ᩿䛧䛯䚹䛺䛚 EZR 䛸䛿䠈 ⤫ィゎᯒ䝋䝣䝖䜴䜵䜰䛷䛒䜛R 䜢䠈䝇䜽䝸䝥䝖䜢ධຊ䛩䜛䛣䛸䛺䛟⏝䛷䛝䜛䜘䛖⮬་⛉
25 Ꮫ䛻䜘䜚㛤Ⓨ䛥䜜䛯䝋䝣䝖䛷䛒䜛䠄46䠅䚹 ➨ ➨3 ⠇㻌 ⤖ᯝ 3-1. ⿕㦂⪅䛚䜘䜃ゎᯒᑐ㇟⪅䛾㑅ᐃ ゎᯒᑐ㇟⪅䛾㑅ᐃ䛾ὶ䜜䜢Fig. 8 䛻♧䛧䛯䚹15 ᰯ䛾ཧຍᰯ䛾ඣ❺䛿㒊䛷 3356 ྡ䛷䛒䛳䛯䚹ඣ❺䛻ヨ㦂༠ຊ䜢౫㢗䛧䛯⤖ᯝ䠈ಖㆤ⪅䛛䜙䜲䞁䝣䜷䞊䝮䝗䝁䞁䝉䞁䝖䛜 ᚓ䜙䜜䛺䛛䛳䛯421 ྡ䛾ඣ❺䛚䜘䜃㝖እᇶ‽䛻ྜ⮴䛧䛯 9 ྡ䛾ඣ❺䜢㝖እ䛧䛯⤖ᯝ䠈 ᭱⤊ⓗ䛺◊✲ᑐ㇟⪅䛿2926 ྡ䛷䛒䛳䛯䚹◊✲ᑐ㇟⪅䛾䛖䛱䠈௨ୗ䛾䛩䜉䛶䛻ᙜ䛶䛿䜎 䜛1783 ྡ䛾ඣ❺䜢ゎᯒᑐ㇟⪅䛸䛧䛯䚹 䞉㣧⏝ᮇ㛫䛻ヨ㦂㣗ရ䜢5 ᪥/㐌㣧⏝䛧䛯䛸ᅇ⟅ 䞉㠀㣧⏝ᮇ㛫䛻⿕㦂㣗ရ䜢䛟㣧⏝䛧䛺䛛䛳䛯䛸ᅇ⟅ 䞉䛶䛾䜰䞁䜿䞊䝖㡯┠䛻ᅇ⟅ ゎᯒᑐ㇟⪅䛾䛖䛱䠈๓༙㣧⏝⩌䛻ヱᙜ䛩䜛⪅䛿694 ྡ䠈ᚋ༙㣧⏝⩌䛻ヱᙜ䛩䜛⪅ 䛿1089 ྡ䛷䛒䛳䛯䚹䜎䛯䠈ゎᯒᑐ㇟⪅䛛䜙㝖እ䛥䜜䛯 1143 ྡ䛾䛖䛱䠈301 ྡ䛿䜰䞁 䜿䞊䝖䛻グධ₃䜜䛜䛒䛳䛯⪅䛷䛒䜚䠈842 ྡ䛿つᐃ㏻䜚䛻ヨ㦂㣗ရ䜢ᦤྲྀ䛧䛺䛛䛳䛯⪅䛷 䛒䛳䛯䚹 3-2. ᰣᮌ┴ෆ䛾䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜ᗘ ᰣᮌ┴ឤᰁሗ䝉䞁䝍䞊䛻䜘䜚බ㛤䛥䜜䛶䛔䜛䠈ヨ㦂ᮇ㛫୰䛻䛚䛡䜛ᰣᮌ┴ෆ䛾ᐃ Ⅼ䛒䛯䜚䛾䜲䞁䝣䝹䜶䞁䝄ሗ࿌ᩘ䠄100䠅䜢 Fig. 7B 䛻♧䛧䛯䚹2013䡚2014 ᖺ䛾䜲䞁䝣 䝹䜶䞁䝄ὶ⾜䛾䝢䞊䜽䛿2014 ᖺ 1 ᭶ୗ᪪䛛䜙 2 ᭶䛻䛛䛡䛶䛷䛒䜚䠈๓༙ᮇ㛫䛜ヱᙜ䛩 䜛2013 ᖺ䛻䛚䛔䛶䛿䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜䛾⛬ᗘ䛿ప䛛䛳䛯䚹
26 3-3. ゎᯒᑐ㇟⪅䛻䛚䛡䜛䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡ 㻌 ๓༙ᮇ㛫䜎䛯䛿ᚋ༙ᮇ㛫䛻䛚䛔䛶䠈KB290 ᦤྲྀ⩌䛸㠀ᦤྲྀ⩌䛸䛾㛫䛷䜲䞁䝣䝹䜶䞁䝄 ⨯ᝈ⋡䜢ẚ㍑䛧䛯䠄Table 1䠅䚹๓༙ᮇ㛫䛻䛚䛔䛶䜲䞁䝣䝹䜶䞁䝄䛸デ᩿䛥䜜䛯⪅䛿䠈ᦤྲྀ ⩌䛷䛿0 ྡ䠈㠀ᦤྲྀ⩌䛷䛿 2 ྡ䛾䜏䛷䛒䜚䠈2 ⩌㛫䛻⤫ィᏛⓗ䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳 䛯䚹3-2 䛻グ㍕䛾㏻䜚䠈ᰣᮌ┴䛻䛚䛔䛶䛿䠈๓༙ᮇ㛫䛿䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜๓䛷䛒䜚䠈 ᮏᮇ㛫䛿ヨ㦂㣗ရᦤྲྀ䛾ຠᯝ䜢ホ౯䛩䜛䛻䛿㐺䛷䛒䜛䛸⪃䛘䜙䜜䛯䚹୍᪉䠈䜲䞁䝣䝹 䜶䞁䝄䛜ὶ⾜䛧䛯ᚋ༙ᮇ㛫䛻䛚䛔䛶䛿䠈ᦤྲྀ⩌䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛜15.7%䠈㠀ᦤ ྲྀ⩌䛷䛿23.9%䛷䛒䜚䠈ᦤྲྀ⩌䛾⨯ᝈ⋡䛜㠀ᦤྲྀ⩌䛸ẚ㍑䛧䛶᭷ព䛻ప್䜢♧䛧䛯 䠄Table 1䠈p < 0.001䠅䚹 䛺䛚䠈Ꮫᰯ㛫䛷䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜䛾⛬ᗘ䛻ᕪ䛜↓䛛䛳䛯䛣䛸䜢☜䛛䜑䜛䛯䜑䠈ヨ㦂 䛻ཧຍ䛧䛯15 ᰯ䛻ᡤᒓ䛩䜛ඣ❺䠄ᮏヨ㦂䛻ཧຍ䛧䛶䛔䛺䛔ඣ❺䜒ྵ䜐䠅䛻䛚䛡䜛ᚋ༙ ᮇ㛫䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䜢ẚ㍑䛧䛯⤖ᯝ䠈๓༙㣧⏝ᰯ䠄Group A䠅䛻䛚䛔䛶 19.8% 䠄325/1645 ྡ䠅䠈ᚋ༙㣧⏝ᰯ䠄Group B䠅䛻䛚䛔䛶 17.9%䠄306/1711 ྡ䠅䛷䛒䜚䠈⩌㛫䛻 ⤫ィᏛⓗ䛺᭷ពᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䠄┦ᑐ༴㝤⋡ = 1.105, 95%ಙ㢗༊㛫 = 0.960 – 1.272, p = 0.165䠅䚹 㻌 ḟ䛻䠈ᦤྲྀ⩌䛸㠀ᦤྲྀ⩌䛸䛾㛫䛷ㄆ䜑䜙䜜䛯䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛾ᕪ䛜䜲䞁䝣䝹䜶䞁 䝄䛾ண㜵᥋✀䛾᭷↓䛻㉳ᅉ䛩䜛䜒䛾䛷䛿䛺䛔䛣䛸䜢☜䛛䜑䜛䛯䜑䠈ண㜵᥋✀䜢ཷ䛡䛯⪅ 䛸ᮍ᥋✀䛾⪅䛸䛷ᒙู䛧䠈䛭䜜䛮䜜䛻䛚䛔䛶ᦤྲྀ⩌䛸㠀ᦤྲྀ⩌䛸䛾㛫䛷⨯ᝈ⋡䜢ẚ㍑䛧 䛯䠄Table 1䠅䚹䛭䛾⤖ᯝ䠈ண㜵᥋✀䜢ཷ䛡䛯⪅䛻䛚䛔䛶䛿䠈⨯ᝈ⋡䛿⩌㛫䛻⤫ィᏛⓗ䛺 ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹୍᪉ᮍ᥋✀⪅䛻䛚䛔䛶䛿䠈KB290 ᦤྲྀ⩌䛾䜲䞁䝣䝹䜶䞁䝄⨯ ᝈ⋡䛜16.3%䠈㠀ᦤྲྀ⩌䛷䛿 28.7%䛷䛒䜚䠈ᦤྲྀ⩌䛻䛚䛡䜛⨯ᝈ⋡䛾పῶ䛜䜘䜚㢧ⴭ䛻
27 ㄆ䜑䜙䜜䛯䠄Table 1䠈p < 0.001䠅䚹 3-4. ゎᯒᑐ㇟⪅䛻䛚䛡䜛㢼㑧⨯ᝈ⋡䛚䜘䜃⫶⭠⅖⨯ᝈ⋡ ゎᯒᑐ㇟⪅䛻䛚䛡䜛๓༙ᮇ㛫䛚䜘䜃ᚋ༙ᮇ㛫䛾㢼㑧䛚䜘䜃⫶⭠⅖⨯ᝈ⋡䜢Table 2 䛻♧䛧䛯䚹๓༙ᮇ㛫䛻䛚䛔䛶䛿䠈ᦤྲྀ⩌䛾㢼㑧⨯ᝈ⋡䛜㠀ᦤྲྀ⩌䛻ẚ䜉᭷ព䛻㧗್䜢 ♧䛧䛯䚹ᚋ༙ᮇ㛫䛻䛚䛔䛶䛿䠈୧⩌䛾㢼㑧⨯ᝈ⋡䛾㛫䛻⤫ィᏛⓗ䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳 䛯䚹⫶⭠⅖䛾⨯ᝈ⋡䛿୧ᮇ㛫䛸䜒䛻ẚ㍑ⓗప䛟䠈⩌㛫䛻ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹 3-5. ᭷ᐖ㇟ 㻌 ヨ㦂ᮇ㛫୰䠈ヨ㦂㈐௵་ᖌ䛻ᑐ䛧䛶䠈1 ྡ䛾⿕㦂⪅䛛䜙⭡③䠈䜒䛖 1 ྡ䛾⿕㦂⪅䛛䜙㢼 㑧≧䛾ᝏ䛾⏦䛧ฟ䛜ᐤ䛫䜙䜜䛯䚹䛧䛛䛧䠈䛹䛱䜙䛾䜿䞊䝇䜒䠈་ᖌ䛾⒪䛜ᚲせ䛺䜋 䛹㔜⠜䛷䛿䛺䛟୍㐣ᛶ䛷䛒䛳䛯䛣䛸䛛䜙䠈ヨ㦂㈐௵་ᖌ䛿ᮏヨ㦂䛻䛚䛔䛶᭷ᐖ㇟䛿Ⓨ ⏕䛧䛺䛛䛳䛯䛸ุ᩿䛧䛯䚹 ➨ ➨4 ⠇㻌 ⪃ᐹ 㻌 ᮏヨ㦂䛷䛿䠈2013 ᖺ 10䡚12 ᭶䛚䜘䜃 2014 ᖺ 1䡚3 ᭶䛾 2 ᮇ㛫䛷୪⾜⩌㛫ヨ㦂䜢⾜ 䛳䛯䛜䠈๓༙ᮇ㛫䛿䜲䞁䝣䝹䜶䞁䝄䛜䜋䛸䜣䛹ὶ⾜䛫䛪䠈ヨ㦂㣗ရ䛷䛒䜛KB290 䛾ຠᯝ䜢 ホ౯䛷䛝䛺䛛䛳䛯䚹䛧䛯䛜䛳䛶䠈䜲䞁䝣䝹䜶䞁䝄䛾䜘䛖䛺Ⓨ⏕䛻Ꮨ⠇ᛶ䛾䛒䜛ᝈ䜔≧䛻 ᑐ䛩䜛ຠᯝ䜢ホ౯䛩䜛ሙྜ䛻䛿䠈ヨ㦂ᮇ㛫䛾㑅ᐃ䛜㔜せ䛷䛒䜛䛸⪃䛘䜙䜜䛯䚹 䜲䞁䝣䝹䜶䞁䝄䛜ὶ⾜䛧䛯ᚋ༙ᮇ㛫䛻䛚䛔䛶䠈KB290 䜢⥅⥆ⓗ䛻ᦤྲྀ䛧䛯⩌䛾䜲䞁䝣䝹 䜶䞁䝄⨯ᝈ⋡䛿㠀ᦤྲྀ⩌䛸ẚ㍑䛧䛶᭷ព䛻ప䛛䛳䛯䚹䛣䛾⤖ᯝ䛿䠈䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜ ᮇ㛫䛻KB290 䜢⥅⥆ⓗ䛻ᦤྲྀ䛩䜛䛣䛸䛜䜲䞁䝣䝹䜶䞁䝄⨯ᝈ䝸䝇䜽䜢పῶ䛷䛝䜛ྍ⬟ᛶ䜢
28 ♧၀䛧䛶䛔䜛䚹୍᪉䠈ᮏヨ㦂䛻ཧຍ䛧䛯ᑠᏛᰯ䛻䛚䛡䜛ඣ❺䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡ 䛿䠈๓༙㣧⏝ᰯ䠄Group A䠅䛸ᚋ༙㣧⏝ᰯ䠄Group B䠅䛸䛾㛫䛻ᕪ䛿䛺䛛䛳䛯䚹䛣䛾䛣䛸䛿䠈๓ ༙㣧⏝ᰯ䛸ᚋ༙㣧⏝ᰯ䛸䛾㛫䛷䠈ᚋ༙ᮇ㛫䛾䜲䞁䝣䝹䜶䞁䝄䛾ὶ⾜䛾⛬ᗘ䛻䛿ᕪ䛜↓䛛䛳 䛯䛣䛸䜢♧၀䛧䛶䛚䜚䠈ୖグ䛷ᚓ䜙䜜䛯⤖ᯝ䛜KB290 ᦤྲྀ䛻㉳ᅉ䛩䜛䛣䛸䜢ᨭᣢ䛩䜛䜒䛾 䛷䛒䜛䚹 䛥䜙䛻ᮏヨ㦂䛷䛿䠈ண㜵᥋✀䜢ཷ䛡䛯ඣ❺䜘䜚䜒ཷ䛡䛺䛛䛳䛯ඣ❺䛻䛚䛔䛶䠈KB290 ᦤ ྲྀ⩌䛸㠀ᦤྲྀ⩌䛸䛾㛫䛾⨯ᝈ⋡䛾ᕪ䛜㢧ⴭ䛻ㄆ䜑䜙䜜䛯䚹䛧䛯䛜䛳䛶䠈KB290 ᦤྲྀ䛻䜘䜛 䜲䞁䝣䝹䜶䞁䝄⨯ᝈ䝸䝇䜽䛾పῶస⏝䛿䠈ண㜵᥋✀䜢ཷ䛡䛶䛔䛺䛔䜘䛖䛺⨯ᝈ䝸䝇䜽䛾㧗䛔䝠 䝖䛻䛚䛔䛶䜘䜚㢧ⴭ䛻ㄆ䜑䜙䜜䜛ྍ⬟ᛶ䛜䛒䜛䚹 ங㓟⳦䜢䛿䛨䜑䛸䛩䜛䝥䝻䝞䜲䜸䝔䜱䜽䝇ᦤྲྀ䛾ຠᯝ䛸䛧䛶䠈䜲䞁䝣䝹䜶䞁䝄ண㜵᥋✀䛾ච ཎᛶ䜢㧗䜑䜛䛣䛸䠄䜰䝆䝳䝞䞁䝖ຠᯝ䠅䛜ሗ࿌䛥䜜䛶䛔䜛䠄110䠅䚹䛧䛛䛧䛺䛜䜙ᮏ◊✲䛷䛿䠈 ண㜵᥋✀䜢ཷデ䛧䛯⪅䛻䛚䛔䛶䛿㠀ᦤྲྀ⩌䛸ᦤྲྀ⩌䛸䛷䛿ᕪ䛿ㄆ䜑䜙䜜䛪䠈KB290 ᦤྲྀ 䛻䜘䜛ண㜵᥋✀䛾ຠᯝ䛾ቑᙉస⏝䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䚹䜲䞁䝣䝹䜶䞁䝄䛾ண㜵᥋✀䛿12 ᭶䜎䛷䛻ཷデ䛩䜛䛣䛸䛜୍⯡ⓗ䛷䛒䜛䛯䜑䠈ᮏヨ㦂䛾⿕㦂⪅ဨ䛜ண㜵᥋✀䛻KB290 䜢ᦤྲྀ䛧䛶䛔䛯䛸䛿㝈䜙䛺䛔䚹≉䛻ᚋ༙㣧⏝⩌䛻䛴䛔䛶䛿KB290 ᦤྲྀ๓䛻ண㜵᥋✀䜢ཷ デ䛧䛯⪅䛜ከ䛔䛸⪃䛘䜙䜜䜛䚹䛧䛯䛜䛳䛶䠈ᮏヨ㦂䛾タィ䛿䜰䝆䝳䝞䞁䝖ຠᯝ䜢ホ౯䛩䜛䛻 䛿㐺ษ䛷䛿䛺䛛䛳䛯䛸⪃䛘䜙䜜䜛䚹ຍ䛘䛶䠈䛭䛾䛾ཎᅉ䛸䛧䛶䠈ᮏヨ㦂䛷䛿ண㜵᥋✀䛜༑ ศ䛻ຠᯝ䜢Ⓨ䛧䛯䛣䛸䛜ᣲ䛢䜙䜜䜛䚹ᮏヨ㦂䛾ᚋ༙ᮇ㛫䛻䛚䛔䛶䛿䠈㠀ᦤྲྀ⩌䛾䛖䛱䠈ண 㜵᥋✀䜢ཷ䛡䛯ඣ❺䛾䜲䞁䝣䝹䜶䞁䝄䛾⨯ᝈ⋡䛜ཷ䛡䛺䛛䛳䛯ඣ❺䛾⨯ᝈ⋡䜘䜚䜒᭷ព䛻 ప್䜢♧䛧䛯䠄┦ᑐ༴㝤⋡䠖0.686, 95%ಙ㢗༊㛫䠖0.524䠉0.898, p < 0.01)䚹䜎䛯䠈2010 ᖺᗘ䛛䜙2015 ᖺᗘ䛾 6 䝅䞊䝈䞁䛻䛚䛔䛶䠈ᑠᏛ⏕⣙ 5,000 ே䜢ᑐ㇟䛻䜲䞁䝣䝹䜶䞁䝄ண 㜵᥋✀䛾᭷ຠ⋡䜢ホ౯䛧䛯◊✲䠄70䠅䛷䛿䠈᭷ຠ⋡䛾 6 䝅䞊䝈䞁䛾ᖹᆒ್䛿 17.3%䠄᭱㧗
29 38%, ᭱ప䠉6%䠅䛸ሗ࿌䛥䜜䛶䛔䜛䛾䛻ᑐ䛧䠈ᮏヨ㦂䛻䛚䛡䜛ண㜵᥋✀䛾᭷ຠ⋡䠄䠄1䠉┦ ᑐ༴㝤⋡䠅㽢100䠅䛿 31%䛷䛒䜚䠈᪤ሗ䛸ẚ㍑䛧䛶䜒᭷ຠ⋡䛿༑ศ㧗䛛䛳䛯䚹䛧䛯䛜䛳䛶䠈ᮏ ヨ㦂䛷䛿ண㜵᥋✀䛜䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛾పῶ䛻༑ศ䛻ᐤ䛧䠈䛭䛾䛯䜑䠈KB290 䛻 䜘䜛ண㜵᥋✀䜈䛾ຠᯝ䛜ㄆ䜑䜙䜜䛺䛛䛳䛯ྍ⬟ᛶ䛜䛒䜛䚹ᐇ㝿䠈ᑠᏛᰯ䛻䛚䛡䜛䜲䞁䝣䝹 䜶䞁䝄ண㜵᥋✀䛾ཷデ⋡䛸䠈Ꮫ⣭㛢㙐Ⓨ⏕⋡䜔Ḟᖍ⋡䛸䛾㛫䛻䛿㈇䛾┦㛵䛜䛒䜛䛺䛹䠈ᑠ Ꮫ⏕䛻䛚䛡䜛䜲䞁䝣䝹䜶䞁䝄ண㜵䛻ண㜵᥋✀䛜ຠᯝⓗ䛷䛒䜛䛣䛸䜢♧䛩ሗ࿌䛜」ᩘᏑᅾ䛩 䜛䠄47, 101䠅䚹 䝥䝻䝞䜲䜸䝔䜱䜽䝇ᦤྲྀ䛻䜘䜛䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛾పῶస⏝䛻㛵䛩䜛᪤ሗ䛸䛧䛶䠈 Namba 䜙䠄73䠅䛜䠈䝡䝣䜱䝈䝇⳦䛾୍✀䛷䛒䜛Bifidobacterium longum BB536 䛾ᦤྲྀ䛻 䜘䜚㧗㱋⪅䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛜పୗ䛧䛯䛸ሗ࿌䛧䛶䛔䜛䚹䛧䛛䛧䠈䛣䛾◊✲䛷䛿◊✲ ᑐ㇟⪅䛜27 ྡ䛸㠀ᖖ䛻ᑡ䛺䛔䚹䜎䛯䠈638 ྡ䛾ᑠඣ䜢ᑐ㇟䛻䛧䛯◊✲䛻䛚䛔䛶䛿䠈ங㓟 ⳦䛾୍✀䛷䛒䜛Lactobacillus casei DN-114 001 䛾ᦤྲྀ䛻䜘䜚྾ჾឤᰁ䜔ᾘჾ ឤᰁ䛾⨯ᝈ⋡䛜పῶ䛧䛯䛸ሗ࿌䛥䜜䛶䛔䜛䛜䠈䜲䞁䝣䝹䜶䞁䝄䛻㝈ᐃ䛧䛯⨯ᝈ⋡䛿ホ౯ 䛥䜜䛶䛔䛺䛔䠄69䠅䚹୍᪉䠈䝥䝻䝞䜲䜸䝔䜱䜽䝇௨እ䛾㣗ရᡂศ䛻䛚䛔䛶䛿䠈」ᩘ䛾᪤ሗ䛻䛚 䛔䛶䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䜈䛾ຠᯝ䛜♧䛥䜜䛶䛔䜛䚹䛘䜀䠈334 ྡ䛾ᑠᏛ⏕䜢ᑐ㇟䛻䛧 䛯◊✲䛷䛿䝡䝍䝭䞁D 䛾ᦤྲྀ䛜 A ᆺ䜲䞁䝣䝹䜶䞁䝄䛾⨯ᝈ⋡䜢పῶ䛥䛫䛯䛣䛸䜔䠈197 ྡ 䛾ᡂே䜢ᑐ㇟䛻䛧䛯◊✲䛷䛿⥳Ⲕ䜹䝔䜻䞁䛾ᦤྲྀ䛜䜲䞁䝣䝹䜶䞁䝄䛸デ᩿䛥䜜䛯⪅䛾ྜ 䜢పῶ䛧䛯䛣䛸䛺䛹䛜ሗ࿌䛥䜜䛶䛔䜛䠄65, 102䠅䚹䛣䜜䜙䛾◊✲䛸↷䜙䛧ྜ䜟䛫䛶䜒䠈ᮏ◊✲ 䛻䛚䛡䜛◊✲ᑐ㇟⪅䛾つᶍ䛿䜲䞁䝣䝹䜶䞁䝄⨯ᝈ⋡䛻ᑐ䛩䜛ຠᯝ䜢☜ㄆ䛩䜛䛾䛻༑ศ䛰䛸 ⪃䛘䜙䜜䜛䚹䛧䛛䛧䛺䛜䜙䠈䛣䜜䜙䜲䞁䝣䝹䜶䞁䝄䛻ᑐ䛩䜛ຠᯝ䜢ホ౯䛧䛯2 䛴䛾◊✲䛿䛔 䛪䜜䜒↓సⅭ㔜┣᳨䝥䝷䝉䝪ᑐ↷ẚ㍑ヨ㦂䛷䛒䜚䠈ᮏ◊✲䛾ヨ㦂䝕䝄䜲䞁䛷䛒䜛㠀┣ ᳨ヨ㦂䛸ẚ㍑䛧䛶⢭ᗘ䛾㧗䛔䜒䛾䛷䛒䜛䚹䝠䝖䛻䛚䛡䜛KB290 ᦤྲྀ䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ
30 ⋡పῶస⏝䜢⤖ㄽ䛡䜛䛻䛿䠈ᮏヨ㦂䜘䜚䜒⢭ᗘ䛾㧗䛔ヨ㦂䝕䝄䜲䞁䛷䛾ホ౯䛜ᚲせ䛷䛒 䜚䠈ᚋ䛾ㄢ㢟䛷䛒䜛䚹 ᮏ◊✲䛷䛿䠈䜲䞁䝣䝹䜶䞁䝄௨እ䛾ឤᰁ䛸䛧䛶㢼㑧䛚䜘䜃⫶⭠⅖≧䛻ᑐ䛩䜛 KB290 䛾ຠᯝ䜢ணഛⓗ䛻ホ౯䛧䛯䚹䛧䛛䛧䠈䛔䛪䜜䛻ᑐ䛧䛶䜒 KB290 䛾ຠᯝ䜢☜ㄆ䛩䜛 䛣䛸䛿䛷䛝䛺䛛䛳䛯䚹KB290 䛿⣽⬊യᐖάᛶ䜢ୖ᪼䛥䛫䜛䛣䛸䛜♧䛥䜜䛶䛔䜛䛜䠈ྠᵝ䛻䠈 ⣽⬊യᐖάᛶୖ᪼స⏝䛜♧䛥䜜䛶䛔䜛ங㓟⳦䛷䛒䜛Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 䜢ྵ䜐䝶䞊䜾䝹䝖ᦤྲྀ䛜㢼㑧䛾䝸䝇䜽䜢పῶ䛥䛫䜛䛣䛸䛜ሗ࿌䛥 䜜䛶䛔䜛䠄60䠅䚹䛧䛯䛜䛳䛶䠈KB290 䜒㢼㑧䛻ᑐ䛧䛶ண㜵ຠᯝ䜢♧䛩䛣䛸䛜ᮇᚅ䛥䜜䛯䜒䛾 䛾䠈ᮏヨ㦂䛷䛿☜ㄆ䛷䛝䛺䛛䛳䛯䚹䛭䛾ཎᅉ䛸䛧䛶䠈㢼㑧䛾ὶ⾜䛾⛬ᗘ䛜⩌㛫䛷␗䛺䛳䛶 䛔䛯䠈㢼㑧䜢䜂䛔䛶䜒㝔䛻⾜䛛䛺䛔⪅䛜Ꮡᅾ䛧䛯䠈㢼㑧䛾デ᩿ᇶ‽䛜་ᖌ䛻䜘䜚␗䛺䛳 䛶䛔䛯䠈䛺䛹䛾ྍ⬟ᛶ䛜⪃䛘䜙䜜䜛䚹KB290 䛾㢼㑧䛻ᑐ䛩䜛ຠᯝ䜢ุ᩿䛩䜛䛯䜑䛻䛿䠈䛣 䜜䜙䜢᫂☜䛻ᢕᥱ䞉つᐃ䛧䛯ヨ㦂䛜ᚲせ䛷䛒䜛䚹 㻌 ௨ୖ䜘䜚䠈䜲䞁䝣䝹䜶䞁䝄ὶ⾜ᮇ䛻KB290 䜢⥅⥆ⓗ䛻ᦤྲྀ䛩䜛䛣䛸䛿䠈ᑠඣ䛾䜲䞁䝣䝹䜶 䞁䝄⨯ᝈ䝸䝇䜽䜢పῶ䛷䛝䜛䛣䛸䛜♧၀䛥䜜䛯䚹ᮏヨ㦂䛾䜘䛖䛺1000 ே䜢㉸䛘䜛◊✲ᑐ㇟⪅ 䛻䛚䛔䛶䝥䝻䝞䜲䜸䝔䜱䜽䝇䛾䜲䞁䝣䝹䜶䞁䝄⨯ᝈ䝸䝇䜽పῶస⏝䜢♧䛧䛯ሗ࿌䛿ึ䜑䛶䛷 䛒䜛䚹䛧䛛䛧䛺䛜䜙䠈KB290 䛾ຠᯝ䜢䜘䜚᫂☜䛻♧䛩䛯䜑䛻䛿↓సⅭ㔜┣᳨䝥䝷䝉䝪 ᑐ↷ẚ㍑ヨ㦂䛺䛹䛾䠈䜘䜚⢭ᗘ䛾㧗䛔ヨ㦂䝕䝄䜲䞁䛷䛾ホ౯䛜ᚲせ䛷䛒䜚䠈ᚋ䛾ㄢ㢟䛷 䛒䜛䚹
31
F
Figure 7. Clinical trial schedule (A) and number of people diagnosed with influenza in Tochigi prefecture (B). For subjects in Group A, test drink containing KB290 was provided 5 d per week for 2 months in the first period (consumption period; solid arrow) and no treatment in the second period (non-consumption period; dotted arrow), while those in Group B were defined vice versa (A). Data of number of people diagnosed with influenza in Tochigi prefecture were derived from the Tochigi Prefectural Infectious Disease Surveillance Centre (B).
32
F
33
T
Table 1. Incidence of influenza
RR: relative risk, CI: confidence interval, Non-consumption group: the group which did NOT consume test drink including KB290, Consumption group: the group which consumed test drink including KB290
Non-consumption group
Consumption group
RR
95% CI
P value
First period
All subjects analysed
2/1089 (0.2)
0/694 (0.0)
䠉
䠉
0.259
Second period
All subjects analysed
166/694 (23.9)
171/1089 (15.7)
0.656
0.542, 0.795 < 0.001
- Vaccinated subjects
70/356 (19.7)
70/466 (15.0)
0.764
0.565, 1.032
0.079
- Unvaccinated subjects
96/335 (28.7)
101/620 (16.3)
0.568
0.445, 0.727 < 0.001
34
T
Table 2. Incidence of common cold and gastroenteritis
RR: relative risk, CI: confidence interval, Non-consumption group: the group which did NOT consume test drink including KB290, Consumption group: the group which consumed test drink including KB290
Non-consumption group
Consumption group
RR
95% CI
P value
First period
Common cold
131/1089 (12.0)
108/694 (15.6)
1.294
1.021, 1.638
0.033
Gastroenteritis
19/1089 (1.7)
17/694 (2.4)
1.404
0.735, 2.682
0.302
Second period
Common cold
62/694 (8.9)
117/1089 (10.7)
1.203
0.897, 1.612
0.215
Gastroenteritis
27/694 (3.9)
63/1089 (5.8)
1.487
0.957, 2.310
0.075
Subjects diagnosed with
35
➨
➨3 ❶㻌 Citrobacter rodentiumឤᰁ䝬䜴䝇䛻ᑐ䛩䜛Lactobacillus brevis KB290 䛾ឤ
ᰁ㜵ᚚస⏝
➨1 ⠇㻌 ᑠᗎ
⭠⟶ཎᛶ⭠⳦䠄enteropathogenic Escherichia coli ; EPEC䠅䜔⭠⟶ฟ⾑ᛶ⭠
⳦䠄enterohemorrhagic Escherichia coli ; EHEC䠅䛿䠈䛻 2 ṓ௨ୗ䛾Ꮚ౪䛻ୗ⑩䜢㉳
䛣䛩ཎయ䛷䛒䜛䚹≉䛻Ⓨᒎ㏵ୖᅜ䛷䛿䠈ஙᗂඣୗ⑩䛾30䡚40䠂䛜䛣䜜䜙䛾ཎయ䛻
䜘䜛䜒䛾䛸䜒䛔䜟䜜䛶䛚䜚䠈䛣䛾ୗ⑩䛻䜘䜛ஙඣ䛾Ṛஸ⋡䛾పῶ䛿䠈≉䛻Ⓨᒎ㏵ୖᅜ䛻䛚
䛔䛶㠀ᖖ䛻㔜せ䛸䛥䜜䛶䛔䜛䠄17䠅䚹
EPEC 䜔 EHEC 䛜⭠䛾⢓⭷ୖ⓶⣽⬊䛻᥋╔䛧ቑṪ䛩䜛䛸䠈attaching䠄⳦䛾᥋╔䠅
/effacing䠄ᚤ⤧ẟ䛾㏥⦰䠅䠄A/E䠅 lesions 䛸䜀䜜䜛䠈ᚤ⤧ẟ䛾◚ቯ䛸㏥⦰䠈ୖ⓶⣽⬊㦵 ᱁䛾യᐖ䠈⣽⬊⭷䛾㝗ἐ䛚䜘䜃◚ቯ䠈⢓⭷䛾⫧ཌ䛺䛹䛾≉ᚩⓗ䛺ኚ䜢ㄏᑟ䛩䜛䠄92䠅䚹 䛭䛧䛶䠈䛣䜜䜙䛾ཎయ䛾ឤᰁ䝰䝕䝹䛸䛧䛶Citrobacter (C.) rodentiumឤᰁື≀䛜୍⯡ ⓗ䛻⏝䛔䜙䜜䛶䛔䜛䠄71䠅䚹䛭䛾⌮⏤䛸䛧䛶䠈C. rodentium䛿䝬䜴䝇䛾⤖⭠䛻᥋╔䛧A/E lesions 䛸ྠᵝ䛾ኚ䜢ᘬ䛝㉳䛣䛩䛣䛸䛜▱䜙䜜䛶䛚䜚䠄5, 58, 87, 88䠅䠈 䛣䛾ኚ䛜 EPEC 䜔EHEC 䛜䝠䝖䛻ᑐ䛧䛶ㄏⓎ䛩䜛䜒䛾䛸㓞ఝ䛧䛶䛔䜛䛣䛸䛜ᣲ䛢䜙䜜䜛䠄103䠅䚹䛣䜜䛿䠈
C. rodentium䛜EPEC 䜔 EHEC 䛸ྠᵝ䛻 locus for enterocyte effacement䠄LEE䠅䛸
䛔䛖㑇ఏᏊᗙ䜢ᣢ䛴䛯䜑䠈ཎᛶ䛻㛵䛩䜛䜶䝣䜵䜽䝍䞊䝍䞁䝟䜽㉁䛜㢮ఝ䛧䛶䛔䜛䛣䛸䛻
36 EHEC 䛺䛹䛾⭠⟶ឤᰁ⣽⳦䛻ᑐ䛧䛶 KB290 䛜ᢠឤᰁస⏝䜢Ⓨ䛩䜛䛛ྰ䛛᳨ウ䛧䛯䚹 ๓㏙䛾㏻䜚C. rodentium䛻䜘䜛ែ䛿䠈C. rodentium䛜⭠ୖ⓶䛻᥋╔䠈ቑṪ䛩䜛 䛣䛸䛛䜙ጞ䜎䜛䚹䛭䛾䛯䜑䠈⭠ෆ䛷䛾C. rodentium䛷䛾ᣲື䜢ᢕᥱ䛩䜛䛣䛸䛿ᢠឤᰁస ⏝䛾᭷↓䜔䛭䛾䝯䜹䝙䝈䝮䜢ホ౯䛩䜛ୖ䛷㔜せ䛷䛒䜛䚹C. rodentium䛾⭠ෆ䛷䛾ᣲື䛾 ホ౯ᣦᶆ䛸䛧䛶䛿⣅౽୰䜎䛯䛿⭠⤌⧊୰䛾C. rodentiumᩘ䛜୍⯡ⓗ䛻⏝䛔䜙䜜䛶䛔 䜛䚹⣅౽୰䛾⳦ᩘ䛿㠀くⓗ䛛䛴⤒ⓗ䛻 ᐃ䛷䛝䜛䜒䛾䛾䠈C. rodentium䛜⭠䛻ᙉ 䛟᥋╔䛧ἥ䛥䜜䛺䛔ሙྜ䠈౽䛻C. rodentium䛜Ṛ⁛䛧䛯ሙྜ䠈䛒䜛䛔䛿ᦤ㣵┤ᚋ 䛸✵⭡䛺䛹⣅౽䛾⤌ᡂ䛜䛝䛟␗䛺䜛ሙྜ䛺䛹䠈ᚲ䛪䛧䜒⣅౽୰䛾⳦ᩘ䛜⭠ෆ䛾⳦ᩘ䜢 ᫎ䛧䛶䛔䜛䛸䛿㝈䜙䛺䛔䚹୍᪉䠈⭠⤌⧊୰䛾⳦ᩘ䛿䠈⭠䛻Ꮡᅾ䛩䜛⳦ᩘ䜢┤᥋ⓗ䛻 ィ 䛩䜛䛣䛸䛜ྍ⬟䛷䛒䜛䛜䠈 ᐃ䛩䜛䛯䜑䛻䛿䝬䜴䝇䜢ᏳᴦṚ䛥䛫䜛ᚲせ䛜䛒䜛䛯䜑䠈⤒ ⓗ䛻 ᐃ䛩䜛ሙྜ䛿ྛホ౯䝍䜲䝭䞁䜾䛾䛯䜑䛾⩌タᐃ䛜ᚲせ䛷䛒䜛䚹䜎䛯䠈⣅౽䠈⭠⤌ ⧊䛾䛔䛪䜜䛾ሙྜ䛻䛚䛔䛶䜒䠈ホ౯䝃䞁䝥䝹䜢⭠ෆ⣽⳦ศ㞳⏝ᇵᆅ䛻䛶24 㛫⛬ᗘᇵ 㣴䛩䜛ᚲせ䛜䛒䜛䛯䜑⤖ᯝ䛜ฟ䜛䜎䛷㛫䜢せ䛩䜛䚹䛭䛣䛷ᮏ◊✲䛷䛿䠈㠀くⓗ䛛䛴㎿ ㏿䛻⭠ෆ䛾C. rodentium䛾ᣲື䜢ᢕᥱ䛩䜛᪉ἲ䛸䛧䛶⏕≀Ⓨග䜲䝯䞊䝆䞁䜾
䠄bioluminescent imaging; BLI䠅ἲ䜢⏝䛔䛯䚹
BLI ἲ䛿䠈⏕యෆ䛻䛚䛡䜛㑇ఏᏊ䜔䝍䞁䝟䜽㉁䛾Ⓨ⌧䠈⣽⳦ឤᰁ䛾⛬ᗘ䛺䛹䜢యእ䛛䜙
䝸䜰䝹䝍䜲䝮䛻ホ౯䛷䛝䜛ᡭἲ䛷䛒䜛䚹㑇ఏᏊ䜔䝍䞁䝟䜽㉁䠈⣽⳦䛺䛹䛻Ⓨග䝬䞊䜹䞊䜢
37
䛿⌧ᅾ䠈䛜䜣㌿⛣䠈⸆ຠ◊✲䜢䛿䛨䜑䠈ឤᰁ䠈⛣᳜䞉⏕་⒪䠈㑇ఏᏊ⒪䛺䛹䛾in
vivo◊✲䛻ᖜᗈ䛟⏝䛔䜙䜜䛶䛚䜚䠈C. rodentium䜢ྵ䜑䠈ᵝ䚻䛺⣽⳦䛾⏕యෆ䛷䛾ᣲື
䜢ᢕᥱ䛩䜛┠ⓗ䛷䜒ከ䛟䛾◊✲䛷⏝䛔䜙䜜䛶䛔䜛ᐇ⦼䛜䛒䜛䠄82, 107䠅䚹୍⯡ⓗ䛻Ⓨග⣽
⳦䛾స〇᪉ἲ䛸䛧䛶䠈䝩䝍䝹⏤᮶䛾䝩䝍䝹䝹䝅䝣䜵䝷䞊䝊㑇ఏᏊ䜢ᑟධ䛩䜛᪉ἲ䛸䠈Ⓨග⣽
⳦䛷䛒䜛Photorhabdus luminescens⏤᮶䛾Ⓨග㑇ఏᏊ⩌䛷䛒䜛lux operon 䜢⤌䜏㎸
䜐᪉ἲ䛜▱䜙䜜䛶䛔䜛䚹๓⪅䛿䝹䝅䝣䜵䝷䞊䝊䛾ᇶ㉁䛷䛒䜛䝹䝅䝣䜵䝸䞁䜢እⓗ䛻ῧຍ䛧䛺
䛡䜜䜀䛺䜙䛺䛔䛾䛻ᑐ䛧䠈ᚋ⪅䛾lux operon 䜢⏝䛔䛯᪉ἲ䛷䛿ෆᅾᛶ䛾≀㉁䛜ᇶ㉁䛻⏝
䛔䜙䜜䜛䛯䜑ᇶ㉁䛾ῧຍ䛜ᚲせ䛺䛔䚹䛧䛯䛜䛳䛶䠈Ⓨග⣽⳦䛾ከ䛟䛿ᚋ⪅䛾lux operon ᑟ
ධ䛜᥇⏝䛥䜜䛶䛔䜛䠄40, 45, 62, 83, 94䠅䚹䛭䛣䛷ᮏ◊✲䛷䜒䛣䛾᪉ἲ䜢᥇⏝䛩䜛䛣䛸䛸䛧䠈
ឤᰁ䛥䛫䜛C. rodentium䛸䛧䛶䠈lux operon 䜢┦ྠ⤌䛘䛻䜘䜚 16S rDNA 䛻⤌䜏㎸䜣
䛰C. rodentium ICC169::p16Slux (C. rodentium lux) 䜢⏝䛔䛯䠅䚹䛣䛾⳦ᰴ䛿Ⓨග䛩
䜛䛸䛔䛖Ⅼ௨እ䛿䠈㔝⏕ᰴ䛸ẚ㍑䛧䛶⏕⫱㏿ᗘ䠈䝬䜴䝇䜈䛾ឤᰁ⬟ຊ䠈ឤᰁ㒊䛻ᕪ␗䛜䛺
䛔䛣䛸䛜ド᫂䛥䜜䛶䛔䜛䠄82䠅䚹
௨ୖ䜘䜚ᮏ◊✲䛷䛿䠈C. rodentiumឤᰁ䝬䜴䝇䛻ᑐ䛩䜛KB290 䛾ຠᯝ䜢䠈Ⓨග䛩䜛
C. rodentiumᰴ䛷䛒䜛C. rodentium lux 䜢⏝䛔䛯 BLI ἲ䛻䜘䜚᳨ウ䛧䛯䚹䛥䜙䛻䛭䛾䝯
䜹䝙䝈䝮䜢᳨ウ䛩䜛䛯䜑䠈⤖⭠䛻䛚䛡䜛C. rodentiumឤᰁ㜵ᚚ䛻㛵䜟䜛ྛ㑇ఏᏊⓎ⌧㔞
䜢ホ౯䛧䛯䚹䛺䛚䠈C. rodentiumឤᰁ䛻⏝䛔䜛⁐፹䛸䛧䛶୍⯡ⓗ䛻䛿1×PBS 䛜⏝䛔䜙䜜
38 య䛜ㄆ䜑䜙䜜䛯䠄䝕䞊䝍♧䛥䛪䠅䚹䛣䜜䛿䠈䝬䜴䝇䛾⫶㓟䛻䜘䛳䛶C. rodentium䛜Ṛ⁛䛧䛯 䛣䛸䛜ཎᅉ䛸ண䛥䜜䛯䚹䛭䛣䛷ᮏ◊✲䛷䛿䠈➨୍䛻Ᏻᐃ䛧䛶C. rodentiumឤᰁ䜢ᡂ❧䛥 䛫䜛䛾䛻㐺䛧䛯⁐፹䜢᳨ウ䛧䠈ḟ䛻䛭䛾᮲௳䜢⏝䛔䛶C. rodentium ឤᰁ䛻ᑐ䛩䜛 KB290 䛾ຠᯝཬ䜃䝯䜹䝙䝈䝮䜢ホ౯䛧䛯䚹 ➨ ➨2 ⠇㻌 ᮦᩱ䛸᪉ἲ 2-䠍䠊⏝ື≀ 5 㐌㱋䛾䝯䝇䛾 C57BL/6 䝬䜴䝇䠄᪥ᮏ䜽䝺䜰ᰴᘧ♫䠈ᮾி䠅䜢⏝䛔䠈1 㐌㛫䛾㥆ᮇ 㛫䛾ᚋ䠈ྛヨ㦂䛻⏝䛧䛯䚹䛺䛚䠈ඹྠ䛷ヨ㦂䜢ᐇ䛧䛯㟼ᒸ┴❧Ꮫ䛾㐣ཤ䛾᳨ウ䛻 䜘䜚䠈䛾⣔⤫䛻ẚ䜉䛶C57BL/6 䝬䜴䝇䛻䛚䛔䛶C. rodentiumឤᰁ䛜᭱䜒Ᏻᐃⓗ䛻 ᡂ❧䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛔䛯䛣䛸䠈䛥䜙䛻䝯䝇䛷䛿䜸䝇䛸ẚ㍑䛧䛶䝣䜯䜲䝔䜱䞁䜾䛜㉳䛣 䜚䛻䛟䛔䛣䛸䛛䜙䠈ᮏ⣔⤫䛾䝯䝇䜢㑅ᢥ䛧䛯䚹 㣫⫱ᮇ㛫୰䠈⢭〇㣫ᩱ䠄AIN-93䠈᪥ᮏ䜽䝺䜰ᰴᘧ♫䠈ᮾி䠅䛸Ỉ䜢⮬⏤ᦤྲྀ䛷 䛘䠈12 㛫䛾᫂ᬯ䝃䜲䜽䝹䠈 ᗘ 23 ± 2Υ䠈‵ᗘ 50 ± 10䠂䛾⎔ቃ䛷㣫⫱䛧䛯䚹䛺䛚䠈 ᮏヨ㦂䛿㟼ᒸ┴❧Ꮫ㣗ရᰤ㣴⛉Ꮫ㒊㣗ရ⏕⛉Ꮫ⛉ᚤ⏕≀Ꮫ◊✲ᐊ䛻䛶ᐇ䛧䠈 䛭䛾ィ⏬䛿㟼ᒸ┴❧Ꮫື≀ᐇ㦂ጤဨ䛷ᢎㄆ䜢ཷ䛡䛯䠄ᢎㄆ␒ྕ䠖135006䠅䚹
39
2-2䠊ⓎගCitrobacter rodentium ICC169::p16Slux 䛾ㄪ〇
䜾䝸䝉䝻䞊䝹䝇䝖䝑䜽䛸䛧䛶ಖᏑ䛧䛯C. rodentium lux 䜢 250 μg/mL 䜶䝸䝇䝻䝬䜲䝅䞁 䠄ග⣧⸆ᕤᴗᰴᘧ♫䠈㜰䠅ῧຍLB ᐮኳᇵᆅ䛻ሬᕸ䛧䠈䜲䞁䜻䝳䝧䞊䝍䞊䛻䛶 37ΥዲẼ᮲௳ୗ䛷୍ᬌᇵ㣴䛧䛯䚹ᚓ䜙䜜䛯䝁䝻䝙䞊䜢 4 mL 䛾 250 μg/mL 䜶䝸䝇䝻䝬䜲 䝅䞁ῧຍLB ᾮయᇵᆅ䛻᳜⳦䛧䠈37Υ䛷୍ᬌ䛸䛖ᇵ㣴䛧䛯䚹䛥䜙䛻䠈᪂䛧䛔 250 μg/mL 䜶䝸䝇䝻䝬䜲䝅䞁ῧຍLB ᾮయᇵᆅ 50 ml 䛻ᇵ㣴ᾮ䜢 2%䠄v/v䠅ῧຍ䛧䠈3 㛫䠈37Υ䛷 䛸䛖ᇵ㣴䛧䛯䚹ᇵ㣴ᾮ䜢4Υ䠈2,500 × g䠈10 ศ㛫㐲ᚰ䛧䠈ୖΎ䜢㝖ཤ䛧䛯䚹ᚓ䜙䜜䛯 C. rodentium lux 䛾䝨䝺䝑䝖䜢 1×PBS䠄0.2 g KH2PO4䠈0.2 g KCl䠈1.15 g Na2HPO4䠈8.0 g NaCl 䜢 1.0 L 䛾␃Ỉ䛻⁐ゎ䠅䛷 2 ᅇὙί䛧䠈1×PBS䠈10×PBS 䠄2.0 g KH2PO4䠈0.2 g KCl䠈11.5 g Na2HPO4䠈8.0 g NaCl 䜢 1.0 L 䛾␃Ỉ䛻⁐ ゎ䠅䠈䜎䛯䛿3%䠄w/v䠅Ⅳ㓟Ỉ⣲䝘䝖䝸䜴䝮䠄NaHCO3䠅䠄ග⣧⸆ᕤᴗᰴᘧ♫䠅Ỉ⁐ᾮ 䛷᭱⤊ⓗ䛻2×1010 cfu/mL 䛸䛺䜛䜘䛖䛻ㄪ〇䛧䛯䚹 2-3䠊KB290 䛾ㄪ〇 䜾䝸䝉䝻䞊䝹䝇䝖䝑䜽䛸䛧䛶ಖᏑ䛧䛯KB290 䜢 MRS ᐮኳᇵᆅ䠄䝯䝹䜽ᰴᘧ♫䠈ᮾி䠅 䛻ሬᕸ䛧䠈30Υ䠈ዲẼ᮲௳ୗ䛷 48 㛫ᇵ㣴䛧䛯䚹䛥䜙䛻䠈ᚓ䜙䜜䛯䝁䝻䝙䞊䜢 50 mL 䛾 MRS ᾮయᇵᆅ䠄䝯䝹䜽ᰴᘧ♫䠅䛻᳜⳦䛧䠈30Υ䠈ዲẼ᮲௳ୗ䛷୍ᬌᇵ㣴䛧䛯䚹ᇵ㣴ᾮ 䜢4Υ䠈2,500 × g䠈10 ศ㛫㐲ᚰ䛧䠈1×PBS 䛷 2 ᅇὙί䛧䛯ᚋ䠈1×1010 cfu/mL 䛸䛺䜛
40
䜘䛖䛻1×PBS 䛷ㄪ〇䛧䛯䚹
2-4䠊ື≀ヨ㦂
2-4-1. C. rodentium lux ᢞ䛻⏝䛔䜛⁐፹䛾᳨ウ
᳨ウ⁐፹䛸䛧䛶䠈1×PBS䠈10×PBS䠈䛺䜙䜃䛻⭠⟶ឤᰁ⣽⳦䛷䛒䜛Salmonella
enterica serovar Enteritidis 䜔Escherichia coli䛾ឤᰁᐇ㦂䛻⏝䛥䜜䛯ሗ࿌
䠄36䠅䛜䛒䜛 3%䠄w/v䠅NaHCO3Ỉ⁐ᾮ䜢⏝䛧䛯䚹䝬䜴䝇䜢1 㐌㛫㡰䛧䛯
ᚋ䠈1×PBS 䠈10×PBS 䜎䛯䛿 3%䠄w/v䠅 NaHCO3Ỉ⁐ᾮ䛷2 × 1010 cfu/mL 䛾⃰
ᗘ䛻ᠱ⃮䛧䛯C. rodentium lux ᾮ䜢 1 ༉䛒䛯䜚 100 μL䠄2×109 cfu/䝬䜴䝇䠅ᙉไ⤒ཱྀ
ᢞ䛧䠈䛭䛾┤ᚋ䛻䠈䜲䝋䝣䝹䝷䞁䠄䝬䜲䝷䞁〇⸆ᰴᘧ♫䠈ᮾி䠅䛾྾ධ㯞㓉䠄ᑟධ⃰
ᗘ4䡚5%䠈⥔ᣢ⃰ᗘ 1䡚3%䠅䛻䜘䜚䝬䜴䝇䜢㙠㟼䛥䛫䛯䚹䝡䝙䞊䝹䝔䞊䝥䛷ᡭ㊊䜢
ᅛᐃ䛧䛶䠈䝬䜴䝇⤌⧊䜢㏱㐣䛧䛯ᚤᙅ䛺C. rodentium lux ⏤᮶䛾Ⓨග䜢䠈⏕≀Ⓨග
ᐃ⨨䠄IVIS Imaging System Xenogen Co., Alameda, CA, USA䠅䛷᳨ฟ䛧䛯䚹
ᮏヨ㦂䛷䛿䠈䝬䜴䝇⭡㒊䛾㛗᪉ᙧ䛷ᅖ䛳䛯㒊ศ䠄⦪⣙3 cm×ᶓ⣙ 2 cm䠅䜢㛵ᚰ㡿ᇦ
䠄Region of Interest; ROI䠅䛸䛧䠄Fig. 9䠅䠈ROI 䛾 1 ศ㛫䛾Ⓨගᙉᗘ䛛䜙䠈Living
Image Software䠄version 3.0䠈Xenogen Co.䠅䜢⏝䛔䛶䠈Ⓨගᙉᗘ䠄photons/s/cm2䠅
41 2-4-2. KB290 䛜C. rodentiumឤᰁ䛻䛘䜛ᙳ㡪䛾᳨ウ ື≀ヨ㦂䛾ヨ㦂䝇䜿䝆䝳䞊䝹䜢Fig. 10 䛻♧䛧䛯䚹ヨ㦂䛿ヨ㦂 1 䛸ヨ㦂 2 䛾 2 ᅇᐇ 䛧䠈ヨ㦂1 䛷䛿 KB290 䛾ᢞᮇ㛫䜢 10 ᪥㛫䛸䛧䠈ᢞ䛛䜙 7 ᪥┠䛻 C. rodentiumឤᰁ䠈10 ᪥┠䠄ឤᰁ 3 ᪥ᚋ䠅䛻ゎ๗䜢⾜䛳䛯䚹ヨ㦂 2 䛷䛿ᢞᮇ㛫 䜢15 ᪥㛫䛸䛧䠈ᢞ䛛䜙 7 ᪥┠䛻C. rodentiumឤᰁ䠈15 ᪥┠䠄ឤᰁ 8 ᪥ᚋ䠅䛻ゎ๗ 䜢⾜䛳䛯䚹ヲ⣽䜢௨ୗ䛻♧䛩䚹 ᢞᮇ㛫୰䛻䛿䠈㠀ឤᰁ⩌䛚䜘䜃ᑐ↷⩌䛻䛿1×PBS 䜢䠈KB290 ᢞ⩌䛻䛿 KB290 ⁐ᾮ䜢䠈1 ༉䛒䛯䜚 100 μL䠄1 × 109 cfu/䝬䜴䝇䠅䛪䛴䠈ẖ᪥ᙉไ⤒ཱྀᢞ䛧 䛯䚹1×PBS 䛚䜘䜃 KB290 ⁐ᾮᢞ㛤ጞ䛛䜙 7 ᪥ᚋ䛻䠈ᑐ↷⩌䛚䜘䜃 KB290 ᢞ
⩌䛻䛿3%䠄w/v䠅NaHCO3Ỉ⁐ᾮ䛷2×1010 cfu/mL 䛻ᠱ⃮䛧䛯C. rodentium lux
ᾮ100 μL䠄2×109 cfu/䝬䜴䝇䠅䜢ᙉไ⤒ཱྀᢞ䛧ឤᰁ䛥䛫䛯䚹㠀ឤᰁ⩌䛻䛿 3%䠄w/v䠅
NaHCO3Ỉ⁐ᾮ䜢100 μL ᢞ䛧䛯䚹ឤᰁ᪥䛛䜙ゎ๗᪥䜎䛷䠈య㔜 ᐃ䛚䜘䜃⏕≀
Ⓨග ᐃ⨨䛻䜘䜛Ⓨගほᐹ䜢ẖ᪥⾜䛳䛯䚹ヨ㦂1 䛷䛿ឤᰁ 3 ᪥ᚋ䛻䠈ヨ㦂 2 䛷䛿
ឤᰁ8 ᪥ᚋ䛻䠈䝬䜴䝇䜢䜲䝋䝣䝹䝷䞁䛷㯞㓉ᚋ㢕᳝⬺⮻䛻䛶ᏳᴦṚ䛥䛫䠈┣⭠䛛䜙⫠㛛
㒊䜎䛷䜢ฟ䛧䠈㐲⤖⭠䠄⫠㛛㒊䛛䜙20䡚30 mm 䛾㒊ศ䠅䜢⤌⧊⏝ RNA Ᏻᐃ
⁐ᾮ䠄RNA later® Solution, Ambion, USA䠅䛻ᾐₕ䛧䛯䚹ᾐₕ䛧䛯⤖⭠䛿䠈4Υ䛷 24
42
2-5䠊㑇ఏᏊⓎ⌧ゎᯒ
Total RNA 䛾ᢳฟ䛻䛿 NucleoSpin RNAϩ䠄MACHEREY-NAGEL GmbH & Co.
KG, Düren, Germany䠅䜢⏝䛔䠈ᒓ䛾䝥䝻䝖䝁䞊䝹䛻ᚑ䛳䛶ᐇ䛧䛯䚹ᅇ䛧䛯 RNA
䛿⏝䛩䜛䜎䛷-80Υ䛻䛶ಖᏑ䛧䛯䚹cDNA 䛾ㄪ〇䛻䛿 PrimeScript RT reagent Kit
䠄䝍䜹䝷䝞䜲䜸ᰴᘧ♫䠈㈡䠅䜢⏝䛔䠈ᒓ䛾䝥䝻䝖䝁䞊䝹䛻ᚑ䛳䛶⾜䛳䛯䚹㏫㌿ᛂ
䛿䠈37Υ䛷 15 ศ䠈㏫㌿㓝⣲䛾⇕ኚᛶ䜢 85Υ䛷 5 ⛊䛾᮲௳䛷⾜䛳䛯䚹
⤖⭠䛻䛚䛡䜛⅖ᛶ䝃䜲䝖䜹䜲䞁䛚䜘䜃䝍䜲䝖䝆䝱䞁䜽䝅䝵䞁㛵㐃ᅉᏊ䛾mRNA 㔞䜢䠈
Thermal Cycler Dice Real Time System䠄䝍䜹䝷䝞䜲䜸ᰴᘧ♫䠅䜢⏝䛔䛯䝸䜰䝹䝍䜲
䝮PCR 䛻䛶 ᐃ䛧䛯䚹⅖ᛶ䝃䜲䝖䜹䜲䞁㑇ఏᏊ䛸䛧䛶 tumor necrosis factor-alpha
䠄TNF-α䠅䠈interleukin-1 beta䠄IL-1β䠅䠈interleukin-6䠄IL-6䠅䠈interleukin-12 p40
䠄IL-12 p40䠅䠈interferon-gamma䠄IFN-γ䠅䜢䠈䝍䜲䝖䝆䝱䞁䜽䝅䝵䞁㛵㐃ᅉᏊ䛸䛧䛶
occludin 䛚䜘䜃 zona occludens protein 1䠄ZO-1䠅䜢 ᐃ䛧䛯䚹Table 3 䛻䛿ྛ㑇ఏᏊ
Ⓨ⌧䛾 ᐃ䛻⏝䛔䛯䝥䝷䜲䝬䞊㓄ิ䜢♧䛧䛯䚹ෆ㒊ᶆ‽䛻䛿β-actin 䜢⏝䛔䠈2-ΔΔCtἲ
䠄57䠅䛻䛶㠀ឤᰁ⩌䛾್䛻ᑐ䛩䜛┦ᑐ್䛸䛧䛶ྛ⩌䛾Ⓨ⌧㔞䜢⟬ฟ䛧䛯䚹
2-6䠊⤫ィゎᯒ
⤖ᯝ䛿䛩䜉䛶ᖹᆒ್䠇ᶆ‽ㄗᕪ䛸䛧䛶♧䛧䠈n ᩘ䛿ື≀䛾ᩘ䛸䛧䛯䚹C. rodentium
43 㑇ఏᏊⓎ⌧㔞䛾ゎᯒ䛻䛿 Steel-Dwass ἲ䜢䛭䜜䛮䜜⏝䛔䛯䚹P ್䛜 0.05 ᮍ‶䛾䛻 ⤫ィᏛⓗ䛻᭷ពᕪ䛜䛒䜛䛸ุ᩿䛧䛯䚹 ➨ ➨3 ⠇㻌 ⤖ᯝ 3-䠍䠊C. rodentium lux ᢞ䛻⏝䛔䜛⁐፹䛾᳨ウ ྛ⁐፹䛷ㄪ〇䛧䛯C. rodentium lux 䜢ᢞ䛧䛯┤ᚋ䛾䝬䜴䝇䜢⏕≀Ⓨග ᐃ⨨䛻 䛶ほᐹ䛧䛯⏬ീ䜢Fig. 11A 䛻♧䛧䛯䚹1×PBS 䜎䛯䛿 10×PBS 䜢⏝䛔䛯⩌䛾୰䛻䛿Ⓨ ග䛜ほᐹ䛥䜜䛺䛔ಶయ䛜Ꮡᅾ䛧䛯䛾䛻ᑐ䛧䠈3%䠄w/v䠅NaHCO3Ỉ⁐ᾮ䜢⏝䛔䛯⩌䛷䛿 䛔䛪䜜䛾䝬䜴䝇䛷䜒Ⓨග䛜☜ㄆ䛥䜜䛯䚹䜎䛯䠈Ⓨගᙉᗘ䜢⩌㛫䛷ẚ㍑䛩䜛䛸䠈᭷ពᕪ䛿ㄆ 䜑䜙䜜䛺䛛䛳䛯䜒䛾䛾䠈3%䠄w/v䠅NaHCO3Ỉ⁐ᾮ⩌䛿䛭䛾䛾⩌䜘䜚䜒㧗䛔್䜢♧䛧䛯
䠄Fig. 11B䠅䚹௨ୖ䜘䜚䠈C. rodentium lux 䜢ᠱ⃮䛩䜛⁐፹䛸䛧䛶 3%䠄w/v䠅NaHCO3Ỉ
⁐ᾮ䛜㐺䛧䛶䛔䜛䛸ุ᩿䛧䠈௨㝆䛾ヨ㦂䛷䛿3%䠄w/v䠅NaHCO3Ỉ⁐ᾮ䜢⁐፹䛻⏝䛔
䛯䚹
3-2䠊KB290 䛜C. rodentiumឤᰁ䛻䛘䜛ᙳ㡪䛾᳨ウ
3-2-1. C. rodentium lux Ⓨගᙉᗘ
ヨ㦂䠍䠄ឤᰁ3 ᪥ᚋ䛻ゎ๗䠅䛻䛚䛡䜛C. rodentium lux ឤᰁ 3 ᪥ᚋ䜎䛷䛾⏕≀Ⓨ
44 ᑐ↷⩌䛚䜘䜃KB290 ᢞ⩌䛾䛔䛪䜜䛻䛚䛔䛶䜒ឤᰁ᪥䛻䛚䛔䛶ಶయ䛾⭡㒊䛷Ⓨ ග䛜☜ㄆ䛥䜜䛯䛣䛸䛛䜙䠈C. rodentium lux 䜢Ᏻᐃⓗ䛻ឤᰁ䛥䛫䜛䛣䛸䛜䛷䛝䛯䛸⪃䛘 䜙䜜䛯䚹 ឤᰁ᪥䛻ẚ䜉ឤᰁ1 ᪥ᚋ䛻Ⓨගᙉᗘ䛜㢧ⴭ䛻పୗ䛧䠈䛭䛾ᚋᚎ䚻䛻ୖ᪼䛧䛯⌮⏤ 䛸䛧䛶䛿䠈䜋䛸䜣䛹䛾C. rodentium lux 䛜ឤᰁ┤ᚋ䛻ἥ䛥䜜䠈ἥ䛥䜜䛪䛻⭠䛻 ᐃ╔䛧䛯C. rodentium lux 䛜ᚎ䚻䛻ቑṪ䛧䛯䛯䜑䛰䛸⪃䛘䜙䜜䛯䚹䛧䛛䛧䛺䛜䜙䠈䛣䛾 Ⓨගᙉᗘୖ᪼䛾⛬ᗘ䛿⩌㛫䛻ᕪ䛜䛒䜚䠈ឤᰁ2䠈3 ᪥ᚋ䛻䛚䛔䛶 KB290 ᢞ⩌ࡢⓎ ගᙉᗘ䛜ᑐ↷⩌䛻ᑐ䛧᭷ព䛻ప್䜢♧䛧䛯䚹 ヨ㦂2䠄ឤᰁ 8 ᪥ᚋ䛻ゎ๗䠅䛻䛚䛡䜛 IVIS 䛾⏬ീ䛚䜘䜃Ⓨගᙉᗘ䜢䠈Fig. 12 䛸ྠ ᵝ䛻Fig. 13A, B 䛻♧䛧䛯䚹ヨ㦂䠍䛸ྠᵝ䠈ឤᰁึᮇ䠄ឤᰁ 1䡚3 ᪥ᚋ䠅䛻䛚䛔䛶 KB290 ᢞ⩌ࡢⓎගᙉᗘ䛜ᑐ↷⩌䛸ẚ㍑䛧䛶᭷ព䛻ప್䜢♧䛧䠈ᮏ⤖ᯝ䛾⌧ᛶ䛜 ☜ㄆ䛷䛝䛯䚹䛥䜙䛻䠈ឤᰁ4 ᪥ᚋ௨㝆䛻䛚䛔䛶䜒䠈᭷ពᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䜒䛾䛾 KB290 ᢞ⩌䛾Ⓨගᙉᗘ䛿ᑐ↷⩌䛸ẚ㍑䛧䛶ప䛔ഴྥ䜢♧䛧䛯䠄Fig. 13B, p < 0.1 on Day 6, 8䠅䚹 3-2-2. య㔜 ឤᰁᚋ䛾య㔜ኚ䛻䛴䛔䛶䠈ヨ㦂1 䛚䜘䜃ヨ㦂 2 䛾⤖ᯝ䜢ᅗ 14A 䛚䜘䜃 14B 䛻 䛭䜜䛮䜜♧䛧䛯䚹ヨ㦂1 䛻䛚䛔䛶䛿䠈C. rodentium lux ឤᰁᚋ䛾య㔜䛿⩌㛫䛷᭷ព
45 䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䠄ᅗ14A䠅䚹୍᪉䠈ヨ㦂 2 䛻䛚䛔䛶䛿⩌㛫䛻ᕪ䛜ㄆ䜑䜙䜜䠈ឤ ᰁ3 ᪥ᚋ䛻䛚䛔䛶䛿ᑐ↷⩌䛜䛾 2 ⩌䛻ẚ䜉䛶᭷ព䛻ప್䜢♧䛧䠈4, 5 ᪥ᚋ䛻䛚䛔 䛶䛿KB290 ᢞ⩌䛜ᑐ↷⩌䛻ẚ䜉䛶᭷ព䛻㧗್䜢♧䛧䛯䠄ᅗ 14B䠅䚹 3-2-3. 㐲⤖⭠䛻䛚䛡䜛⅖ᛶ䝃䜲䝖䜹䜲䞁㑇ఏᏊⓎ⌧ 3-2-1 䛷ㄆ䜑䜙䜜䛯 KB290 䛾C. rodentium lux ቑṪᢚไస⏝䛾ᶵᗎ䜢᳨ド䛩䜛 䛯䜑䠈C. rodentium䛾ᶆⓗ⮚ჾ䛾୍䛴䛷䛒䜛㐲⤖⭠䛻䛚䛡䜛䠈⅖ᛶ䝃䜲䝖䜹䜲䞁 䠄Fig. 15䠅䛚䜘䜃䝍䜲䝖䝆䝱䞁䜽䝅䝵䞁㛵㐃ᅉᏊ䠄Fig. 16䠅䛾㑇ఏᏊⓎ⌧䜢 ᐃ䛧䛯䚹⅖ ᛶ䝃䜲䝖䜹䜲䞁䛻䛴䛔䛶䛿䠈ឤᰁ3 ᪥ᚋ䛻䛚䛔䛶䠈KB290 ᢞ⩌䛻䛚䛡䜛 TNF-α
䛚䜘䜃IFN-γ Ⓨ⌧㔞䛜䠈䛾⩌䛻ᑐ䛧᭷ព䛻㧗䛔್䜢♧䛧䛯䠄Fig. 15A䠅䚹୍᪉䠈ឤᰁ
8 ᪥ᚋ䛷䛿䠈IFN-γ Ⓨ⌧㔞䛜 KB290 ᢞ⩌䛻䛚䛔䛶䛾⩌䛻ᑐ䛧㧗䛔ഴྥ䛜ㄆ䜑 䜙䜜䛯䜒䛾䛾䠈᭷ព䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯䠄Fig. 15B䠅䚹䜎䛯䠈䛭䛾䛾 3 ✀䛾䝃䜲䝖 䜹䜲䞁䛻䛴䛔䛶䛿䠈ឤᰁ3 ᪥ᚋ䛚䜘䜃 8 ᪥ᚋ䛾䛔䛪䜜䛻䛚䛔䛶䜒䠈⩌㛫䛻᭷ព䛺ᕪ䛿 ㄆ䜑䜙䜜䛺䛛䛳䛯䚹୍᪉䠈䝍䜲䝖䝆䝱䞁䜽䝅䝵䞁㛵㐃ᅉᏊ䛷䛒䜛occludin 䛚䜘䜃 ZO-1 䛻 䛴䛔䛶䛿䠈ឤᰁᚋ3 ᪥ᚋ䠈8 ᪥ᚋ䛾䛔䛪䜜䛻䛚䛔䛶䜒⩌㛫䛻᭷ព䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛 䛳䛯䠄Fig. 16A, B䠅䚹
