上原記念生命科学財団研究報告集, 34 (2020)ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧

33. ᪂つⓎග࢖࣓࣮ࢪࣥࢢࢆ⏝࠸ࡓ⭘⒆ෆᚤᑠ⎔ቃࡢゎᯒ㏆⸨ ⛉Ụᮾிᕤᴗ኱Ꮫ ⏕࿨⌮ᕤᏛ㝔Key words㸸⏕యⓎග࢖࣓࣮ࢪࣥࢢ㸪㛫㉁⣽⬊㸪ᝏᛶ໬ᶵᵓ㸪ప㓟⣲ㄏᑟᅉᏊ㸦HIF㸧⥴ ゝప㓟⣲ㄏᑟᅉᏊ㸦HIF㸧ࡣࠊ⏕యࡢప㓟⣲ᛂ⟅ࢆྖࡿ㌿෗ᅉᏊ࡛ࠊⓎ⏕ࡸศ໬࡟ࡶ㔜せ࡞ᙺ๭ࢆᢸ࠺ᚲ㡲ࡢᅉᏊ࡛࠶ࡿࠋ୍᪉࡛ࠊ⭘⒆࡟࠾࠸࡚ࡣࠊ୙Ṛ໬ࠊᮍศ໬ࡢ⥔ᣢࠊ⾑⟶᪂⏕ࠊ௦ㅰࢩࣇࢺࠊቑṪᅉᏊࡢ⮬ᕫศἪࠊ៏ᛶ⅖⑕ࠊ
㌿⛣࣭ᾐ₶ࠊච␿ᢚไࠊ἞⒪᢬ᢠᛶ࡞࡝࡟㛵୚ࡍࡿከࡃࡢᅉᏊࡀ HIF ࡟ࡼࡗ࡚ㄏᑟࡉࢀࠊணᚋ୙Ⰻ࡟῝ࡃ㛵ಀࡋ࡚࠸ࡿ㹙1㹛ࠋࡀࢇ⣽⬊࡟࠾ࡅࡿ HIF άᛶࡀࠊࡀࢇࡢቑṪࡸᝏᛶ໬㐣⛬࡛㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࡇ࡜ࡣࠊከࡃࡢ◊✲࡟ࡼࡾ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࡀࠊ㛫㉁⣽⬊࡟࠾ࡅࡿᙺ๭ࡣ᫂☜࡛ࡣ࡞࠸ࠋ⭘⒆ෆ࡟ࡣࠊ⾑⟶࣭ࣜࣥࣃ⟶ࢆᙧᡂࡍࡿෆ⓶⣽⬊ࡸ࿘⓶⣽⬊ࠊ⥺⥔ⱆ⣽⬊ࠊ࠾ࡼࡧච␿⣽⬊࡞࡝ࡢከ✀ከᵝ࡞㛫㉁⣽⬊ࡀᏑᅾࡋ࡚࠸ࡿࠋ≉࡟ࠊච␿⣔ࡢ⣽⬊࡛ࡣࠊᮏ᮶ࡣ⭘⒆ࢆᨷᧁࡋᾘ⁛ࡉࡏࡿ࡭ࡁච␿ᶵ⬟ࡀᢚไࡉࢀࠊ㏫࡟⭘⒆ቑṪࡸᝏᛶ໬ࢆᡭຓࡅࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟
ࡉࢀ࡚ࡁ࡚࠸ࡿ㹙2㹛ࠋᡃࠎࡣࠊHIF άᛶࢆ Firefly luciferase-D-luciferin㸦Fluc-luci㸧ࡢ⣔ࢆ⏝࠸ࡓ⏕యⓎග࢖࣓࣮ࢪࣥࢢ࡛ྍど໬ࡍࡿࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ㸦Tg㸧࣐࢘ࢫ HOL-Tg ࢆᵓ⠏ࡋࠊⓎࡀࢇ๣࡟ࡼࡿㄏⓎࡀࢇ࡛ࡣࠊHIF άᛶࡀࠊࡀࢇ⑓ኚ⑓⌮ീࢆ࿊ࡍࡿ⤌⧊࡟࠾࠸࡚ࠊ⭘⒆Ⓨ⏕᪩ᮇ࠿ࡽ᳨ฟࡉࢀࠊ⭘⒆ࡢቑṪ࡜࡜ࡶ࡟ቑຍࡍࡿࡇ࡜ࢆሗ࿌ࡋࡓ㹙3㹛ࠋࡇࢀࡽࡢゎᯒ࡛ࠊࡀࢇࡢቑṪࡸᝏᛶ໬㐣⛬࠾࠸࡚ࠊࡀࢇ⣽⬊ࡔࡅ࡛࡞ࡃࠊࡀࢇ⣽⬊ࢆྲྀࡾᕳࡃ㛫㉁⣽⬊࡟࠾ࡅࡿ HIF άᛶࡢᐤ୚ࡀᙉࡃ♧၀ࡉࢀࡓࠋࡇࢀࡽࡢ㛫㉁⣽⬊࡜ࡀࢇ⣽⬊࡜ࡢ┦஫స⏝ࡀ⭘⒆ᝏᛶ໬࡟኱ࡁࡃᐤ୚ࡋ࡚࠾ࡾࠊච␿ᢚไ⎔ቃࡢᵓ⠏࡟ࡶ῝ࡃ㛵ࢃࡗ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࡶࡢࡢࠊ⭘⒆ᙧᡂ㐣⛬ࡢ࡝ࡢẁ㝵࡛ࠊ࡝ࡢࡼ࠺࡞┦஫స⏝ࡀ㉳ࡇࡾࠊࡑࡢ㐣⛬࡛ HIF άᛶࡀ࡝ࡢࡼ࠺࡟㛵୚ࡋ࡚࠸ࡿࡢ࠿ࡣ᫂☜࡛ࡣ࡞࠸ࠋᡃࠎࡣ᭱㏆ࠊHIF άᛶࢆྍど໬࡛ࡁࡿ Tg ࣐࢘ࢫࡢ⓶ୗ࡟ࠊ࣏࣮ࣞࢱ࣮㑇ఏᏊࢆᣢࡓ࡞࠸ࡀࢇ⣽⬊ࢆ⛣᳜ࡍࡿ࡜ࠊ⛣᳜ᚋ᪩ᮇ࠿ࡽ⭘⒆ෆ㛫㉁⣽⬊ࡢ HIF άᛶࡀほᐹࡉࢀࠊ⭘⒆ቑṪ࡜࡜ࡶ࡟ HIF άᛶࡀᒁᅾ໬ࡋ࡚ࢡࣛࢫࢱ࣮ࢆࡘࡃࡾࠊ⭘⒆඲య࡟ HIF άᛶࡀᣑ኱ࡋ࡚࠸ࡿࡇ࡜ࢆぢฟࡋࡓ㸦ᢞ✏‽ഛ୰㸧ࠋࡲࡓࠊୡ⏺᭱㧗᳨ฟឤᗘࡢ⏕యⓎග࢖࣓࣮ࢪࣥࢢᢏ⾡ AkaBLI ࢆᵓ⠏ࡋࡓ㹙4㹛ࠋࡇࡢᢏ⾡࡛ࡣࠊୡ⏺ึࡢᐇ⏝ⓗ࡞㏆㉥እⓎගᇶ㉁ Akalumine㹙5㹛࡜Akalumine ࡟᭱㐺໬ࡋࡓⓎග㓝⣲ AkaLuc ࡢ⤌ྜࡏ࡛ࠊFluc-luci ࡢ⤌ྜࡏ࡟ࡼࡿⓎගࡼࡾᩘ༓ಸ᫂ࡿ࠸Ⓨගࢆᐇ⌧ࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡲࡓࠊAkalumine ࡢ㓟ᛶ≉ᛶࢆᨵⰋࡋࡓ᪂つ㏆㉥እⓎගᇶ㉁ࡶ㛤Ⓨࡋࡓ㹙6㹛ࠋࡇࢀࡲ࡛ࡶࠊࡀࢇ㛫㉁⣽⬊࡟ࡘ࠸࡚ࡣከࡃࡢ◊✲ࡀ࡞ࡉࢀ࡚ࡁࡓࡀࠊ㛫㉁⣽⬊ࡢ HIF άᛶ࡟ὀ┠ࡋࡓ◊✲ሗ࿌ࡣ↓࠸ࠋHIF ࡣࡇࢀࡲ࡛ప㓟⣲࡜ࡢ㛵㐃࡛◊✲ࡉࢀࡿࡇ࡜ࡀከ࠸ࡀࠊ⭘⒆ᙧᡂึᮇ࡟࠾ࡅࡿ HIF άᛶ໬ᶵᵓࡣࡲࡔࡼࡃࢃ࠿ࡗ࡚࠸࡞࠸ࠋࡲࡓࠊ㛫㉁⣽⬊ࡢ౵ධࢆ⤒᫬ⓗ࡟ほᐹࡋࠊࡑࢀࡽࡢ㛫㉁⣽⬊ࢆ⥙⨶ⓗ࡟ゎᯒࡋࡓ◊✲ࡶ↓࠸ࠋᮏ◊✲ࡣࠊ࡝ࡢࡼ࠺࡞㛫㉁⣽⬊ࡀ⭘⒆ᙧᡂࡢ࡝ࡢ᫬ᮇ࡟ HIF άᛶࢆᣢࡕࠊ࡝ࡢࡼ࠺࡟⭘⒆ࡢቑṪ࣭ᝏᛶ໬࡟㛵୚ࡋ࡚࠸ࡿࡢ࠿ࢆㄪᰝࡋࠊ᭦࡟ྠᐃࡋࡓ㛫㉁⣽⬊࡜ࡀࢇ⣽⬊ࡢ┦஫స⏝ࢆゎᯒࡍࡿࡇ࡜࡛ࠊ⭘⒆ᝏᛶ໬ࡢ࣓࢝ࢽࢬ࣒ࢆ
ศᏊ࡛ࣞ࣋ࣝゎ᫂ࡋࠊ᪂ࡓ࡞἞⒪ᶆⓗࢆྠᐃࡋ࡚ࠊࡀࢇࡢ᰿἞࡟⧅ࡀࡿ἞⒪ἲࡢ㛤Ⓨࢆ⾜࠺ࡇ࡜ࢆ᭱⤊┠ⓗ࡜ࡍࡿࠋึᖺᗘ࡟࠶ࡓࡿ 2019 ᖺᗘ࡛ࡣࠊAkaLuc ࡸ Fluc Ⓨග࣏࣮ࣞࢱ࣮㑇ఏᏊࢆ⏝࠸࡚ Tg ࣐࢘ࢫࢆస〇ࡋࠊ⏕యග࢖࣓࣮ࢪࣥࢢࢆ㥑౑ࡋ࡚ࠊ⭘⒆ෆ࡟౵ධࡋ HIF άᛶࢆ᭷ࡍࡿ㛫㉁⣽⬊ࢆ㠀౵くⓗ࡟ほᐹࡍࡿࡇ࡜࡟ᡂຌࡋࡓࠋࡲࡓࠊ⾑⟶᪂⏕ࡀጞࡲࡿ๓ࡢ᪩ᮇ࡟⭘⒆ෆ࡟౵ධࡋࠊ⭘⒆ቑṪ࡟ᐤ୚ࡍࡿ㛫㉁⣽⬊ࢆྠᐃࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࢆ㋃ࡲ࠼
࡚ࠊ2020 ᖺᗘࡣࡼࡾⰋ࠸ Tg ࣐࢘ࢫࢆస〇ᵓ⠏ࡋࠊ⥙⨶ⓗゎᯒࢆ㧗࠸ಙ㢗ᛶࡶࡗ࡚㐙⾜࡛ࡁࡿ⣽⬊ᅇ཰ἲࡢ☜❧࡜ࠊin vitro ホ౯⣔ࡢ඘ᐇࢆᅗࡾࠊ᭱⤊┠ⓗࡢ㐩ᡂࢆࡵࡊࡍࠋ1ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌᪉ἲ࠾ࡼࡧ⤖ᯝ1㸬 AkaBLI ࢆ⏝࠸ࡿ HVA-Tg ࣐࢘ࢫࡢᵓ⠏AkaBLI ࢆ⏝࠸࡚ࠊ⭘⒆ෆ࡟౵ධࡋࡓ㛫㉁⣽⬊ࡢ HIF άᛶࢆࣜ࢔ࣝࢱ࢖࣒࡛㠀౵くⓗ࡟ࣔࢽࢱࣜࣥࢢࡍࡿࡓࡵ࡟ࠊHIF ౫Ꮡⓗ࡟ AkaLuc ࢆⓎ⌧ࡍࡿ Tg ࣐࢘ࢫࢆᵓ⠏ࡋࡓࠋ
1㸧࣏࣮ࣞࢱ࣮㑇ఏᏊࡢᵓ⠏࡜ホ౯HOL-Tg ࡟࠾ࡅࡿゎᯒ㹙3㹛࡛ࡣࠊⓎගᙉᗘࡀ୙༑ศ࡛࠶ࡗࡓࡇ࡜࡜ࠊex vivo ゎᯒ࡟㐺ࡋࡓ⺯ග࣏࣮ࣞࢱ࣮㑇ఏᏊࡀ↓࠿ࡗࡓࡓࡵ࡟ࠊ㛫㉁⣽⬊ࡢ༑ศ࡞ゎᯒࡀ࡛ࡁ࡞࠿ࡗࡓࠋࡑࡢࡓࡵࠊTg ࣐࢘ࢫࢆᵓ⠏ࡍࡿࡓࡵࡢⓎ⌧ࣉࣛࢫ࣑ࢻ࡜
ࡋ࡚ࠊ௒ᅇࡣ AkaLuc ࡜ Venus ࡢ⼥ྜࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ cDNA㹙4㹛ࢆ HIF ౫Ꮡⓗ࡞ࣉ࣮ࣟࣔࢱ࣮㸦pHRE㸧
ࡢୗὶ࡟⧅࠸ࡓ pHRE/ Venus-AkaLuc㸦HVA㸧ࢆᵓ⠏ࡋࡓࠋࡇࡢࣉࣛࢫ࣑ࢻࢆ HeLa ⣽⬊࡟୍㐣ᛶ࡟ᑟධࡋ࡚ࠊHIF౫Ꮡⓗ࡞࣏࣮ࣞࢱ࣮㑇ఏᏊࡢⓎ⌧ࢆ᳨ドࡋࡓࠋࡑࡢ⤖ᯝࠊప㓟⣲㸦1㸣O㸰㸧ᇵ㣴࡛ HIF άᛶࡀㄏᑟࡉࢀࡓ⣽⬊≉␗ⓗ࡟Venus ࡢ⺯ග㸦⥳㸧࡜㧗࠸Ⓨගࡀほᐹࡉࢀ㸦ᅗ 1㸧
ࠊHVA-Tg ࡢᵓ⠏࡟౑⏝࡛ࡁࡿࣉࣛࢫ࣑ࢻࡀ᏶ᡂࡋࡓࠋa.

HVA ( 5HRE-Venus-AkaLuc )৅ਠউছ५঑ॻ
pHREVenusc.

21% O2

1% O2᫂ど㔝30 µm㼂㼑 㼚 㼡 㼟

BL intensity(¼107 p / s / cm² / sr )b.

AkaLucpٛ0.01*

21% O2 1% O2

ᅗ 1㸬HVA ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢᵓ⠏࡜ホ౯a㸧  HVA ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢᵓ㐀 pHRE, HIF ౫Ꮡⓗࣉ࣮ࣟࣔࢱ࣮㸹Venus ࡜ AkaLuc ࡢ cDNA ࠿ࡽ⼥ྜࢱࣥࣃࢡ㉁࡜ࡋ࡚Ⓨ⌧ࡍࡿࠋbࠊc㸧 HVA ࢆ୍㐣ᛶ࡟ᑟධࡋࡓ HeLa ⣽⬊ࢆ㏻ᖖ㓟⣲㸦21㸣O㸰㸧࡜ప㓟⣲㸦1㸣O㸰㸧࡛ᇵ㣴ࡋ Venus ࡢⓎ⌧ࢆ⺯ග㢧ᚤ㙾㸦b㸧࡛ࠊAkaLuc ࡢⓎ⌧ࢆ(c) IVIS ࡛ほᐹࡋࡓࠋᐇ㦂ࡣ 3 ᅇ⧞ࡾ㏉ࡋࠊ௦⾲ⓗ࡞⤖ᯝࢆ♧ࡋࡓࠋྛᐇ㦂ࡣ n = 3 ࡛ᐇ᪋ࡋࠊmeansSD ࡛⤖ᯝࢆ♧ࡋࡓࠋ᭷ពᕪ᳨ᐃࡣ two-side student’s t-test ࡛ᐇ᪋ࡋࡓࠋ2㸧HVA-Tg ࡢᵓ⠏࡜ホ౯
HVA Ⓨ⌧ࣉࣛࢫ࣑ࢻࢆࠊி㒔኱Ꮫ࡟࡚ C57BL/6 background ࡢ Tg ࣐࢘ࢫࢆᵓ⠏ࡋࡓࠋ᭱ึ࡟స〇ࡋࡓ 3 ࣛ࢖ࣥࢆᮾᕤ኱࡟㍺㏦ࡋࠊ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢゎᯒࢆ⾜ࡗࡓࠋHIF ᛂ⟅ᛶࢆ☜ㄆࡍࡿࡓࡵ࡟ HIF-1Șࢱࣥࣃࢡ㉁ࢆᏳᐃ໬ࡍࡿ
ᢠ㓟໬๣ Propyl gallate㹙7㹛ࢆ⓶ୗ࡟ᢞ୚ࡋ࡚ࠊHIF άᛶ౫Ꮡⓗ࡞ AkaLuc ࡢⓎගࢆࠊⓎග࢖࣓࣮ࢪࣥࢢ⿦⨨ IVISτࢆ⏝࠸࡚ほᐹࡋࡓࠋࡑࡢ⤖ᯝࠊTg ࣐࢘ࢫࡢࡳ࡛ࠊPG ౫Ꮡⓗ࡞ⓎගࡀほᐹࡉࢀࠊHIF άᛶ౫Ꮡⓗ࡞ AkaLuc ࡢᛂ⟅ᛶࡀ☜ㄆ࡛ࡁࡓ㸦ᅗ 2㸧
ࠋ3 ࣛ࢖ࣥࡢ࠺ࡕࠊ᭱ࡶⓎගࡀᙉ࠿ࡗࡓࣛ࢖ࣥࢆ⏝࠸࡚⛣᳜ࣔࢹࣝᐇ㦂ࢆ⾜࠺ࡇ࡜࡜ࡋࡓࠋ2ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌ᅗ 2㸬HVA-Tg ࡢ HIF ᛂ⟅ᛶホ౯࣐࢘ࢫࡢ⮌㒊࡟ 10 mM PG ࢆ 100ȣL ⓶ୗ࡟ᢞ୚ࡋ 4 ᫬㛫ᚋ࡟ IVIS™࡛Ⓨග࢖࣓࣮ࢪࣥࢢࢆ⾜ࡗࡓࠋ3㸧Cre-lox ࢩࢫࢸ࣒ࢆ⏝࠸ࡓㄏᑟᆺ HVA Ⓨ⌧ࣉࣛࢫ࣑ࢻࡢᵓ⠏Venus-AkaLuc ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢୖὶ࡟ lox-STP-lox㸦LSL㸧㓄ิࢆධࢀࡿࡇ࡜࡛ࠊCre recombinase㸦CRE㸧ࡀⓎ⌧ࡋࡓ⣽⬊≉␗ⓗ࡟ࠊHIF ౫Ꮡⓗ࡟࣏࣮ࣞࢱ࣮㑇ఏᏊࡢⓎ⌧ࡀㄏᑟࡉࢀࡿ conditional reporter ࣉࣛࢫ࣑ࢻࡢᵓ⠏ࢆ⾜ࡗࡓࠋࡇࡢࣉࣛࢫ࣑ࢻࢆ HeLa ⣽⬊࡟୍㐣ᛶ࡟ᑟධࡋࠊప㓟⣲࡜ CoCl2 ࢆ⏝࠸࡚ HIF άᛶ໬ࢆࡋࡓ࡜ࡇࢁࠊ㏻ᖖ㓟⣲ୗ࡛ᇵ㣴ࡋࡓ⣽⬊㸦21㸣 O2㸧ࡸ CRE ࡢⓎ⌧ࡀ↓࠸⣽⬊㸦CRE㸦㸫㸧㸧࡜ẚ㍑ࡋ࡚ࠊCRE㸦㸩㸧ࡢ⣽⬊ࡢ᪉ࡀࠊࡑࢀࡒࢀ 18 ಸ࡜ 70 ಸ㧗࠸Ⓨගᙉᗘࢆ♧ࡋࡓ㸦ᅗ 3㸧ࠋࡋ࠿ࡋࠊlox-STP-lox 㓄ิࢆධࢀࡿ๓ࡢ HVA ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢప㓟⣲࡟࠾ࡅࡿⓎ⌧ࡢ᪉ࡀ 1.5 ಸ࡯࡝㧗ࡃ࡞ࡗ࡚࠾ࡾࠊࣉࣛࢫ࣑ࢻ࡟ᨵⰋࡢవᆅࡀ࠶ࡿ࠿ࢆ᳨ウࡋࡓᚋࠊᑗ᮶≉ᐃࡢ㛫㉁⣽⬊ࡢᶵ⬟ࢆࣔࢽࢱࣜࣥࢢࡍࡿᚲせࡀฟ࡚ࡁࡓ᫬ࡢࡓࡵ࡟ࠊconditional Tg ࣐࢘ࢫࢆస〇ࡍࡿࡇ࡜࡜ࡋࡓࠋ

ᅗ 3㸬HVA ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢᵓ⠏࡜ホ౯a)conditional reporter ࣉࣛࢫ࣑ࢻࡢᵓ㐀ࠋb)a ࡢࣉࣛࢫ࣑ࢻࢆ HeLa ⣽⬊࡟୍㐣ᛶ࡟ᑟධࡋ࡚ప㓟⣲࡜ CoCl㸰ࢆ⏝࠸࡚ HIF άᛶ໬ࡋࠊAkaLuc
ࡢⓎගࢆ IVIS™࡛ࡢほᐹ㸦ୗᅗ㸧࡜Ⓨගᙉᗘ ᐃࢆ⾜ࡗࡓࠋྛᐇ㦂ࡣ n = 3 ࡛ᐇ᪋ࡋࠊmeansSD࡛⤖ᯝࢆ♧ࡋࡓࠋⓎග࢖࣓࣮ࢪᅗࡣࠊ௦⾲ⓗ࡞⤖ᯝࢆ♧ࡋࡓࠋ3ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌2㸬⭘⒆࡟ᾐ₶ࡋࡓ㛫㉁⣽⬊ࡢ㠀౵くⓗⓎග࢖࣓࣮ࢪࣥࢢHVA-Tg ࡟࣐࢘ࢫ⫵ࡀࢇ⣽⬊ᰴ LLC ࢆྠ⣔⛣᳜ࡋ࡚ࠊ⤒᫬ⓗ࡟ IVIS™࡛⏕యⓎග࢖࣓࣮ࢪࣥࢢࢆ⾜ࡗࡓ࡜ࡇࢁࠊ⛣᳜ᚋ 7 ᪥࡟ࡣࠊAkaBLI ࡛᫂☜࡞Ⓨගࢩࢢࢼࣝࡀ☜ㄆࡉࢀࡓࠋⓎගࢩࢢࢼࣝࡣࠊ࠸ࡗࡓࢇῶᙅࡋࡓࡶࡢࡢࠊ⭘⒆ࡢ
ᡂ㛗࡜࡜ࡶ࡟ࢩࢢࢼࣝࡣᙉࡃ࡞ࡾࠊ⭘⒆ࡢ୍㒊࡛㧗࠸ࢩࢢࢼࣝࡀほᐹࡉࢀࡓ㸦ᅗ 4㸧ࠋ⭘⒆ᡂ㛗ึᮇ࡟ HIF άᛶࡀ㧗ࡃ࡞ࡿࡢࡣࠊ⾑⟶᪂⏕ࡀ┒ࢇ࡟⾜ࢃࢀࡿ࠿ࡽ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࡀࠊ௒ᚋ⾑⟶᪂⏕ᅉᏊࡢゎᯒ➼ࢆ⾜࠺ࡇ࡜࡛ࠊHIF άᛶࡀ㧗࠸㛫㉁⣽⬊ࢆྠᐃࡋ࡚࠸ࡁࡓ࠸ࠋ௒ᅇࡢゎᯒ࡛ࡣࠊ⭘⒆ෆ࡛ᒁᅾ໬ࡋࡓ㛫㉁⣽⬊࡛ HIF άᛶࡀ㧗ࡃ࡞ࡿࡇ࡜ࡀศ
࠿ࡗࡓࠋྠᵝࡢ⤖ᯝࡣࠊ௨๓ Fluc ࢆ⏝࠸ࡓ ex vivo ゎᯒ࡛ࡶほᐹࡉࢀ࡚࠾ࡾࠊ⭘⒆ቑṪึᮇ࡟ࡣ㛫㉁⣽⬊ࡀ⭘⒆ෆ࡛
ᒁᅾ໬ࡋ࡚࠸ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋᒁᅾࡍࡿ㛫㉁⣽⬊࡜ᒁᅾ໬ࡍࡿ⭘⒆ෆࡢᵓ㐀ⓗ≉ᚩࢆㄪ࡭ࡿࡇ࡜࡛ࠊ⭘⒆ᝏᛶ໬
࣓࢝ࢽࢬ࣒ࡢ᪂ࡓ࡞▱ぢࡀᚓࡽࢀࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋᅗ 4㸬㛫㉁⣽⬊࡟࠾ࡅࡿ HIF άᛶࡢྍど໬࣏࣮ࣞࢱ࣮㑇ఏᏊࡀᑟධࡉࢀ࡚࠸࡞࠸࣐࢘ࢫ⫵ࡀࢇ⣽⬊ᰴ LLC ࢆୗ⫥ࡢ⓶ୗ࡟⛣᳜ࡋࠊ⛣᳜ᚋ 7ࠊ18ࠊ28 ᪥ᚋ࡟ IVISτ࡛⭘⒆㒊఩ࡢⓎගࢆほᐹࡋࡓࠋ3㸬⭘⒆࡟ᾐ₶ࡋࡓ㛫㉁⣽⬊ࡢ⺯ග࢖࣓࣮ࢪࣥࢢⓎගࢩࢢࢼࣝࡀ㠀౵くⓗ࡟ほᐹ࡛ࡁࡓࡇ࡜࡛ࠊ⭘⒆ෆ㛫㉁⣽⬊ࡢ⤒᫬ⓗ࡞ゎᯒࢆ⾜࠸ࠊ⺯ගࢩࢢࢼࣝࢆ⏝࠸࡚ HIFάᛶࢆ᭷ࡍࡿ㛫㉁⣽⬊ࢆ FACS ࡛ࢯ࣮ࢺࡋࠊ⥙⨶ⓗ࡞ゎᯒࢆࡍࡿணᐃ࡛࠶ࡗࡓࠋࡋ࠿ࡋࠊ௒ᅇ౑⏝ࡋࡓ HVA-Tg
ࣛ࢖࡛ࣥࡣࠊ༑ศ࡞ Venus ࡢ⺯ගࢩࢢࢼࣝࢆ☜ㄆ࡛ࡁ࡞࠿ࡗࡓࠋ஦๓ࡢᇵ㣴⣽⬊ࢆ⏝࠸ࡓ࣏࣮ࣞࢱ࣮ホ౯⤖ᯝ㸦ᅗ 1㸧
࡛ࡣࠊVenus ࡢ HIF ౫Ꮡⓗ࡞Ⓨ⌧ㄏᑟ࡟ၥ㢟ࡀ↓࠿ࡗࡓࡇ࡜࠿ࡽ Tg ࡢᵓ⠏ࡢ㝿࡟ Venus ࡢ࣏࣮ࣞࢱ࣮㡿ᇦ࡟ఱࡽ࠿
ࡢኚ␗ࡀධࡗࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ࣏࣮ࣞࢱ࣮㑇ఏᏊࡀ୧᪉࡜ࡶᶵ⬟ࡍࡿ Tg ࡢస〇ࢆ⥅⥆ࡋ࡚⾜࠺ࡇ࡜࡜ࡋࡓࠋ4㸬⤌⧊㛫㉁⣽⬊ࡢゎᯒTg ࣐࢘ࢫࡢ෌ᵓ⠏ࢆ⾜࠺㛫࡟ࠊ᪤Ꮡࡢ Tg ࢆ⏝࠸࡚⛣᳜᪩ᮇ࡟⭘⒆ෆ࡟౵ධࡍࡿ⤌⧊㛫㉁⣽⬊ࡢゎᯒࢆ⾜ࡗࡓࠋලయⓗ࡟ࡣࠊGFP-Tg ࣐࢘ࢫ㹙8㹛࡟ Fluc Ⓨග࣏࣮ࣞࢱ࣮ࢆࡶࡗࡓࡀࢇ⣽⬊ࢆྠ⣔⛣᳜ࡋࠊ⭘⒆࡟౵ධࡍࡿ㛫㉁⣽⬊ࡢ⭘⒆ቑṪ࡬ࡢᙳ㡪ࢆゎᯒࡋࡓ㸦ᅗ 5㸧ࠋࡇࡢᐇ㦂⣔࡟࠾࠸࡚ࡣࠊHIF άᛶࡢゎᯒࡣ⾜࠼࡞࠸ࡀࠊ⭘⒆⾑⟶ࡀᙧᡂࡉࢀࡿ๓ࡢ⛣᳜ᚋ 3 ᪥௨ෆࡢ⭘⒆࡟౵ධࡍࡿ㛫㉁⣽⬊ࢆ GFP ⺯ග࡛ほᐹࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡗࡓࠋࡲࡓࠊ⭘⒆ᡂ㛗ࢆⓎග࡛ᐃ㔞ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ⭘⒆ษ∦ࢆస〇ࡋࠊ㛫㉁⣽⬊࣐࣮࣮࢝ࢆ⏝࠸࡚ゎᯒࡋࡓ࡜ࡇࢁࠊGFP 㝧ᛶࡢ㛫㉁⣽⬊ A ࡣࠊྠᡤ⛣᳜⭘⒆࡛⮌㒊ࡢ⓶ୗ⭘⒆࡟ẚ࡭࡚᭷ព࡟ከࡃ⭘⒆ෆ࡟౵ධࡋ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ
㛫㉁⣽⬊ A ࢆ≉␗ⓗ㜼ᐖ๣࡛Ṛ⁛ࡉࡏࠊࡀࢇ⣽⬊ࡢቑṪࢆㄪ࡭ࡿ࡜ࠊ㛫㉁⣽⬊ A ࡀ↓࠸ሙྜࡣࠊ⭘⒆ࡢᡂ㛗ࡀⴭࡋࡃ㐜ࡃ࡞ࡿࡇ࡜ࡀศ࠿ࡗࡓ㸦ᅗ 6㸧ࠋࡇࡢࡇ࡜ࡣࠊ⤌⧊㛫㉁⣽⬊ A ࡀ⭘⒆ቑṪ࡟኱ࡁࡃ㈉⊩ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ4ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌ᅗ 5㸬⭘⒆ᙧᡂึᮇ࡟⭘⒆࡟౵ධࡍࡿ㛫㉁⣽⬊ࡢゎᯒa㸧 ⭘⒆ࡢ⛣᳜㒊఩࡟࠾ࡅࡿቑṪ㏿ᗘࡢࡕࡀ࠸ࢆ⏕యⓎග࢖࣓࣮ࢪࣥࢢࢆ⏝࠸࡚ ᐃࡋࡓࠋb㸧 ⭘⒆ษ∦࡟࠾ࡅࡿ GFP 㝧ᛶࡢ㛫㉁⣽⬊ࢆච␿⤌⧊ᰁⰍࡋ࡚㆑ูࡋᩘ࠼ࡓࠋᐇ㦂ࡣ n = 5 ࡛ᐇ᪋ࡋࠊmeansSD ࡛⤖ᯝࢆ♧ࡋࡓࠋ᭷ពᕪ᳨ᐃࡣ two-side student’s t-test ࡛ᐇ᪋ࡋࡓࠋ

ᅗ 6㸬㛫㉁⣽⬊ࡢ⭘⒆ቑṪ࡬ࡢᙳ㡪a㸧 㛫㉁⣽⬊ A ࢆ≉␗ⓗ㜼ᐖ๣㸦INH㸧࡛ฎ⌮ࡋࡓ⤌⧊࡟⛣᳜ࡋࡓࡀࢇ⣽⬊ࡢቑṪࢆ 1 ᪥ᚋ࡟Ⓨග࢖࣓࣮ࢪࣥࢢ࡛ほᐹࡋࡓࠋb㸧 PBSࠊࡲࡓࡣ INH ࡛ฎ⌮ࡋࡓሙྜࡢ⭘⒆࠿ࡽࡢⓎගᙉᗘࢆࠊ1 ᪥ᚋ࡜ 3 ᪥ᚋ࡟ ᐃࡋࠊPBS ࡜ࡢẚࢆࢢࣛࣇෆ࡟♧ࡋࡓࠋᐇ㦂ࡣ 3 ᅇ⧞ࡾ㏉ࡋࠊ௦⾲ⓗ࡞⤖ᯝࢆ♧ࡋࡓࠋᐇ㦂ࡣ n = 3 ࡛ᐇ᪋ࡋࠊ
meansSD ࡛⤖ᯝࢆ♧ࡋࡓࠋ5ୖཎグᛕ⏕࿨⛉Ꮫ㈈ᅋ◊✲ሗ࿌㞟, 34㸦2020㸧㻌⪃ ᐹ㉸㧗ឤᗘ AkaBLI ࢆ⏝࠸ࡿࡇ࡜࡛ࠊHVA-Tg ࡛ࡣࠊ⛣᳜ᚋ 5 ᪥⛬ᗘࡢ⭘⒆ࡀ☜ㄆ࡛ࡁ࡞࠸᫬ᮇ࠿ࡽࠊ౵ධࡋࡓ
㛫㉁⣽⬊ࡢ HIF άᛶ࡟㉳ᅉࡍࡿⓎගࢩࢢࢼࣝࢆほᐹࡍࡿࡇ࡜࡟ᡂຌࡋࡓࠋࡋ࠿ࡋࠊ⭘⒆ෆ㛫㉁⣽⬊ࢆ FACS ᅇ཰ࡍࡿ㝿࡟ᚲせ࡞ Venus ⺯ගࡀ᳨ฟࡉࢀࡎࠊ෌ᗘ Tg ࣐࢘ࢫࡢᵓ⠏ࢆ⾜࠺ࡇ࡜࡟ࡋࡓࠋࡑࡢ㛫ࠊ⭘⒆⛣᳜┤ᚋ࠿ࡽ⾑⟶᪂⏕ࡀጞࡲࡿ⛣᳜ᚋ 3 ᪥ࡲ࡛ࡢ⭘⒆ቑṪ࡟㛵୚ࡍࡿ㛫㉁⣽⬊ࡢ᳨ฟࢆヨࡳࠊྠᐃࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡇࡢ㛫㉁⣽⬊࡜ࡀࢇ⣽⬊ࡢ┦஫స⏝ࢆゎᯒࡍࡿࡓࡵ࡟ࠊ⌧ᅾᇵ㣴⣽⬊⣔ࢆ⏝࠸ࡓࢧ࢖ࢺ࢝࢖ࣥࡸ࢚࢟ࢯࢰ࣮࣒ࡢゎᯒࢆィ⏬ࡋ࡚࠾ࡾࠊ㛫㉁⣽⬊ࡀࡀࢇ⣽⬊ࡢቑṪಁ㐍࡟స⏝ࡍࡿ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ⾜࠺ணᐃ࡛࠶ࡿࠋTg ࣐࢘ࢫࡀᵓ⠏࡛ࡁࡓࡽࠊ⾑ὶ࠿ࡽ౵ධࡍࡿ㛫㉁⣽⬊ࢆࡼࡾヲ⣽࡟ゎᯒࡍࡿࡇ࡜࡛ࠊ㛫㉁⣽⬊ࡢᝏᛶ໬࡬ࡢᐤ୚ࡀゎ࡛᫂ࡁࡿࠋᮏ◊✲ࡢᙜึࡢィ⏬㏻ࡾࠊ㛫㉁⣽⬊ࡢ⥙⨶ⓗ࡞ゎᯒࢆᐇ᪋ࡍࡿࡇ࡜࡛ࠊ⭘⒆ෆࡢᝏᛶ໬࣓࢝ࢽࢬ࣒࡟῝ࡃ࠿࠿ࢃࡗ࡚࠸ࡿ㛫㉁⣽⬊ࢆྠᐃࡋࠊච␿ᢚไ⎔ቃࡢᵓ⠏ࡢ࣓࢝ࢽࢬ࣒ࡶ᫂ࡽ࠿࡟࡞ࡿ࡜ᮇᚅࡉࢀࡿࠋࡇࢀࡽࡢ▱ぢ࡟ᇶ࡙࠸ࡓ᪂つ἞⒪ἲࡢ㛤Ⓨࡀḟࡢࢫࢸࢵࣉ࡜࡞ࡿࠋඹྠ◊✲⪅࣭ㅰ㎡ᮏ◊✲ࡢඹྠ◊✲⪅ࡣࠊி㒔኱Ꮫ࢘࢖ࣝࢫ࣭෌⏕་⛉Ꮫ◊✲ᡤ㝃ᒓ෌⏕ᐇ㦂ື≀᪋タ⤫ྜ⏕యࣉࣟࢭࢫศ㔝ࡢ㏆⸨⋞ᩍᤵ࡛࠶ࡿࠋື≀ᐇ㦂࠾ࡼࡧࠊ㑇ఏᏊ⤌᥮࠼ᐇ㦂ࡣࠊࡑࢀࡒࢀࠊᮾிᕤᴗ኱Ꮫࠊ࠾ࡼࡧி㒔኱Ꮫࡢື≀ᐇ㦂ጤဨ఍ࠊ࠾ࡼࡧ㑇ఏᏊ⤌᥮࠼ᐇ㦂➼Ᏻ඲ጤဨ఍ࡢᢎㄆࢆᚓ࡚⾜ࡗࡓࠋᩥ ⊩
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