Expression and functional role of sclerostin in the fibrocartilaginous enthesis

概要




Ꮫ ఩ ㄽ ᩥ ඲ ᩥ せ ᪨
Expression and functional role of sclerostin in the
fibrocartilaginous enthesis

㸦⥺⥔㌾㦵ᛶ࢚ࣥࢸ࣮ࢩࢫ࡟࠾ࡅࡿࢫࢡࣞࣟࢫࢳࣥࡢ
Ⓨ⌧࡜ᶵ⬟ⓗᙺ๭㸧



୺ᣦᑟᩍဨ㸸㇂ᮏ ᖾኴ㑻 ᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ṑ⛉▹ṇᏛ㸧
๪ᣦᑟᩍဨ㸸ᐟ༡ ▱బ ᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ⏕యศᏊᶵ⬟Ꮫ㸧
๪ᣦᑟᩍဨ㸸ຍ᮶ ┿ே ᩍᤵ
㸦་⣔⛉Ꮫ◊✲⛉ ⏕యᵓ㐀࣭ᶵ⬟ಟ᚟Ꮫ㸧


ụ⏣ ࿴㝯
㸦་ṑ⸆ಖ೺Ꮫ◊✲⛉ ་ṑ⸆Ꮫᑓᨷ㸧



 ࠙⫼ᬒࠚ
㌾㦵ࡣࠊ⤌⧊Ꮫⓗ࡟ࡣࠊ◪Ꮚ㌾㦵ࠊ⥺⥔㌾㦵ࠊᙎᛶ㌾㦵ࡢ 3 ✀㢮࡟ศ㢮ࡉࢀࡿࠋ㌾㦵ᛶ㦵ཎᇶ
ࡸ㛵⠇㌾㦵ࡣࠊType II collagen ࡜ Aggrecan ࡞࡝ࢆ㇏ᐩ࡟ྵࡴࠋ୍᪉ࠊType II collagen ࡔࡅ࡛࡞
ࡃ Type I collagen ࢆⓎ⌧ࡍࡿ⥺⥔㌾㦵ࡣࠊ᳝㛫ᯈࠊ㢡㛵⠇ࡢ㛵⠇෇ᯈࠊ༙᭶ᯈࠊ⭝࣭㠎ᖏࡢ㦵
࡬ࡢ௜╔㒊࡛࠶ࡿ࢚ࣥࢸ࣮ࢩࢫ࡞࡝࡟Ꮡᅾࡋࠊྍືᛶࡢ኱ࡁ࡞⁥⭷㛵⠇ࢆそ࠺◪Ꮚ㌾㦵࡜ྠ
ᵝࠊ◳⤌⧊㐃⤖࡟ࡣḞ࠿ࡏ࡞࠸⤌⧊࡛࠶ࡿࠋ⥺⥔㌾㦵ࡣࠊ⭝㺃㠎ᖏ࡞࡝ࡢᐦᛶ⥺⥔⤌⧊࡜◪Ꮚ㌾
㦵࡜ࡢ୰㛫ⓗ࡞⤌⧊࡛ࠊ⭝㺃㠎ᖏ࡜㦵ࡢ㐃⤖ࢆᶫΏࡋࡍࡿᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿࠋ᳝㛫ᯈࠊ㛵⠇෇
ᯈࠊ༙᭶ᯈࡣࠊ⬇Ⓨ⏕ࡢ㐣⛬࡛ᙧᡂࡉࢀࡿࡀࠊ࢚ࣥࢸ࣮ࢩࢫ࡛ࡣࠊ⏕ᚋࠊ◪Ꮚ㌾㦵ࡀ㦵࡬⨨᥮
ࡉࢀࡿ㐣⛬࡛ࠊຊᏛⓗ㈇Ⲵ࡟ᛂ⟅ࡍࡿࡼ࠺࡟⥺⥔㌾㦵ࡀᙧᡂࡉࢀࡿࠋ᭱⤊ⓗ࡟ࡣࠊ㦵㺃▼⅊໬⥺
⥔㌾㦵㺃㠀▼⅊໬⥺⥔㌾㦵㺃⭝/㠎ᖏ࡜࠸࠺ 4 ᒙᵓ㐀ࡀᵓ⠏ࡉࢀࡿࠋ࢚ࣥࢸ࣮ࢩࢫཎᇶࡣࠊ
Scx+/Sox9+๓㥑⣽⬊࡟⏤᮶ࡋࠊⓎ⏕㐣⛬࡛ࠊ⭝㺃㠎ᖏ࡜◪Ꮚ㌾㦵ࡢ㐃⤖㒊࡜ࡋ࡚ᙧᡂࡉࢀࡿࠋ㛵
⠇㌾㦵௨እࡢ◪Ꮚ㌾㦵࡛ࡣࠊ⫧኱໬㺃▼⅊໬ࡍࡿ࡜⾑⟶ࡀ౵ධࡋ㦵࡬⨨᥮ࡉࢀࡿࡀࠊ▼⅊໬⥺⥔
㌾㦵ࡣ↓⾑⟶࡟ಖࡓࢀࠊ㦵࡬ࣜࣔࢹࣜࣥࢢࡉࢀ࡞࠸ࠋ⥺⥔㌾㦵ᛶ࢚ࣥࢸ࣮ࢩࢫ࡟࠾ࡅࡿ⤌⧊⤫
ྜᛶࡣࠊෆᅉᛶᅉᏊ࡜⭝࠿ࡽఏࢃࡿᶵᲔⓗ่⃭࡟ࡼࡗ࡚༠ㄪⓗ࡟ไᚚࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚
࠸ࡿࡀࠊࡑࡢศᏊ࣓࢝ࢽࢬ࣒ࡣ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣࠊ㦵⣽⬊࡛Ⓨ⌧ࡋ㦵ᙧᡂࢆ
ᢚไࡍࡿࢫࢡࣞࣟࢫࢳࣥࡀࠊ▼⅊໬⥺⥔㌾㦵࡛ࡶⓎ⌧ࡋ࡚࠸ࡿࡇ࡜ࢆ᪂ࡓ࡟ぢฟࡋࡓࡢ࡛ࠊ࢔
࢟ࣞࢫ⭝ࡢ࢚ࣥࢸ࣮ࢩࢫ࡟࠾ࡅࡿࡑࡢᶵ⬟ⓗᙺ๭ࢆࠊ㑇ఏᏊᨵኚື≀ࢆ㥑౑ࡋ࡚᫂ࡽ࠿࡟ࡍࡿ
ࡇ࡜ࢆ┠ᣦࡋࡓࠋ

࠙᪉ἲࠚ
࢚ࣥࢸ࣮ࢩࢫࡢゎᯒ࡟ࡣࠊ㔝⏕ᆺࠊScxGFP transgenic ࣐࢘ࢫࠊScx Ḟኻ࣐࢘ࢫࠊPlatinum
TALEN ࢆ⏝࠸࡚స〇ࡋࡓ Sost Ḟኻ࣐࢘ࢫࢆ⏝࠸ࡓࠋSox9CreERT2

knock-in (KI)࣐࢘ࢫ࡜

RosaTomato ࣐࢘ࢫࢆ஺㓄ࡋ࡚ᚓࡽࢀࡓ Sox9CreERT2/+;RosaTomato ࣐࢘ࢫࢆ⏝࠸ࠊ⣔㆕ゎᯒࢆ
⾜࠺ࡓࡵ࡟ࠊ4-ࣄࢻࣟ࢟ࢩࢱࣔ࢟ࢩࣇ࢙ࣥࢆᢞ୚ࡋࡓࠋࡇࢀࡽࡢ࣐࢘ࢫࢆࠊ4% PFA/PBS ࠶ࡿ
࠸ࡣ 20% Sucrose ࢆྵࡴ 4% PFA/PBS ࡛℺ὶᚋࠊ࢔࢟ࣞࢫ⭝௜╔㒊఩ࢆᅛᐃࡋࠊ㠀⬺⅊ᶆᮏ
ࡣ SCM ࡛ࠊMorse ᾮ࡟ࡼࡗ࡚⬺⅊ࡋࡓᶆᮏࡣࣃࣛࣇ࢕࡛ࣥໟᇙࡋࡓࠋ㠀⬺⅊ᶆᮏࡣࠊࢡࣜ࢜
ࢫࢱࢵࢺ࡛ࢡࣛ࢖࢜ࣇ࢕࣒ࣝ࡜ࢱࣥࢢࢫࢸ࣮ࣥ࢝ࣂ࢖ࢻลࢆ⏝࠸࡚ࠊ⬺⅊ࣃࣛࣇ࢕ࣥᶆᮏࡣࠊ
࣑ࢡࣟࢺ࣮࣒ࢆ⏝࠸࡚ⷧษࡋࡓࠋᚓࡽࢀࡓษ∦ࢆ⏝࠸࡚ࠊࢺࣝ࢖ࢪࣥࣈ࣮ࣝᰁⰍࠊALP/࢔ࣜࢨ
ࣜࣥᰁⰍࠊච␿ᰁⰍࢆ⾜ࡗࡓࠋࡲࡓࠊSost Ḟኻ࣐࢘ࢫ࡜㔝⏕ᆺ࣐࢘ࢫࡢ μCT ⏬ീࢆඖ࡟ࠊ3D
ྍど໬ゎᯒࢯࣇࢺ࢙࢘࢔ Amira ࢆ⏝࠸࡚୕ḟඖ෌ᵓ⠏ࢆ⾜࡞ࡗ࡚ࠊᙧែせ⣲ࢆྍど໬ࡋࡓࠋ

࠙⤖ᯝ࡜⪃ᐹࠚ
Wnt ཬࡧ BMP ࢩࢢࢼࣝఏ㐩⤒㊰ࡢ antagonist ࡛࠶ࡿࢫࢡࣞࣟࢫࢳࣥࡣࠊ▼⅊໬⥺⥔㌾㦵࡟࠾
࠸࡚ࠊ࢜ࢫࢸ࢜࢝ࣝࢩࣥࢆ⏘⏕ࡍࡿ⥺⥔㌾㦵⣽⬊࡛Ⓨ⌧ࡋ࡚࠾ࡾࠊ᭱⤊ศ໬ࡋࡓ⥺⥔㌾㦵⣽⬊
ࡢⰋ࠸࣐࣮࣮࢝ศᏊ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ࢚ࣥࢸ࣮ࢩࢫࡢ㠀⬺⅊ษ∦࡟࠾࠸࡚࣓ࢱࢡ࣐ࣟࢪ࣮
ࢆ♧ࡍ㡿ᇦ࡜࢔ࣜࢨ࡛ࣜࣥᰁⰍࡉࢀࡿ▼⅊໬㡿ᇦࢆẚ㍑ࡋࡓ࡜ࡇࢁࠊ▼⅊໬⥺⥔㌾㦵ᒙࡢᣑ኱

ᚋࠊ▼⅊໬๓⥺ࡢ ALP 㝧ᛶ⣽⬊ࡀῶᑡ࡟కࡗ࡚ࠊ㠀▼⅊໬⥺⥔㌾㦵ᒙࡀᣑ኱ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿
࡟࡞ࡗࡓࠋච␿ᰁⰍ࡜⣔㆕ゎᯒࡢ⤖ᯝࠊࢫࢡࣞࣟࢫࢳࣥ㝧ᛶ⣽⬊ࡣࠊSox9 㝧ᛶ⣽⬊࡟⏤᮶ࡍ
ࡿࡇ࡜ࡀ♧ࡉࢀࡓࡀࠊSox9 ࡢⓎ⌧ࡣ⥺⥔㌾㦵⣽⬊ࡢᡂ⇍㐣⛬࡛ᛴ㏿࡟పୗࡋࡓࠋ࢔࢟ࣞࢫ⭝
ࡢᙧᡂ୙඲ࢆ࿊ࡍࡿ Scx Ḟኻ࣐࢘ࢫ࡛ࡣࠊ⥺⥔㌾㦵ࡀ࡯࡜ࢇ࡝ᙧᡂࡉࢀࡎ㦵➃◪Ꮚ㌾㦵⣽⬊ࡢ
ศ໬ࡶ㐜ᘏࡋ࡚࠾ࡾࠊ࣐࣍ࣔ࢘ࢫ࡛ࡣ㔝⏕ᆺ࣐࢘ࢫ࡛ほᐹࡉࢀࡿࢫࢡࣞࣟࢫࢳࣥࡢⓎ⌧ࡀ᳨ฟ
ࡉࢀ࡞࠿ࡗࡓࠋᚑࡗ࡚ࠊ➽཰⦰࡛⏕ࡌ⭝ࢆ௓ࡋ࡚ఏ㐩ࡉࢀࡿᶵᲔⓗ่⃭ࡀࠊ⥺⥔㌾㦵ࡢᙧᡂࡔ
ࡅ࡛࡞ࡃ㦵➃◪Ꮚ㌾㦵ࡢᡂ⇍࡟ࡶ୙ྍḞ࡛࠶ࡿ࡜♧၀ࡉࢀࡓࠋࢤࣀ࣒⦅㞟࡟ࡼࡗ࡚సᡂࡋࡓ
Sost Ḟኻ࣐࢘ࢫ࡛ࡣࠊ26 ሷᇶḞኻ࡟ࡼࡿ frameshift ࡟ࡼࡗ࡚୰㏵࡛⩻ヂࡀ೵Ṇࡍࡿ⤊Ṇࢥࢻࣥ
ࡀ⏕ࡌࠊ࣐࣍ࣔ࢘ࢫ࡛ࡣࢫࢡࣞࣟࢫࢳࣥࡢᒁᅾࡀᾘኻࡋ࡚࠸ࡓࠋ࣐࣍ࣔ࢘ࢫ࡛ࡣࠊALP 㝧ᛶ⣽
⬊ࡢቑຍ࡟కࡗ࡚ࠊ㌾㦵ୗ㦵ࡢ㦵໬ࡔࡅ࡛࡞ࡃ⥺⥔㌾㦵ࡢ▼⅊໬ࡀஹ㐍ࡋ࡚࠸ࡓࠋ௨ୖࡢ⤖ᯝ
ࡼࡾࠊ᭱⤊ศ໬ࡋࡓ▼⅊໬⥺⥔㌾㦵⣽⬊࡛Ⓨ⌧ࡍࡿࢫࢡࣞࣟࢫࢳࣥࡣࠊ⥺⥔㌾㦵ࡢ▼⅊໬ࡸ㦵
ᙧᡂࡢஹ㐍ࢆ㜵ࡂࠊ࢚ࣥࢸ࣮ࢩࢫࡢ⤌⧊⤫ྜᛶࢆ⥔ᣢࡍࡿᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀ
ࡓࠋ

࠙⤖ㄽࠚ
ᮏ◊✲࡟ࡼࡗ࡚ࠊࢫࢡࣞࣟࢫࢳࣥࡣࠊ㌾㦵ୗ㦵ࡢ㦵໬ࡔࡅ࡛࡞ࡃ▼⅊໬⥺⥔㌾㦵ࡢ▼⅊໬ࢆ
ᢚไⓗ࡟ไᚚࡍࡿࡇ࡜࡟ࡼࡾ࢚ࣥࢸ࣮ࢩࢫࡢ⤌⧊⤫ྜᛶࢆㄪ⠇ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ