内容の要旨, 審査の要旨広島大学(Hiroshima University)博士(歯学)Doctor of Philosophy in Dental Sciencedoctora

Yashima, Yuka

内容の要旨, 審査の要旨博士(歯学)Doctor of Philosophy in Dental Science広島大

Hiroshima University Institutional Repository

ูグᵝᘧ➨ ྕ㸦➨ ᮲➨ 㡯㸪➨ ᮲➨ 㡯㛵ಀ㸧ㄽ ᩥ ᑂ ᰝ ࡢ ⤖ ᯝ ࡢ せ ᪨༤ኈࡢᑓᨷศ㔝ࡢྡ⛠ ༤ኈ㸦 ṑᏛ 㸧Ặྡ ඵᓥ ⏤ెᏛ ఩ ᤵ ୚ ࡢ ᮲ ௳ Ꮫ఩つ๎➨ ᮲➨ ࣭㡯ヱᙜㄽ ᩥ 㢟 ┠ᣢ⥆ⓗᅽ⦰่⃭ࡀ࣐࢘ࢫ㦵⣽⬊࡟࠾ࡅࡿ RANKLࠊOPG ࠾ࡼࡧ VEGF Ⓨ⌧࡟ཬࡰࡍᙳ㡪ㄽᩥᑂᰝᢸᙜ⪅୺  ᰝ       ᩍᤵ ຍ⸨ ຌ୍        ༳ᑂᰝጤဨ       ᩍᤵ ஧ᕝ ᾈᶞ        ᑂᰝጤဨ       ᩍᤵ ᐟ༡ ▱బ        ࠝㄽᩥᑂᰝࡢ⤖ᯝࡢせ᪨ࠞṑ࡟▹ṇຊࢆ㈇Ⲵࡍࡿ࡜ࠊṑ᰿⭷ࠊ࠾ࡼࡧṑᵴ㦵࡟≀⌮ⓗ่⃭ࡀຍࢃࡾࠊᅽ㏕ഃ࡛ࡣ◚㦵⣽⬊࡟ࡼࡿ㦵྾཰ࡀࠊ≌ᘬഃ࡛ࡣ㦵ⱆ⣽⬊࡟ࡼࡿ㦵ῧຍࡀ⏕ࡌࡿࠋ◚㦵⣽⬊ࡢศ໬࡟ࡣreceptor activator of nuclear factor kappa-B ligand / osteoprotegerin  (RANKL / OPG) ⣔ࡀ㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾࠊࡲࡓ vascular endothelial growth factor (VEGF) ࡣ㦵௦ㅰ᫬࡟࠾ࡅࡿ⾑⟶᪂⏕࡜㦵྾཰ࡢ୧㠃࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿࠋ㦵ⱆ⣽⬊࡟ᑐࡍࡿᶵᲔⓗ่⃭ࡢᙳ㡪࡟ࡘ࠸࡚ࡣከࡃࡢሗ࿌ࡀ࠶ࡾࠊᮏᕝࡽࡣᶵᲔⓗఙᒎ่⃭ࡀᶵᲔཷᐜయ࡛࠶ࡿ stretch-activated channel (S-A channel)ࢆ௓ࡋ࡚㦵ⱆ⣽⬊ࡢ VEGF࠾ࡼࡧ macrophage colony-stimulating factor (M-CSF) Ⓨ⌧ࢆಁ㐍ࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡜ࡋࡓࠋ୍᪉ࠊ㦵⣽⬊ࡣ࣓࢝ࢽ࢝ࣝࢫࢺࣞࢫࢆឤ▱ࡋࠊ◚㦵⣽⬊ࡸ㦵ⱆ⣽⬊ࢆ௓ࡋࡓ㦵㔞ㄪᩚࡀ⾜ࢃࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀࠊ㦵⣽⬊࡬ࡢᅽ⦰่⃭ (compressive force : CF) ࡀࢧ࢖ࢺ࢝࢖ࣥⓎ⌧࡟୚࠼ࡿᙳ㡪ࡣᮍࡔሗ࿌ࡀ࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ࣐࢘ࢫ㦵⣽⬊ (MLO-Y4⣽⬊) ࡟࠾ࡅࡿ RANKLࠊOPG࠾ࡼࡧ VEGFࡢⓎ⌧ࢆ࣐࢘ࢫ㦵ⱆ⣽⬊ (MC3T3-E1⣽⬊) ࡜ẚ㍑ࡍࡿ࡜࡜ࡶ࡟ࠊࡑࢀࡒࢀࡢ⣽⬊࡟ᑐࡍࡿ CFࡢ㈇ⲴࡀྛᅉᏊࡢⓎ⌧࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋࡲࡓࠊS-A channelࡣᶵᲔ่⃭࡟ࡼࡗ࡚⣽⬊⭷ࡀఙᒎࡉࢀࡿࡇ࡜࡛άᛶ໬ࡉࢀࡿ࢖࢜ࣥࢳࣕࢿ࡛ࣝ࠶ࡾࠊS-A channelࡢ㜼ᐖࡀ CF㈇Ⲵ࡟ࡼࡗ࡚ RANKLࠊOPG࠾ࡼࡧ VEGFࡢⓎ⌧࡟୚࠼ࡿᙳ㡪ࢆほᐹࡋࡓࠋࡲࡎࠊMLO-Y4⣽⬊࠾ࡼࡧ MC3T3-E1⣽⬊࡟࠾ࡅࡿྛᅉᏊࡢⓎ⌧ࢆᐃ㔞ࣜ࢔ࣝࢱ࢖࣒ PCRἲ࡜ ELISAἲࢆ⏝࠸᳨࡚ウࡋࡓࠋᐃ㔞ࣜ࢔ࣝࢱ࢖࣒ PCRゎᯒࡣ୧⣽⬊࡜ࡶ࡟ 6 well plate࡟ 5.0×104 / wellࡢᐦᗘ࡛᧛✀ࡋࠊ4᪥ᚋ 80㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡗࡓ᫬Ⅼ࡛ࢺ࣮ࢱࣝ RNAࢆᅇ཰ࡋゎᯒࢆ⾜ࡗࡓࠋELISAศᯒࡣ 80㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡗࡓ୧⣽⬊ࢆ↓⾑Ύᇵᆅ࡟࡚ 24᫬㛫ᇵ㣴ࡋࠊࡑࡢᇵ㣴ୖΎࢆ⏝࠸࡚⾜ࡗࡓࠋḟ࡟ࠊ୧⣽⬊࡟ 0.5 g/cm2ࡲࡓࡣ 1.0 g/cm2ࡢ CFࢆ 1ࠊ3ࠊ6ࠊ12᫬㛫㈇ⲴࡋࡓࠋCF㈇Ⲵࡣ Tripuwabhrutࡽࡢ᪉ἲ࡟ᇶ࡙ࡁࠊ⣽⬊ᐦᗘࡀ 80㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟㐩ࡋࡓ 6 well plateᇵᆅ࡟┤ᚄ 30 mmࡢ࢞ࣛࢫࣉ࣮ࣞࢺࢆ⨨ࡁࠊࡑࡢୖ࡟ศ㖡ࢆ஌ࡏࡿࡇ࡜࡛⾜ࡗࡓࠋࡲࡓࠊ㦵⣽⬊ࡢ CFឤཷ࡟㝿ࡍࡿࢩࢢࢼࣝఏ㐩⤒㊰ࡢゎ᫂ࡢࡓࡵ S-A channelࡢ㜼ᐖ๣࡛࠶ࡿ Gd3+ࢆ⏝࠸࡚ 1.0 g/cm2CF㈇Ⲵ᫬࡛ࡢ RANKLࠊOPG࠾ࡼࡧ VEGFⓎ⌧࡟୚࠼ࡿᙳ㡪ࢆ᳨ウࡋࡓࠋGd3+ࡢ⃰ᗘࡣ㐣ཤࡢሗ࿌ࢆࡶ࡜࡟ 10 ǍMࢆ⏝࠸ࡓࠋMLO-Y4⣽⬊ࡀ 80㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟㐩ࡋࡓᚋࠊ10 ǍM Gd3+࡜ඹ࡟ 30ศ㛫ᇵ㣴ࡋࠊࡑࡢᚋ 1.0 g/cm2CFࢆ㈇Ⲵࡋࡓࠋ3᫬㛫 CFࢆ㈇Ⲵࡋࡓࡶࡢࡣࢺ࣮ࢱࣝ RNAࢆᅇ཰ࡋࡓᚋࠊᐃ㔞ࣜ࢔ࣝࢱ࢖࣒ PCRゎᯒࢆ⾜࠸ࠊ12᫬㛫 CFࢆ㈇Ⲵࡋࡓࡶࡢࡣᇵ㣴ୖΎࢆᅇ཰ᚋࠊELISAศᯒࢆ⾜ࡗࡓࠋ ࡑࡢ⤖ᯝࠊ௨ୗࡢࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ 1. MLO-Y4⣽⬊ࡣ MC3T3-E1⣽⬊࡜ẚ㍑ࡋ࡚ RANKLࠊVEGFࡢ㑇ఏᏊ࠾ࡼࡧࢱࣥࣃࢡⓎ⌧ࡀ᭷ព࡟㧗ࡃࠊOPGⓎ⌧ࡣ᭷ព࡟ప࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ 2. MLO-Y4⣽⬊࡟࠾ࡅࡿ RANKLࡢ㑇ఏᏊ࠾ࡼࡧࢱࣥࣃࢡⓎ⌧ࡣࠊ0.5 g/cm2CF⩌࡛᭷ព࡞ቑຍࢆㄆࡵࡓࡀࠊMC3T3-E1⣽⬊࡛ࡣࢥࣥࢺ࣮ࣟࣝ⩌࡜ 0.5 g/cm2CF⩌࡜ࡢ㛫࡛᭷ពᕪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ 3. MLO-Y4࠾ࡼࡧ MC3T3-E1⣽⬊࡜ࡶ࡟ 1.0 g/cm2 CF㈇Ⲵ࡟ࡼࡾ RANKLࠊVEGFࡢ㑇ఏᏊ࠾ࡼࡧࢱࣥࣃࢡⓎ⌧ࡢ᭷ព࡞ቑຍࢆㄆࡵࠊOPGⓎ⌧ࡣ᭷ព࡟ῶᑡࡋࡓࠋMLO-Y4⣽⬊࡟࠾࠸࡚ 1.0 g/cm2ࡢ CF㈇Ⲵ᫬ࡢ RANKLࠊOPGࠊVEGFࡢ㑇ఏᏊ࠾ࡼࡧࢱ ࣥࣃࢡⓎ⌧ࡣ 10 ǍMࡢ Gd3+ῧຍ࡟ࡼࡾ᭷ព࡟ᢚไࡉࢀࡓࠋ  ௨ୖࡢ⤖ᯝࡼࡾࠊᮏㄽᩥࡣ㦵⣽⬊ࡀ࣓࢝ࢽ࢝ࣝࢫࢺࣞࢫࢆ S-A channel ࢆ௓ࡋ࡚ឤ▱ࡋࠊRANKLࠊOPG ࠾ࡼࡧ VEGF ࡢⓎ⌧ࢆㄪ⠇ࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡜ࡋࠊࡑࡢ཯ᛂࡣ㦵ⱆ⣽⬊࡜ẚ㍑ࡋ࡚㗦ᩄ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋࡋࡓࡀࡗ࡚ࠊ㦵⣽⬊ࡣእ㒊࠿ࡽࡢ࣓࢝ࢽ࢝ࣝࢫࢺࣞࢫࢆཷࡅࡿࡇ࡜࡟ࡼࡾ㦵௦ㅰ࡜⾑⟶᪂⏕ࢆಁࡍୖ࡛㔜せ࡞ᙺ๭ࢆᯝࡓࡍࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ ࡼࡗ࡚ᑂᰝጤဨ఍ጤဨ඲ဨࡣࠊᮏㄽᩥࡀඵᓥ⏤ె࡟༤ኈ㸦ṑᏛ㸧ࡢᏛ఩ࢆᤵ୚ࡍࡿ࡟༑ศ࡞౯್࠶ࡿࡶࡢ࡜ㄆࡵࡓࠋูグᵝᘧ➨ ྕ㸦➨ ᮲➨ 㡯㛵ಀ㸧᭱⤊ヨ㦂ࡢ⤖ᯝࡢせ᪨༤ኈࡢᑓᨷศ㔝ࡢྡ⛠ ༤ኈ㸦 ṑᏛ 㸧Ặྡ ඵᓥ ⏤ెᏛ ఩ ᤵ ୚ ࡢ ᮲ ௳ Ꮫ఩つ๎➨ ᮲➨ ࣭㡯ヱᙜㄽ ᩥ 㢟 ┠ᣢ⥆ⓗᅽ⦰่⃭ࡀ࣐࢘ࢫ㦵⣽⬊࡟࠾ࡅࡿ RANKLࠊOPG ࠾ࡼࡧ VEGF Ⓨ⌧࡟ཬࡰࡍᙳ㡪᭱⤊ヨ㦂ᢸᙜ⪅୺  ᰝ        ᩍᤵ ຍ⸨ ຌ୍       ༳ᑂᰝጤဨ        ᩍᤵ ஧ᕝ ᾈᶞ       ᑂᰝጤဨ        ᩍᤵ ᐟ༡ ▱బ         ࠝ᭱⤊ヨ㦂ࡢ⤖ᯝࡢせ᪨ࠞ  ุ  ᐃ  ྜ  ᱁ୖグ 3 ྡࡢᑂᰝጤဨ఍ጤဨ඲ဨࡀฟᖍࡢ࠺࠼㸪ᖹᡂ 30 ᖺ 11 ᭶ 21 ᪥ࡢ➨ 4 ᅇᗈᓥ኱Ꮫ◊✲⛉Ⓨ⾲఍㸦ṑᏛ㸧ཬࡧᖹᡂ 31 ᖺ 2 ᭶ 6 ᪥ᮏጤဨ఍࡟࠾࠸࡚᭱⤊ヨ㦂ࢆ⾜࠸㸪୺࡜ࡋ࡚ḟࡢヨၥࢆ⾜ࡗࡓࠋ 1 ศ㖡࡟ࡼࡿᇵ㣴⣽⬊࡬ࡢᅽ㏕่⃭㈇Ⲵࡢጇᙜᛶ࡟ࡘ࠸࡚ 2 ⏕య࡟࠾ࡅࡿᅽ㏕ഃࡢ⣽⬊࡟⏕ࡌࡿ㈇Ⲵ࡜ࡢᩚྜᛶ࡟ࡘ࠸࡚ 3 S-A channel௨እࡢ࣓࢝ࣀࣞࢭࣉࢱ࣮ࡢᙳ㡪࡜㜼ᐖ๣ࡢ≉␗ᛶ࡟ࡘ࠸࡚ 4 㦵⣽⬊ࡢᶵᲔⓗ㈇Ⲵ࡬ࡢឤཷᛶࡀ㦵ⱆ⣽⬊ࡼࡾ㧗࠸ࡇ࡜ࡢ⏕≀Ꮫⓗព⩏ 5 ᐇ㦂࡟⏝࠸ࡓ㦵⣽⬊ᰴ࡜㦵ⱆ⣽⬊ᰴ࡜ࡢ㐪࠸ 6 ௒ᚋࡢ◊✲࡟ࡘ࠸࡚  ࡇࢀࡽ࡟ᑐࡋ࡚ᴟࡵ࡚㐺ษ࡞ゎ⟅ࢆ࡞ࡋ㸪ᮏጤဨ఍ࡀᮏேࡢᏛ఩⏦ㄳㄽᩥࡢෆᐜཬࡧ㛵ಀ஦㡯࡟㛵ࡍࡿᮏேࡢᏛ㆑࡟ࡘ࠸࡚ヨ㦂ࡋࡓ⤖ᯝ㸪඲ဨ୍⮴ࡋ࡚࠸ࡎࢀࡶᏛ఩ࢆᤵ୚ࡍࡿ࡟ᚲせ࡞Ꮫ㆑ࢆ᭷ࡍࡿࡶࡢ࡜ㄆࡵࡓࠋ

持続的圧縮刺激がマウス骨細胞におけるRANKL、OPGおよびVEGF発現に及ぼす影響

http://ir.lib.hiroshima-u.ac.jp/files/public/4/47907/20190718103519472434/k7883_2.pdf

持続的圧縮刺激がマウス骨細胞におけるRANKL、OPGおよびVEGF発現に及ぼす影響

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