(1)ᖹᡂ27 ᖺᗘ ಟኈㄽᩥ GFP ᶆ㆑㓝ẕࢆ⏝࠸ࡓ ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࡢ ᐃ Measurements of Phagocytic Activities of Paramecium bursaria Using GFP-labeled Yeast ᘯ๓኱Ꮫ኱Ꮫ㝔 ᩍ⫱Ꮫ

ᖹᡂ27 ᖺᗘ ಟኈㄽᩥ

GFP ᶆ㆑㓝ẕࢆ⏝࠸ࡓ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࡢ ᐃ

Measurements of Phagocytic Activities of Paramecium bursaria Using GFP-labeled Yeast

ᘯ๓኱Ꮫ኱Ꮫ㝔 ᩍ⫱Ꮫ◊✲⛉ ᩍ⛉ᩍ⫱ᑓᨷ ⌮⛉ᩍ⫱ᑓಟ

13GP211 ୕ᾆ㈗ኈ

ᣦᑟᩍဨ ᒾ஭ⲡ௓

┠ḟ

1 ᗎㄽ ... 3

2 ᮦᩱ࡜᪉ἲ ... 6

2.1 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᇵ㣴 ... 6

2.2 ⓑ໬ᰴࡢసᡂ ... 7

2.3 GFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳࡢほᐹ࡜ ᐃ ... 7

2.4 GFPᶆ㆑㓝ẕࡢᾘ໬ࡢ ᐃ ... 8

2.5 GFPᶆ㆑㓝ẕࡢ⣽⬊እ࡬ࡢ᤼ฟࡢほᐹ ... 9

2.6 㓝ẕࡢ⣽⬊ཱྀ࡬ࡢ㐠ᦙࡢほᐹ ... 9

3 ⤖ᯝ ... 11

3.1 㓝ẕࢆ㣵࡜ࡋࡓሙྜࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢቑṪ᭤⥺ ... 11

3.2 GFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳཬࡧ᤼ฟࡢほᐹ ... 12

3.3 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶ(1)ඹ⏕⸴ࡢ᭷↓࡟ࡼࡿ㐪࠸ ... 15

3.4 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶ(2)ቑṪ᫬ᮇ࡟ࡼࡿ㐪࠸ ... 17

3.5 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᾘ໬άᛶ ... 19

3.6 ࢰ࣒࢘ࣜࢩࡢ㣗స⏝ ... 21

4 ⪃ᐹ ... 23

4.1 GFPᶆ㆑㓝ẕࢆ⏝࠸ࡓ⧄ẟ⹸ࡢ㣗స⏝ࡢ ᐃ ... 23

4.2 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊ෆඹ⏕࡜㣗స⏝ ... 24

ཧ⪃ᩥ⊩ ... 26 ㅰ㎡

3 1 ᗎㄽ

༢⣽⬊ࡢཎ⏕⏕≀࡛࠶ࡿ࣑ࢻࣜࢰ࣒࢘ࣜࢩ(Paramecium bursaria)ࡣ⧄ẟ⹸

ࢰ࣒࢘ࣜࢩ㢮ࡢ㢮⦕✀࡛࠶ࡿࡀࠊ኱ࡁ࡞≉ᚩ࡜ࡋ࡚⣽⬊ෆ࡟⣙500㹼700ಶࡢࢡ

ࣟࣞࣛᵝ⸴㢮ࡀඹ⏕㸦஧ḟඹ⏕㸧ࡋ࡚࠸ࡿࠋᐟ୺ࡢ⣽⬊ෆ࡛ࡣࠊඹ⏕⸴ࡣ 1 ⣽

⬊ࡎࡘ㣗⬊⭷⏤᮶ࡢඹ⏕⬊⭷࡟ໟࡲࢀ࡚࠾ࡾࠊࡑࡢ୰࡛⣽⬊ศ⿣ࡋ࡚ቑṪࡍࡿ

㸦1㸧ࠋ㏻ᖖࡢග᮲௳࡛ࡣࠊᐟ୺ࡢ⣽⬊ෆ࡛ẚ㍑ⓗᏳᐃ࡟⥔ᣢࡉࢀ࡚࠸ࡿ㸦2㸧ࠋ ᐟ୺ࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀ㣗స⏝࡛ᚓࡓ❅⣲※࡞࡝ࢆඹ⏕⸴࡟౪⤥ࡍࡿ୍᪉࡛ࠊ ඹ⏕⸴ࡣ࣐ࣝࢺ࣮ࢫࡸ㓟⣲࡞࡝ࡢගྜᡂ⏘≀ࢆᐟ୺࡟౪⤥ࡋ࡚࠸ࡿࡇ࡜࠿ࡽࠊ ୧⪅ࡣ┦฼ඹ⏕ࡢ㛵ಀ࡟࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ㸦1㸧ࠋඹ⏕⣔඲య࡜ࡋ࡚ࡣࠊඹ

⏕⸴࡟ࡼࡿගྜᡂ࡜ᐟ୺࡟ࡼࡿ㣗స⏝ࡢ 2 ✀㢮ࡢ␗࡞ࡿ࢚ࢿࣝࢠ࣮㸦ᰤ㣴㸧⋓

ᚓᶵᵓࢆᣢࡗ࡚࠸ࡿࡇ࡜࠿ࡽࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡣ⊂❧ᰤ㣴࡜ᚑᒓᰤ㣴ࡢ୧᪉

ࢆවࡡഛ࠼ࡓ͆ΰྜᰤ㣴⏕≀͇࡜࠸࠺ࡇ࡜࡟࡞ࡿ㸦3,4㸧ࠋᐟ୺࡜ඹ⏕⸴ࡣࡑࢀࡒ

ࢀ༢⊂࡛ࡶᇵ㣴࡛ࡁࠊᐟ୺࡜ඹ⏕⸴ࢆ⤌ࡳྜࢃࡏࡿࡇ࡜࡟ࡼࡗ࡚ඹ⏕ࡢ෌ᵓᡂ

ࡶྍ⬟࡞ࡇ࡜࡞࡝࠿ࡽࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡣྂࡃ࠿ࡽ஧ḟඹ⏕ࡢ◊✲࡟ࡼࡃ⏝

࠸ࡽࢀ࡚ࡁࡓࠋ

㣗స⏝࡜ࡣࠊ⣽⬊ࡀ⣽⳦ࡸ␗≀ࠊ㣵࡞࡝ࢆእ㒊⎔ቃ࠿ࡽ⣽⬊ෆ࡟ྲྀࡾ㎸ࡳࠊ ศゎࡍࡿάື࡛࠶ࡿࠋ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡗ࡚⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ≀య ࡣ⭷࠿ࡽ࡞ࡿ㣗⬊࡟ໟࡲࢀࠊࡇࢀ࡟ࡉࡲࡊࡲ࡞ศゎ㓝⣲ࢆྵࡴࣜࢯࢯ࣮࣒ࡀ⼥

ྜࡍࡿࡇ࡜࡟ࡼࡗ࡚ศゎࡉࢀࡿࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝࡟ࡘ࠸࡚ࡣࠊ㓝ẕ

ࡸඹ⏕⸴ࢆ㣵࡜ࡋ࡚⏝࠸ࡿ◊✲࡟ࡼࡗ࡚ࠊ௨ୗࡢࡼ࠺࡟㐍⾜ࡍࡿࡇ࡜ࡀศ࠿ࡗ

࡚࠸ࡿ㸦1㸧ࠋࡲࡎ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡗ࡚⣽⬊ཱྀ࠿ࡽྲྀࡾ㎸ࡲࢀࡓ㣵ࡣࠊ 0.5 ศ௨ෆ࡟㣗⬊࡟ໟࡲࢀࠊ࢔ࢩࢻࢯ࣮࣒ࡀ㣗⬊࡟⼥ྜࡋ࡚㣗⬊ෆࡢ pH ࡣ 2.4-3.0ࡲ࡛పୗࡍࡿࠋࡑࡢᚋࣜࢯࢯ࣮࣒ࡢ⼥ྜࡀ㉳ࡇࡾࠊpHࡣ6.4-7.0ࡲ࡛ୖ

᪼ࡋ࡚㣵ࡣᾘ໬ࡉࢀࡿࠋᾘ໬ࡉࢀ࡞࠿ࡗࡓࡶࡢࡣ⣽⬊⫠㛛࠿ࡽ᤼ฟࡉࢀࡿࠋ௨

ୖࡢࡼ࠺࡟ࠊ㣗స⏝ࡣྲྀࡾ㎸ࡳ࠿ࡽศゎ࡟⮳ࡿ㐣⛬࡛ࡉࡲࡊࡲ࡞ࢱࣥࣃࢡ㉁ࡀ ᶵ⬟ࡍࡿࡓࡵࠊࡑࢀࢆ⾜࠺࡟ࡣᑡ࡞ࡃ࡞࠸㔞ࡢ⣽⬊ෆࡢ㈨※㸦ᰤ㣴ศ㸧ࢆせࡍ

ࡿ࡜⪃࠼ࡽࢀࡿࠋ

୍⯡࡟ࠊගྜᡂ࡜㣗స⏝ࡢ୧᪉ࢆ⾜࠺ΰྜᰤ㣴⏕≀࡟࡜ࡗ࡚ࡣࠊ⏕ᏑࡸቑṪ ࡢຠ⋡ࢆ㧗ࡵࡿࡓࡵ࡟㝈ࡾ࠶ࡿ⣽⬊ෆࡢ㈨※㸦ᰤ㣴ศࠊ≉࡟ࢱࣥࣃࢡ㉁㸧ࢆග

4

ྜᡂ࡜㣗స⏝ࡢ࡝ࡕࡽ࡟࡝ࢀࡔࡅ᣺ࡾศࡅࡿࡢࡀ᭷฼࠿ࠊ࡜࠸࠺኱ࡁ࡞ၥ㢟ࡀ

࠶ࡿ㸦3㸧ࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡑࡢࡼ࠺࡞ΰྜᰤ㣴⏕≀࡟࠾ࡅࡿ⣽⬊ෆ㈨※ࡢ᭱㐺

࡞ศ㓄ᡓ␎࡟ࡘ࠸࡚ࡣࠊࡲࡔศ࠿ࡗ࡚࠸࡞࠸ࡇ࡜ࡀከ࠸ࠋࡑࡢ୰࡛ࡶ≉࡟ࠊ⸴

㢮ࡀ⣽⬊ෆ࡟ඹ⏕ࡋ࡚࠸ࡿ⏕≀࡟࠾࠸࡚ࡑࡢ⣽⬊ෆඹ⏕ࡀᐟ୺ࡢ㣗స⏝࡟୚࠼

ࡿᙳ㡪࡟ࡘ࠸࡚ࡣ࡯࡜ࢇ࡝ศ࠿ࡗ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊࡑࢀࡽ࡟ࡘ࠸

࡚▱ぢࢆᚓࡿࡓࡵ࡟ࠊࢡࣟࣞࣛᵝ⸴㢮ࡀ⣽⬊ෆ࡟ඹ⏕ࡋ࡚࠸ࡿ࣑ࢻࣜࢰ࣒࢘ࣜ

ࢩࡢ㣗స⏝ࢆᐃ㔞ⓗ࡟ㄪ࡭ࡿࡇ࡜࡟ࡋࡓࠋ

⧄ẟ⹸ࡢ㣗స⏝ࡢ ᐃ࡟࠾࠸࡚ࡣᚑ᮶ࠊୖグࡢࡼ࠺࡞㓝ẕࡸඹ⏕⸴ࡢ࡯࠿ࠊ

⣽⳦ࡸ⺯ගࣅ࣮ࢬ࡞࡝ࡀ㣵࡜ࡋ࡚⏝࠸ࡽࢀ࡚ࡁࡓ㸦5,6㸧ࠋࡋ࠿ࡋࡇࢀࡽࡢ᪉ἲࡣࠊ 㣵ࡢ⣽⬊ෆ࡬ࡢྲྀࡾ㎸ࡳࢆ ᐃࡍࡿࡇ࡜ࡣ࡛ࡁࡿࡶࡢࡢࠊ㣵ࡀศゎࡉࢀ࡞࠸࠿

ࡶࡋࡃࡣศゎࡀほᐹࡋ࡟ࡃ࠸ࡓࡵ࡟ྲྀࡾ㎸ࡲࢀࡓ㣵ࡢᾘ໬ࢆㄪ࡭ࡿࡇ࡜ࡣ㞴ࡋ

࠸ࠋ᭱㏆ࠊ⧄ẟ⹸ࡢ 1 ✀࡛࠶ࡿࢸࢺࣛࣄ࣓ࢼࡢ㣗స⏝ ᐃ࡟࠾࠸࡚ࠊ࢜࣡ࣥࢡ

ࣛࢤ⏤᮶ࡢ⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁GFPࢆⓎ⌧ࡋ࡚࠸ࡿ኱⭠⳦ࡀ㣵࡜ࡋ࡚⏝࠸ࡽࢀ

ࡓ㸦7㸧ࠋGFP࡟ࡣpHࡀపࡃ࡞ࡿ࡜⺯ගࡀῶᑡࡍࡿ࡜࠸࠺ᛶ㉁ࡀ࠶ࡿࡓࡵ㸦8㸧ࠊ

኱⭠⳦ࡀ㣗⬊ෆ࡛ᾘ໬ࡉࢀ࡚ࡑࡢ⣽⬊ቨ࡜⭷ࡀኻࢃࢀࡿ࡜ࠊGFP ࡣpH ࡢప࠸

㣗⬊ෆ࡟ᣑᩓࡋ࡚ࡑࡢ⺯ගࡣ኱ࡁࡃῶᑡࡍࡿࠋࡑࡢࡓࡵࠊ⺯ග㢧ᚤ㙾࡞࡝ࢆ⏝

࠸ࡿࡇ࡜࡟ࡼࡗ࡚㣵ࡢᾘ໬ࢆ᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋࡓࡔࡋ኱⭠⳦ࡣࠊ㛗ࡉࡀ

⣙2-4 Pmࡢ⣽㛗࠸ᱰ⳦࡛࠶ࡿࡓࡵ㢧ᚤ㙾࡟ࡼࡿほᐹࡀᐜ࡛᫆ࡣ࡞ࡃࠊࡲࡓ┠ⓗ

ࡢ⧄ẟ⹸࡟࡜ࡗ࡚ᚲࡎࡋࡶࡼ࠸㣵࡛ࡣ࡞࠸ྍ⬟ᛶࡶ࠶ࡿࠋ

ࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࢆᐃ㔞ⓗ࡟ ᐃࡍࡿࡓࡵ࡟ࠊ GFPࢆ㐣๫Ⓨ⌧ࡋ࡚࠸ࡿฟⱆ㓝ẕ㸦Saccharomyces cerevisiae^{㸧ࢆ⏝࠸ࡓ㸦௨} 㝆ࡇࢀࢆGFPᶆ㆑㓝ẕ࡜࿧ࡪ㸧ࠋฟⱆ㓝ẕࡢ⣽⬊ࡣ┤ᚄ⣙5 Pmࡢᴃ෇ᙧ࡛࠶ࡾࠊ

኱⭠⳦࡜ẚ࡭࡚㢧ᚤ㙾ほᐹࡀᐜ࡛᫆࠶ࡿࠋGFP ᶆ㆑㓝ẕࡣࠊ㐣ཤ࡟㉥⑩࢔࣓࣮

ࣂࡢ㣗స⏝ࡢ ᐃ࡟⏝࠸ࡽࢀࡓ౛ࡀ࠶ࡿࡀ㸦9㸧ࠊ⧄ẟ⹸ࡢ㣗స⏝ࡢ ᐃ࡟ࡣ⏝

࠸ࡽࢀࡓࡇ࡜ࡣ࡞࠸ࠋᮏ◊✲࡛ࡣࡇࡢGFPᶆ㆑㓝ẕࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ࠊྲྀ

ࡾ㎸ࢇࡔ㣵࡜ඹ⏕⸴ࡢ༊ู࠾ࡼࡧ㣵ࢆྲྀࡾ㎸ࢇࡔᚋࡢᾘ໬࡜᤼ฟࡢ༊ูࡀྍ⬟

࡟࡞ࡾࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࢆᐃ㔞ⓗ࡟ ᐃࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋ ᐃ࡟

ࡼࡗ࡚ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝࡟ࡘ࠸࡚௨ୗࡢࡼ࠺࡞᪂ࡓ࡞⤖ᯝࡀᚓࡽࢀ

ࡓࠋ

5

1㸧 ඹ⏕⸴ࡢᏑᅾࡣࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊ෆ࡟࠾ࡅࡿᾘ໬άᛶ࡟ࡣᙳ㡪ࡋ

࡞࠸ࠋ

2㸧 㣵ࡢྲྀࡾ㎸ࡳάᛶࡣⓑ໬ᰴࡢ᪉ࡀ㔝⏕ᰴࡼࡾ㧗࠿ࡗࡓࡇ࡜࠿ࡽࠊඹ⏕⸴

ࡢᏑᅾࡣ㣵ࡢྲྀࡾ㎸ࡳάᛶࢆపୗࡉࡏࡿࠋ

3㸧 ྠࡌ㔝⏕ᰴ࡛ࡶࠊᐃᖖᮇ࡟ධࡿ࡜ᑐᩘቑṪᮇࡼࡾࡶྲྀࡾ㎸ࡳάᛶࡀపୗ

ࡍࡿࠋࡇࢀࡣࠊᐃᖖᮇ࡟ධࡿ࡜㣵ࢆྲྀࡾ㎸ࡴࡓࡵࡢࢱࣥࣃࢡ㉁ࡢⓎ⌧ࡀ ᢚไࡉࢀࡿࡓࡵ࡛࠶ࡿࠋ

ࡇࢀࡽࡢ⤖ᯝ࡟ࡘ࠸࡚ࠊΰྜᰤ㣴⏕≀࡟࠾ࡅࡿ⣽⬊ෆ㈨※ࡢ᭱㐺࡞ศ㓄ᡓ␎ࡢ ほⅬ࠿ࡽ⪃ᐹࡍࡿࠋ

6 2 ᮦᩱ࡜᪉ἲ

2.1 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᇵ㣴

ᮏ◊✲ࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᐇ㦂ࡣࠊᙜ◊✲ᐊ࡟ࡼࡗ࡚2010ᖺ࡟㟷᳃┴ᘯ๓ ᕷෆࡢụࡼࡾ᥇㞟ࡉࢀࡓHA1ᰴࢆ㔝⏕ᰴ࡜ࡋ࡚⏝࠸ࡓࠋ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᇵ㣴ࡣ௨ୗࡢࡼ࠺࡟⾜ࡗࡓࠋࡲࡎ50 mlࡢ୕ゅࣇࣛࢫࢥ

࡟ࠊᕷ㈍ࡢ࣑ࢿ࢛࣮ࣛࣝ࢘ࢱ࣮ࡸἙᕝ࡞࡝ࡢ࢖࢜ࣥ⤌ᡂࢆཧ⪃࡟ᙜ◊✲ᐊ࡛స ᡂࡋࡓ͆ேᕤụỈ͇ 㸦0.3 mM NaCl, 0.1 mM MgSO4, 0.1 mM KCl, 0.5 mM CaCl2㸹௨ୗAW࡜࿧ࡪ㸧ࢆ19 mlධࢀࠊࢩࣜࢥࢭࣥ㸦ಙ㉺࣏࣐࣮ࣜ㸹T-24㸧࡛

ᰦࢆࡋ࡚㧗ᅽ⵨Ẽ⁛⳦ࡋ࡚࠿ࡽࠊᐊ ࡲ࡛෭ࡲࡋࡓࠋ㣵࡜ࡋ࡚ࠊYPD ᇵᆅ࡛ 1 ᬌᇵ㣴ࡋࡓฟⱆ㓝ẕSaccharomyces cerevisiae BY4741ᰴࡢᇵ㣴ᾮ1 mlࢆẖศ 3000ᅇ㌿࡛5ศ㛫㐲ᚰࡋୖΎࢆ྾࠸ྲྀࡗ࡚࠿ࡽࠊAW 1 ml࡛ᠱ⃮ࡋ࡚ࡍ࡭࡚ࣇ

ࣛࢫࢥ࡟ධࢀࡓࠋࡉࡽ࡟ᢠ⏕≀㉁࢔ࣥࣆࢩࣜࣥࢆ᭱⤊⃰ᗘࡀ 500 Pg/ml ࡜࡞ࡿ

ࡼ࠺࡟ຍ࠼࡚࠿ࡽࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ๓ᇵ㣴ᾮࢆ500 Plຍ࠼ࡓࠋࣇࣛࢫࢥࡣ 24Υࡢᜏ ᇵ㣴ᶵ㸦ࢧ࣮ࣥࣚ㸹MIR-152㸧࡟㟼⨨ࡋࠊ᳜≀⫱ᡂ⏝⺯ගⅉFL20SS࣭ BRN/18㸦ᮾⰪ㸧࡟ࡼࡗ࡚⣙ 1000 lux ࡢගࢆ12:12 ᫬㛫ࡢ᫂ᬯࢧ࢖ࢡ࡛ࣝ↷ᑕ ࡋࡓࠋ࡜ࡁ࡝ࡁࣇࣛࢫࢥࢆࡺࡍࡗ࡚ࠊᗏ࡟ỿࢇࡔ㓝ẕࢆ⯙࠸ୖࡀࡽࡏࡓࠋᚋ㏙

ࡍࡿHA1w3ᰴࡣඹ⏕⸴࠿ࡽගྜᡂ⏘≀ࢆᚓࡽࢀ࡞࠸ࡢ࡛ࠊ඲⁛ࡋ࡞࠸ࡼ࠺࡟2

㐌㛫௨ෆ࡟᳜࠼⥅࠸ࡔࠋ

HA1ᰴ࡜HA1w3ᰴ࡟ࡘ࠸࡚ࠊୖグࡢࡼ࠺࡟ᇵ㣴ࢆ㛤ጞࡋ࡚࠿ࡽ1㐌㛫௨ୖ

ᚋ࡟ࠊᇵ㣴ᾮࡢ୍㒊ࢆᶆ‽ᐮኳᇵᆅ㸦ࢢࣝࢥ࣮ࢫ1 g/L, Bacto Tryptone 5 g/L, Yeast Extract 2.5 g/L, Bacto Agar 15 g/L㸧࡟ሬᕸࡋࠊ30Υ࡛㟼⨨ࡋࡓࠋ2ᬌ௨

ୖᇵ㣴ࡋ࡚ࡶ㣵࡜ࡋࡓ㓝ẕ௨እࡢᚤ⏕≀ࡀቑṪࡋ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊ୧ᇵ㣴ᰴ

ࡣ↓⳦ⓗ࡛࠶ࡿ࡜ุ᩿ࡋࡓࠋ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊⃰ᗘࡢ ᐃࡣ௨ୗࡢࡼ࠺࡟⾜ࡗࡓࠋࡲࡎᇵ㣴ᾮࢆ

100 Pl᥇ྲྀࡋࠊ⣽⬊ࡢ㐠ືࢆ೵Ṇࡉࡏࡿࡓࡵ࡟᭱⤊⃰ᗘࡀ 1 mM࡜࡞ࡿࡼ࠺࡟

NiCl2ࢆຍ࠼࡚࠿ࡽᙜ◊✲ᐊస〇ࡢ࢔ࢡࣜࣝ〇ࡢᐜჾ࡟ධࢀࠊಽ❧㢧ᚤ㙾㸦ࢽࢥ

ࣥ㸹TMS-F㸧࡛⣽⬊ᩘࢆィ ࡋࡓࠋ

7 2.2 ⓑ໬ᰴࡢసᡂ

ⓑ໬㸦㠀ඹ⏕㸧ᰴࡣࠊᩥ⊩㸦10㸧࡟ᚑ࠸௨ୗࡢࡼ࠺࡟ࢱࣥࣃࢡ㉁ྜᡂ㜼ᐖ๣

ࢩ ࢡ ࣟ ࡬ ࢟ ࢩ ࣑ ࢻ ࢆ ⏝ ࠸ ࡚ స ᡂ ࡋ ࡓ ࠋ ࡲ ࡎ ヨ 㦂 ⟶ ࡟ 0.5%㟷 Ồ/0.005%

YeastExtract ࢆ1.8 mlධࢀࠊKlebsiella ^ࢆຍ࠼ࡓ0.5%㟷Ồ㸦ྠோᇽ㸹኱㯏ⱝ ⴥ100㸣㟷Ồ㸧/0.005% YeastExtract࡛ᇵ㣴ࡋࡓ HA1ᰴࡢᇵ㣴ᾮ200 Plࢆຍ

࠼ࠊࡇࡇ࡟ࢩࢡࣟ࣊࢟ࢩ࣑ࢻࢆ᭱⤊⃰ᗘࡀ10 Pg/ml࡜࡞ࡿࡼ࠺࡟ຍ࠼ࡓࠋヨ㦂

⟶ࡣ24Υ࡛㟼⨨ࡋࠊ⣙1000 luxࡢගࢆ12:12᫬㛫ࡢ᫂ᬯࢧ࢖ࢡ࡛ࣝ↷ᑕࡋࡓࠋ 7᪥ᚋࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊ෆࡢඹ⏕⸴ࡀ᏶඲࡟㝖ཤࡉࢀࡓࡇ࡜ࢆ㢧ᚤ㙾ほᐹ

࡛☜ㄆࡋࡓᚋࠊࢡ࣮ࣟࢽࣥࢢࢆ⾜ࡗࡓࠋࢡ࣮ࣟࢽࣥࢢࡣࠊᐇయ㢧ᚤ㙾ୗ࡛࣑ࢻ

ࣜࢰ࣒࢘ࣜࢩࢆ1༉᥇ྲྀࡋࠊ࢔ࣥࣆࢩࣜࣥࢆྵࡴAW 1ml࡛8ᅇὙίࡋ࡚࠿ࡽ

࣑ࢽࢩ࣮࡛ࣕࣞᇵ㣴ࡋࡓࠋ࣑ࢽࢩ࣮࡛ࣕࣞ༑ศ࡟ቑṪࡋࡓࡽࠊ୕ゅࣇࣛࢫࢥ࡟

⛣ࡋ࡚ࡉࡽ࡟ᇵ㣴ࡋࡓࠋᚓࡽࢀࡓⓑ໬ᰴࢆHA1w㸦white㸧3࡜࿨ྡࡋࠊ௨㝆ࡢ ᐇ㦂࡟⏝࠸ࡓࠋ

2.3 GFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳࡢほᐹ࡜ ᐃ

GFPᶆ㆑㓝ẕࡣࠊ௨ୗࡢࡼ࠺࡟ㄪᩚࡋࡓࠋࡲࡎࠊከࢥࣆ࣮ࡢࣉࣛࢫ࣑ࢻ࠾ࡼ

ࡧ TEF1 ࣉ࣮ࣟࣔࢱ࣮ࡢไᚚ࡟ࡼࡗ࡚ GFP ࢆ㐣๫Ⓨ⌧ࡋ࡚࠸ࡿฟⱆ㓝ẕ Saccharomyces cerevisiae pTOWѸpTEF1::GFP/BY4741ᰴ㸦ᒸᒣ኱ᏛᏲᒇ༤ኈ

ࡼࡾ౪୚㸧ࢆSCѸUraᇵᆅ㸦ࢢࣝࢥ࣮ࢫ 20 g/L, Yeast nitrogen base without amino acids 6.7 g/L, ѸUracil drop out mix 0.77 g/L㸧࡛4ᬌ᣺࡜࠺ᇵ㣴ࡋࡓࠋ ࡑࡢᇵ㣴ᾮ1 mlࢆẖศ3000ᅇ㌿࡛5ศ㛫㐲ᚰࡋࠊୖΎࢆ྾࠸ྲྀࡗ࡚࠿ࡽࠊ150 Pl ࡢAW࡛ᠱ⃮ࡋ࡚ࠊGFPᶆ㆑㓝ẕᠱ⃮ᾮ࡜ࡋࡓࠋ

࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟ࡘ࠸࡚ࡣࠊ㐺ᙜ࡞ቑṪ᫬ᮇࡢᇵ㣴ᾮ5 mlࢆ✰ᚄ11 Pmࡢ ࢼ࢖ࣟࣥࢿࢵࢺࣇ࢕ࣝࢱ࣮㸦Millipore㸧࡟㏻ࡋ࡚࠿ࡽࠊAW 1 ml࡛5ᅇὙίࡍ

ࡿࡇ࡜࡟ࡼࡗ࡚ࠊᇵ㣴ᾮ୰࡟ṧࡗ࡚࠸ࡿ㓝ẕࢆ㝖ཤࡋࡓࠋࣆ࣌ࢵࢺ࡛ᅇ཰ࡋࡓ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡣࠊ24Υ࡛ගࢆ↷ᑕࡋ࡞ࡀࡽ 3 ᫬㛫㟼⨨ࡍࡿࡇ࡜࡟ࡼࡗ࡚⣽

⬊ෆ࡟ṧࡗ࡚࠸ࡿᇵ㣴ᾮ⏤᮶ࡢ㓝ẕࢆᾘ໬ࡉࡏࡓࠋ

ࡇࡢ3᫬㛫㟼⨨ࡋࡓ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊ᠱ⃮ᾮࢆࠊGFPᶆ㆑㓝ẕᠱ⃮ᾮ

࡜ࡑࢀࡒࢀࡢ⣽⬊⃰ᗘࡀ1-2™10³ cells/ml࡜5™10⁷ cells/ml࡟࡞ࡿࡼ࠺࡟ΰྜ

ࡍࡿࡇ࡜࡟ࡼࡗ࡚ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࡓࠋࡇࡢ᫬

8

้ࢆ0࡜ࡋ࡚ࠊ5ศ,10ศ, 15ศ, 20ศ, 25ศ, 30ศᚋ࡟⣽⬊ෆࡢGFPᶆ㆑㓝ẕ

ࢆィᩘࡋࡓࠋ

GFPᶆ㆑㓝ẕࡢィᩘࡣࠊ௨ୗࡢࡼ࠺࡟⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚⾜ࡗࡓࠋࡲࡎࠊୖ

グࡢΰྜᾮ࠿ࡽྛ᫬้࡟50 Pl᥇ྲྀࡋࠊ6㸣࣐࣍ࣝࣜࣥࢆ➼㔞ຍ࠼࡚⣽⬊ࢆᅛᐃ ࡋࡓࠋᅛᐃࡋࡓ࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊ࢆࠊ40 ಸࡢ UPlanSApoᑐ≀ࣞࣥࢬ㸦࢜

ࣜࣥࣃࢫ㸧ࢆ⏝࠸࡚ṇ❧ⴠᑕ⺯ග㢧ᚤ㙾BX51㸦࢜ࣜࣥࣃࢫ㸧࡟ࡼࡗ࡚ほᐹࡋࡓࠋ

⺯ගほᐹࡢග※࡜ࡋ࡚ࡣࠊ㧗ᅽỈ㖟ࣛࣥࣉࢆ⏝࠸ࡓࠋGFP ࡢ⺯ගࢆほᐹࡍࡿࡓ

ࡵ࡟ࠊU-FGFP ࣇ࢕ࣝࢱ࣮ࢭࢵࢺ㸦࢜ࣜࣥࣃࢫ㸧ࢆ⏝࠸࡚ヨᩱࢆ㟷Ⰽග࡛ບ㉳

ࡋ࡚⥳Ⰽࡢ⺯ගࢆ᳨ฟࡋࡓࠋ୍᪉ࠊࢡࣟࣟࣇ࢕ࣝ࡟⏤᮶ࡍࡿඹ⏕⸴ࡢ⮬ᐙ⺯ග

ࢆほᐹࡍࡿࡓࡵ࡟ࠊU-MWIG3 ࣇ࢕ࣝࢱ࣮ࢭࢵࢺ㸦࢜ࣜࣥࣃࢫ㸧ࢆ⏝࠸࡚ヨᩱ

ࢆ⥳Ⰽග࡛ບ㉳ࡋࠊ㉥Ⰽࡢ⺯ගࢆ᳨ฟࡋࡓࠋ⣽⬊ෆ࡟ᏑᅾࡍࡿGFPᶆ㆑㓝ẕࡢ ィᩘࡣࠊྛ᫬้࡟ࡘࡁ10⣽⬊௨ୖ࡟ࡘ࠸࡚⾜ࡗࡓࠋ

2.4 GFPᶆ㆑㓝ẕࡢᾘ໬ࡢ ᐃ

๓⠇࡜ྠᵝ࡟⣽⬊ෆࡢ㓝ẕࢆᾘ໬ࡉࡏࡓ࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊ᠱ⃮ᾮࢆࠊ GFP ᶆ㆑㓝ẕᠱ⃮ᾮ࡜ࡑࢀࡒࢀࡢ⣽⬊⃰ᗘࡀ 1-2™10³ cells/ml ࡜ 5™10⁷

cells/ml࡟࡞ࡿࡼ࠺࡟ΰྜࡋ࡚5ศ㛫㟼⨨ࡍࡿࡇ࡜࡟ࡼࡗ࡚ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩ

࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࡓࠋࡇࡢΰྜᾮࢆ✰ᚄ11 Pmࡢࢼ࢖ࣟࣥࢿࢵࢺࣇ

࢕ࣝࢱ࣮࡟㏻ࡋࠊྲྀࡾ㎸ࡲࢀ࡞࠿ࡗࡓ㓝ẕࢆ㝖ཤࡋࡓࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡣࣆ

࣌ࢵࢺ࡛ᅇ཰ࡋࠊ24Υ࡛ගࢆ↷ᑕࡋ࡞ࡀࡽ㟼⨨ࡋࡓࠋ㓝ẕࢆ㝖ཤࡋࡓ᫬้ࢆ 0

࡜ࡋ࡚ࠊ10ศ, 30ศ, 1᫬㛫, 1.5᫬㛫, 2᫬㛫, 2.5᫬㛫, 3᫬㛫ᚋ࡟⣽⬊ෆ࡟ṧᏑ ࡋ࡚࠸ࡿGFPᶆ㆑㓝ẕࢆィᩘࡲࡓࡣ᧜ᙳࡋࡓࠋ

GFPᶆ㆑㓝ẕࡢィᩘ࡜᧜ᙳࡣࠊ௨ୗࡢࡼ࠺࡟⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚⾜ࡗࡓࠋ⣽

⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ㓝ẕ࡟ࡘ࠸࡚ࡣࠊୖグࡢΰྜᾮ࠿ࡽྛ᫬㛫࡟ 7 Pl ᥇ྲྀࡋ࡚

ࢫࣛ࢖ࢻ࢞ࣛࢫ࡟⁲ୗࡋࠊ࢝ࣂ࣮࢞ࣛࢫࢆ࠿ࡪࡏ࡚࡯ࡰືࡁࡀ೵Ṇࡋࡓ⣽⬊ࡢ

ࡶࡢࢆィᩘࡋࡓࠋ⣽⬊እ࡟࠶ࡿ㓝ẕ࡟ࡘ࠸࡚ࡣࠊࡲࡎ┤ᚄ⣙ 5 mm ࡢ✰ࢆ࠶ࡅ ࡓࢩࣜࢥࣥࢦ࣒ࢆࢫࣛ࢖ࢻ࢞ࣛࢫ࡟ᙇࡾ௜ࡅࡓࠋΰྜᾮࢆࣆ࣌ࢵࢸ࢕ࣥࢢ࡟ࡼ

ࡗ࡚ࡼࡃ᧠ᢾࡋ࡚࠿ࡽ 20 Pl ᥇ྲྀࡋࠊ6%࣐࣍ࣝࣜࣥࢆ➼㔞ຍ࠼࡚⣽⬊ࢆᅛᐃࡋ

࡚࠿ࡽࠊࢩࣜࢥࣥࢦ࣒ෆ࡟ධࢀࡓࠋᾮ୰ࡢ඲࡚ࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡜⣽⬊እ࡟

࠶ࡿ㓝ẕࡢ⣽⬊ᩘࢆィᩘࡋࡓࠋࢩࣜࢥࣥࢦ࣒ෆࡢᾮࡣཌࡳࡀ࠶ࡿࡢ࡛ࠊࣆࣥࢺ

9

ࢆ㢖⦾࡟ኚ࠼࡞ࡀࡽィᩘࢆ⾜ࡗࡓࠋࡇࢀࡽࡢヨᩱࡣࠊ๓⠇࡜ྠࡌ᮲௳ࠊྠࡌᑐ

≀ࣞࣥࢬ࡜⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚ほᐹࡋࡓࠋ⺯ගീࡣࣔࣀࢡࣟ CCD ࣓࢝ࣛ

WAT-120N+㸦Watec㸧࡛᧜ᙳࡋࠊUSB᥋⥆ࡢA/Dኚ᥮ჾUSB VideoLink 2.0

㸦Zarbeco㸧࡜௜ᒓࡢࢯࣇࢺ࢙࢘࢔ࢆ⏝࠸࡚ࣃࢯࢥࣥ࡟ྲྀࡾ㎸ࢇࡔࠋྲྀࡾ㎸ࢇࡔ

⏬ ീ ࡣ ࠊ ࢜ ࣮ ࣉ ࣥ ࢯ ࣮ ࢫ ࡢ ⏬ ീ ฎ ⌮ ࢯ ࣇ ࢺ ࢘ ࢙ ࢔ ImageJ (http://rsbweb.nih.gov/ij/)࠾ࡼࡧᙜ◊✲ᐊసᡂࡢࣉࣛࢢ࢖ࣥ㸦ᮍබ㛤㸧࡟ࡼࡗ࡚

ࢥࣥࢺࣛࢫࢺቑᙉ࣭╔Ⰽ࣭ྜᡂࢆ⾜ࡗࡓࠋ

2.5 GFPᶆ㆑㓝ẕࡢ⣽⬊እ࡬ࡢ᤼ฟࡢほᐹ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࢆ⏕ࡁ࡚࠸ࡿ≧ែ࡛㛗᫬㛫ほᐹࡍࡿࡓࡵ࡟ࠊ࣑ࢻࣜࢰ࢘ࣜ

࣒ࢩࢆࢥ࣮ࣥࢫࢱ࣮ࢳ࡜ΰྜࡋࠊࢹࣥࣉࣥ⢏ࡢ㛫࡟㛢ࡌ㎸ࡵࡿࡇ࡜࡟ࡼࡗ࡚ࠊ 㐠ືࢆไ㝈ࡋࡓࠋᐇ㝿࡟ࡣࠊ๓⠇ࡢࡼ࠺࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࡓ࣑ࢻࣜ

ࢰ࣒࢘ࣜࢩࢆᅇ཰ࡋ࡚࠿ࡽ1᫬㛫ᚋ࡟࣑ࢻࣜࢰ࣒࢘ࣜࢩࢆྵࡴᾮ10 Pl࡜80㸣 ࢥ࣮ࣥࢫࢱ࣮ࢳ㸦⣽ᕝ〇㯝㸧⁐ᾮ20 Plࢆΰྜࡋࠊΰྜᾮࢆࢫࣛ࢖ࢻ࢞ࣛࢫ࡟5 Pl

⁲ୗࡋࡓࠋࡑࡢᚋࡍࡄ࡟ࠊ࠶ࡽ࠿ࡌࡵᅄ㝮࡟┿✵⏝ࢢ࣮ࣜࢫ㸦ᮾ࣭ࣞࢲ࢘ࢥ࣮

ࢽࣥࢢ㸹FS50㸧ࢆሬࡗ࡚࠾࠸ࡓ࢝ࣂ࣮࢞ࣛࢫࢆࡍࡤࡸࡃ࠿ࡪࡏࡓࠋࡉࡽ࡟ࠊ࢟

࣒࣡࢖ࣉࢆ࢝ࣂ࣮࢞ࣛࢫ࡟࠿ࡪࡏࠊࢥ࣮ࣥࢫࢱ࣮ࢳࡀࢫࣛ࢖ࢻ࢞ࣛࢫୖ࡟୍ᵝ

࡟ᣑᩓࡍࡿࡼ࠺࡟ୖ࠿ࡽᣦ࡛ᢲࡋࡓࠋ⁐ᾮࡀ⵨Ⓨࡍࡿࡢࢆ㜵ࡄࡓࡵ࡟ࠊ࢝ࣂ࣮

࢞ࣛࢫࡢ࿘ᅖࢆࢩࣜࢥࣥ࢜࢖࡛ࣝᑒධࡋࡓࠋヨᩱࡢほᐹ࡜᧜ᙳࡣࠊ๓⠇࡜ྠᵝ

࡟⺯ග㢧ᚤ㙾࡜CCD࣓࢝ࣛࢆ⏝࠸࡚⾜ࡗࡓࠋࡓࡔࡋࠊᑐ≀ࣞࣥࢬࡣ20ಸࡢࡶ

ࡢࠊග※ࡣ㧗ᅽỈ㖟ࣛࣥࣉࡢගࢆNDࣇ࢕ࣝࢱ࣮࡟ࡼࡗ࡚1.5%࡟ῶගࡋ࡚⏝࠸ࠊ

࣓࢝ࣛࡣࢤ࢖ࣥࢆ᭱኱࡜ࡋ࡚2ࣇ࣮࣒ࣞࡎࡘ✚⟬ࡋ࡚᧜ᙳࡋࡓࠋ

2.6 㓝ẕࡢ⣽⬊ཱྀ࡬ࡢ㐠ᦙࡢほᐹ

CongoRedᰁⰍ㓝ẕࡣࠊḟࡢࡼ࠺࡟ㄪ〇ࡋࡓࠋࡲࡎࠊYPDᇵᆅ࡛1ᬌ᣺࡜࠺

ᇵ㣴ࡋࡓฟⱆ㓝ẕBY4741ᰴࡢᇵ㣴ᾮ200 Plࢆẖศ3000ᅇ㌿࡛5ศ㛫㐲ᚰࡋ

ୖ Ύ ࢆ ྾ ࠸ ྲྀ ࡗ ࡚ ࠿ ࡽ ࠊAW 100 Pl ࡛ ᠱ ⃮ ࡋ ࡓ ࠋ ࡇ ࡢ ᠱ ⃮ ᾮ ࢆ 0.05㸣 CongoRed 500 Pl࡜ΰྜࡋࠊ100Υ࡟1᫬㛫⨨࠸ࡓࠋࡑࡢᚋࠊ㐲ᚰ࡜AW 500 Pl

࡟ࡼࡿὙίࢆ3ᗘ⧞ࡾ㏉ࡋ࡚࠿ࡽࠊ᭱ᚋ࡟ࡶ࠺୍ᗘ㐲ᚰࡋ࡚AW 100 Pl࡛ᠱ⃮

ࡋࡓࠋࡇࡢ CongoRed ᰁⰍ㓝ẕࢆᑐᩘቑṪᮇࡲࡓࡣᐃᖖᮇࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩ

10

࡜ΰྜࡋ࡚࠿ࡽࢫࣛ࢖ࢻ࢞ࣛࢫ࡟7 Pl⁲ୗࡋࠊගᏛ㢧ᚤ㙾࡛ほᐹࡋࡓࠋ

11 3 ⤖ᯝ

3.1 㓝ẕࢆ㣵࡜ࡋࡓሙྜࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢቑṪ᭤⥺

ࡲࡎ㓝ẕࡀࡑࡶࡑࡶ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵࡜ࡋ࡚㐺ࡋ࡚࠸ࡿ࠿ࢆ☜࠿ࡵࡿࡓ

ࡵ࡟ࠊฟⱆ㓝ẕࢆ㣵࡜ࡋ࡚࣑ࢻࣜࢰ࣒࢘ࣜࢩࢆᇵ㣴ࡋࠊࡑࡢቑṪࢆㄪ࡭ࡓࠋᅗ1

࡟ࠊ⣽⬊ෆ࡟ඹ⏕⸴ࢆᣢࡘ㔝⏕ᰴ࠾ࡼࡧேᕤⓗ࡟ඹ⏕⸴ࢆ㝖ཤࡋࡓⓑ໬㸦㠀ඹ

⏕㸧ᰴࡢቑṪ᭤⥺ࢆ♧ࡍࠋࢰ࣒࢘ࣜࢩ㢮ࡢᇵ㣴࡛ࡼࡃ⏝࠸ࡽࢀࡿࠊ⣽⳦ࡢ

Klebsiella ࢆຍ࠼ࡓࣞࢱࢫᾐฟᾮࢆ⏝࠸࡚࣑ࢻࣜࢰ࣒࢘ࣜࢩࢆᇵ㣴ࡋࡓሙྜࡣࠊ

ᇵ㣴㛤ጞ࠿ࡽ1㐌㛫࡛⣙1500 cells/mlࡲ࡛ቑṪࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ(11)ࠋ

୍᪉ࠊ㓝ẕࢆ㣵࡜ࡋࡓሙྜࡣࠊᇵ㣴㛤ጞ࠿ࡽ1㐌㛫࡛⣙2000 cells/mlࡲ࡛ቑṪ ࡋࡓࡇ࡜࠿ࡽࠊ㓝ẕࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵࡜ࡋ࡚㐺ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟

࡞ࡗࡓࠋࡲࡓ௨๓ࡢ◊✲࡜ྠᵝ࡟(11, 12)ࠊ㓝ẕࢆ㣵࡜ࡋࡓሙྜࡶࠊ㔝⏕ᰴࡀ㣵

ࢆᾘ㈝ࡋᑾࡃࡋ࡚ᐃᖖᮇ࡟ධࡗ࡚࠿ࡽࡶ㛗ᮇ㛫⏕Ꮡࡋ⥆ࡅࡿࡢ࡟ᑐࡋ࡚ࠊⓑ໬

ᰴࡣ㣵ࢆᾘ㈝ࡋᑾࡃࡋ࡚ࡋࡤࡽࡃࡍࡿ࡜ᛴ㏿࡟Ṛ⁛ࡋࡓࠋ

ᅗ

ᅗ1. 㓝ẕࢆ㣵࡜ࡋࡓሙྜࡢ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢቑṪ᭤⥺ࠋ㔝⏕ᰴࡣHA1ᰴࠊⓑ໬ᰴࡣHA1 㹵3ᰴࢆ⏝࠸ࡓࠋ

12 3.2 GFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳཬࡧ᤼ฟࡢほᐹ

ḟ࡟ࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓGFPᶆ㆑㓝ẕ࡜ඹ⏕⸴ࢆ༊ู࡛ࡁࡿ࠿࡝࠺࠿ㄪ

࡭ࡿࡓࡵ࡟ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㔝⏕ᰴ⣽⬊࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࠊ⣽

⬊እࡢ㓝ẕࢆ㝖ཤࡋ࡚࠿ࡽ୍ᐃ᫬㛫⤒㐣ᚋ࡟⺯ග㢧ᚤ㙾࡛⣽⬊ෆࡢඹ⏕⸴࡜

GFP ᶆ㆑㓝ẕࢆほᐹࡋࡓࠋ2 ✀㢮ࡢࣇ࢕ࣝࢱ࣮ࢭࢵࢺࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ࠊ ࢡࣟࣟࣇ࢕ࣝ࡟⏤᮶ࡍࡿඹ⏕⸴ࡢ㉥Ⰽࡢ⮬ᐙ⺯ග࡜GFPࡢ⥳Ⰽࡢ⺯ගࢆࡑࢀࡒ

ࢀูࠎ࡟᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡓ㸦ᅗ2㸧ࠋ㓝ẕࡣ1ಶࡲࡓࡣ」ᩘಶࡎࡘ㣗⬊࡟ྲྀ

ࡾ㎸ࡲࢀ࡚࠾ࡾࠊඹ⏕⸴ࡀᏑᅾࡋ࡚࠸ࡿඹ⏕⬊࡜఩⨨ࡀ㔜࡞ࡿࡇ࡜ࡣ࡞࠿ࡗࡓࠋ ᖖ࡟⣽⬊㉁඲య࡟୍ᐃᩘᏑᅾࡋ࡚࠸ࡓඹ⏕⸴࡜ࡣ␗࡞ࡾࠊእ㒊ࡢ㓝ẕࢆ㝖ࡃ࡜

⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿ㓝ẕࡢᩘࡣࠊ᫬㛫࡜࡜ࡶ࡟ῶᑡࡋࡓࠋ௨ୖࡼࡾࠊGFP ᶆ

㆑㓝ẕ࡜⺯ග㢧ᚤ㙾ࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ࠊඹ⏕⸴࡜⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ㓝 ẕࢆ༊ู࡛ࡁࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ

ⓑ໬ᰴ࡟ࡘ࠸࡚ࡶྠᵝ࡟ࠊGFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏ࡚ほᐹࡋࡓ࡜ࡇࢁࠊඹ

⏕⸴ࡢ࡞࠸ⓑ໬ᰴࡶࠊ㔝⏕ᰴ࡜ྠᵝ࡟㓝ẕࢆ⣽⬊ෆ࡟ྲྀࡾ㎸ࡴࡇ࡜ࡀศ࠿ࡗࡓ 㸦ᅗ 3㸧ࠋ࡞࠾ⓑ໬ᰴࡢ⣽⬊ෆ࡟ࡶ㉥Ⰽࡢ⺯ගࢆฟࡍ≀యࡀほᐹࡉࢀࡿࡇ࡜ࡀ࠶

ࡗࡓࡀ㸦▮ᑼ㸧ࠊ㏻ᖖࡢග᮲௳࡛ⓑ໬ᰴࡢᇵ㣴ࢆ⥆ࡅ࡚ࡶࢡࣟࣞࣛᵝࡢ⸴㢮ࡀቑ Ṫࡍࡿࡇ࡜ࡣ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊඹ⏕⸴࡛ࡣ࡞ࡃ㉥Ⰽࡢ⮬ᐙ⺯ගࢆฟࡍఱࡽ࠿

ࡢ⣽⬊ෆᵓ㐀≀࡜⪃࠼ࡽࢀࡿࠋ

13 ᅗ

ᅗ2. 㔝⏕ᰴ㸦ᇵ㣴7᪥┠㸧ࡢ㣗స⏝ࡢほᐹࠋGFPᶆ㆑㓝ẕࠊඹ⏕⸴ࡢ⺯ගീࠊ࠾ࡼ

ࡧ୧⪅ࡢྜᡂ╔Ⰽ⏬ീ㸦GFPᶆ㆑㓝ẕࡣ⥳Ⰽ࡟ࠊඹ⏕⸴ࡣ㉥Ⰽ࡟╔Ⰽࡋࡓ㸧ࢆ♧ࡋࡓࠋ ࢫࢣ࣮ࣝࡣ50 Pmࠋ

50 Pm Ὑ

ί 10 ศ ᚋ

1

᫬ 㛫 ᚋ

2

᫬ 㛫 ᚋ

GFPᶆ㆑㓝ẕ ඹ⏕⸴ ྜᡂ╔Ⰽ⏬ീ

14 ᅗ

ᅗ3. ⓑ໬ᰴ㸦ᇵ㣴7᪥┠㸧ࡢ㣗స⏝ࡢほᐹࠋGFPᶆ㆑㓝ẕࠊඹ⏕⸴ࡢ⺯ගീࠊ࠾ࡼࡧ୧

⪅ࡢྜᡂ╔Ⰽ⏬ീ㸦GFPᶆ㆑㓝ẕࢆ⥳Ⰽ࡟╔Ⰽࡋࡓ㸧ࠋࢫࢣ࣮ࣝࡣ50 Pmࠋⓑ໬ᰴ࡟ඹ⏕

⸴ࡣぢࡽࢀ࡞࠸ࠋ

ࡇࡢGFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࢇࡔ࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊ࢆ᥇ྲྀࡋࠊ⏕ࡁ࡚࠸ࡿ

≧ែ࡛⣙ 100 ศ㛫ほᐹࡋ⥆ࡅࡓ࡜ࡇࢁࠊࡓࡲࡓࡲ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀ㓝ẕࢆ⣽

⬊እ࡬᤼ฟࡍࡿ࡜ࡇࢁࢆほᐹࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋᅗ 4 ࡟㓝ẕࢆ᤼ฟࡍࡿ▐㛫ࡢ 㐃⥆෗┿ࢆ♧ࡍࠋᅗࡢࡼ࠺࡟ࠊ᤼ฟࡉࢀࡓGFPᶆ㆑㓝ẕࡣࡋࡤࡽࡃほᐹࡍࡿࡇ

GFPᶆ㆑㓝ẕ ඹ⏕⸴ ྜᡂ╔Ⰽ⏬ീ

Ὑ ί 10 ศ ᚋ

1

᫬ 㛫 ᚋ

2

᫬ 㛫 ᚋ

50 Pm

15

࡜ࡀྍ⬟࡛࠶ࡾࠊ⺯ගࡀᾘኻࡍࡿᵝᏊࡣ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊᮍᾘ໬ࡢGFPᶆ㆑

㓝ẕࡀ⣽⬊እ࡟᤼ฟࡉࢀ࡚ࡶ⺯ග㢧ᚤ㙾࡟ࡼࡗ᳨࡚ฟྍ⬟࡛࠶ࡿࡇ࡜ࡀศ࠿ࡗ ࡓࠋ

ᅗᅗ 4. 㔝⏕ᰴࡢ⣽⬊ࡀGFPᶆ㆑㓝ẕࢆ᤼ฟࡍࡿ▐㛫ࡢ㐃⥆෗┿ࠋᩘᏐࡣ᫬㛫㸦⛊㸧ࠋ▮ᑼ ࡣ⣽⬊እ࡟᤼ฟࡉࢀࡓGFPᶆ㆑㓝ẕࢆ♧ࡍࠋ

3.3 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶ㸦1㸧ඹ⏕⸴ࡢ᭷↓࡟ࡼࡿ㐪࠸

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝࡟࠾ࡅࡿ㣵ࡢྲྀࡾ㎸ࡳࢆᐃ㔞ⓗ࡟ㄪ࡭ࡿࡓࡵ࡟ࠊ

࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏ࡚࠿ࡽ୍ᐃ᫬㛫ࡈ࡜࡟⣽⬊ࢆᅛ ᐃࡋࠊྲྀࡾ㎸ࡲࢀࡓ㓝ẕࡢᩘࢆィ ࡋࡓ㸦ᅗ 5㸧ࠋᅗ࡟ࡣࡑࢀࡒࢀࡢ᮲௳࡟࠾࠸

࡚ࠊ᭱ࡶྲྀࡾ㎸ࢇࡔ㔞ࡀከ࠿ࡗࡓ᫬ࡢࢹ࣮ࢱࢆ♧ࡋࡓࠋ㔝⏕ᰴ࡜ⓑ໬ᰴࡢ࡝ࡕ

ࡽࡶࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ㓝ẕࡢᩘࡣ᫬㛫࡜࡜ࡶ࡟ቑຍࡋࠊ10-20ศ⛬ᗘ࡛㣬

࿴ࡋࡓࠋྲྀࡾ㎸ࡳ㛤ጞ࠿ࡽ20ศᚋ࡟㔝⏕ᰴ࡜ⓑ໬ᰴࡀྲྀࡾ㎸ࢇࡔ㓝ẕࡢᩘࢆẚ

㍑ࡋࡓ࡜ࡇࢁࠊⓑ໬ᰴࡢ᪉ࡀ㔝⏕ᰴࡼࡾྲྀࡾ㎸ࡴ㓝ẕࡢ㔞ࡀ᭷ព࡟ከ࠿ࡗࡓ 㸦Mann-Whitney ᳨ᐃ, P < 0.01㸧ࠋࡇࡢ⤖ᯝ࠿ࡽࠊ㣵ࡢྲྀࡾ㎸ࡳάᛶࡣⓑ໬ᰴ

ࡢ᪉ࡀ㔝⏕ᰴࡼࡾࡶ㧗ࡃࠊඹ⏕⸴ࡢᏑᅾࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶ

ࢆపୗࡉࡏࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ࡞࠾ࠊⓑ໬ᰴࡢ᪉ࡀ㔝⏕ᰴࡼࡾࡶ㣵ࡢྲྀࡾ㎸ࡳ

㔞ࡀከ࠸ࡇ࡜ࡣࠊ⺯ගࣅ࣮ࢬࢆ⏝࠸ࡓ◊✲࡛᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡿ㸦6㸧ࠋ

0.0 0.6

2.4 3.0 3.6 4.2

1.2 1.8

16

ᅗᅗ 5. 㔝⏕ᰴ࡜ⓑ໬ᰴ࡟ࡼࡿGFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳࠋ᫬㛫ࡣࠊྲྀࡾ㎸ࡳࢆ㛤ጞࡋ࡚࠿ࡽ

⤒㐣ࡋࡓ᫬㛫ࢆ⾲ࡍࠋ⣽⬊ෆࡢGFPᶆ㆑㓝ẕࡢᩘࢆࡑࢀࡒࢀ10ಶయ௨ୖ࡟ࡘ࠸࡚ ᐃࡋࠊ ᖹᆒ࡜ᶆ‽೫ᕪ㸦Წ㸧ࢆ♧ࡋࡓࠋ

ⓑ໬ᰴࡀ㔝⏕ᰴࡼࡾࡶከࡃ㓝ẕࢆྲྀࡾ㎸ࢇࡔ⌮⏤࡜ࡋ࡚ࠊඹ⏕⸴ࡀᏑᅾࡋ࡞

࠸ⓑ໬ᰴࡢ᪉ࡀ༢࡟⣽⬊ෆࡢ✵࠸࡚࠸ࡿ✵㛫ࡀᗈ࠸ࡓࡵከࡃࡢ㓝ẕࢆྲྀࡾ㎸ࡴ

ࡇ࡜ࡀ࡛ࡁࡿ࡜࠸࠺ྍ⬟ᛶࡀ࠶ࡿࠋࡑࡇ࡛ࠊඹ⏕⸴ࡢᩘࢆῶࡽࡋࡓ࣑ࢻࣜࢰ࢘

࣒ࣜࢩࢆ⏝࠸࡚ࠊ⣽⬊ෆࡢඹ⏕⸴ࡢᩘ࡜ྲྀࡾ㎸ࡴ㓝ẕࡢᩘࡢ┦㛵ࢆㄪ࡭ࡓ㸦ᅗ6㸧ࠋ ᜏᬯ᮲௳࡛ 4 ᪥㛫ᇵ㣴ࡍࡿࡇ࡜࡟ࡼࡗ࡚ඹ⏕⸴ࢆῶᑡࡉࡏࡓ㔝⏕ᰴࡢ⣽⬊㸦1

⣽⬊࠶ࡓࡾࡢᖹᆒඹ⏕⸴ᩘ㸸62.8㸧࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࠊ20ศᚋ࡟⣽

⬊ෆࡢඹ⏕⸴ᩘ࡜ྲྀࡾ㎸ࡲࢀࡓ㓝ẕࡢᩘࡢ୧᪉ࢆィᩘࡋࡓࠋࡑࡢ⤖ᯝࠊ࣑ࢻࣜ

ࢰ࣒࢘ࣜࢩ⣽⬊ෆࡢඹ⏕⸴ᩘ࡜ྲྀࡾ㎸ࡴ㓝ẕࡢᩘ࡟ࡣᙅ࠸ṇࡢ┦㛵ࡣ࠶ࡗࡓࡶ

ࡢࡢ㸦┦㛵ಀᩘ㸸㸩0.38㸧ࠊ≉࡟㈇ࡢ┦㛵ࡣ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊ⣽⬊ෆࡢඹ⏕⸴

ࡀᑡ࡞ࡅࢀࡤከࡃࡢ㓝ẕࢆྲྀࡾ㎸ࡴࢃࡅ࡛ࡣ࡞࠸࡜࠸࠺ࡇ࡜ࡀศ࠿ࡗࡓࠋ

17

ᅗᅗ 6. ⣽⬊ෆࡢඹ⏕⸴ᩘ࡜ྲྀࡾ㎸ࢇࡔGFPᶆ㆑㓝ẕࡢᩘࡢ㛵ಀࠋ⣽⬊ࡣ㔝⏕ᰴࢆᜏᬯ᮲௳

࡛4᪥㛫ᇵ㣴ࡋࡓࡶࡢࢆ⏝࠸ࡓࠋ30⣽⬊࡟ࡘ࠸࡚ࡢ⤖ᯝࢆ♧ࡍࠋ

3.4 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶ㸦2㸧ቑṪ᫬ᮇ࡟ࡼࡿ㐪࠸

ḟ࡟ࠊቑṪ᫬ᮇ࡟ࡼࡗ࡚㣵ࡢྲྀࡾ㎸ࡳ࡟㐪࠸ࡀ࠶ࡿ࠿࡝࠺࠿ࢆㄪ࡭ࡿࡓࡵ࡟ࠊ ᑐᩘቑṪᮇ࠾ࡼࡧ㣵ࡢ㓝ẕࡀᯤῬࡋ࡚ᐃᖖᮇ࡟ධࡗࡓ㔝⏕ᰴࡢ⣽⬊࡟ࡘ࠸࡚ࠊ 㓝ẕࡢྲྀࡾ㎸ࡳࢆẚ㍑ࡋࡓ㸦ᅗ5㸧ࠋࡑࡢ⤖ᯝࠊᐃᖖᮇ㸦ᇵ㣴14᪥┠㸧ࡢ⣽⬊ࡣࠊ ᑐᩘቑṪᮇ㸦ᇵ㣴 4 ᪥┠㸧ࡢ⣽⬊࡜ẚ㍑ࡋ࡚᭷ព࡟ྲྀࡾ㎸ࡴ㓝ẕࡢᩘࡀᑡ࡞࠸

ࡇ࡜ࡀศ࠿ࡗࡓ㸦Mann-Whitney᳨ᐃ, P < 0.025㸧ࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀᐃᖖᮇ

࡟ධࡿ࡜㣵ࡢྲྀࡾ㎸ࡳάᛶࡀపࡃ࡞ࡿཎᅉࢆ᥈ࡿࡓࡵ࡟ࠊᐃᖖᮇࡢ⣽⬊࡟㓝ẕ

ࢆ୚࠼࡚ቑṪࢆ෌㛤ࡉࡏ࡚࠿ࡽྲྀࡾ㎸ࡴ㓝ẕࡢᩘࢆㄪ࡭ࡓ㸦ᅗ 7㸧ࠋࡑࡢ⤖ᯝࠊ ᐃᖖᮇࡢ⣽⬊࡟㣵ࡢ㓝ẕࢆ୚࠼ࡿ࡜ࠊ2᪥࡛ྲྀࡾ㎸ࡳάᛶࡀᑐᩘቑṪᮇࡢ⣽⬊࡜

ྠ⛬ᗘ࡟ᡠࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ୍᪉࡛ࠊ㓝ẕࢆ୚࠼ࡿ㝿࡟ྠ᫬࡟࣑ࢻࣜࢰ࢘ࣜ

࣒ࢩࡢࢱࣥࣃࢡ㉁ࡢྜᡂࢆ㜼ᐖࡍࡿPuromycin㸦13㸧ࡶຍ࠼ࡿ࡜ࠊྲྀࡾ㎸ࡳά ᛶࡣᐃᖖᮇࡢ⣽⬊࡜ኚࢃࡽ࡞࠸ࡲࡲࡔࡗࡓ㸦ᅗ 7㸧ࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ࣑ࢻࣜࢰ

࣒࢘ࣜࢩࡣ࿘ᅖ࡟㣵ࡀ࠶ࡗ࡚ቑṪࢆ㛤ጞࡍࡿ࡜ࠊ㣵ࢆྲྀࡾ㎸ࡴࡢ࡟ᚲせ࡞ࢱࣥ

ࣃࢡ㉁ࢆከࡃⓎ⌧ࡍࡿࡼ࠺࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋᐃᖖᮇ࡟ධࡿ࡜ྲྀࡾ㎸ࡳάᛶ ࡀపࡃ࡞ࡿࡢࡣࠊࡑࡢࡼ࠺࡞ࢱࣥࣃࢡ㉁ࡢⓎ⌧ࡀᢚไࡉࢀࡿࡓࡵ࡛࠶ࢁ࠺ࠋ

18

ᅗᅗ 7. 㔝⏕ᰴࡢᑐᩘቑṪᮇ࡜ᐃᖖᮇ࡟࠾ࡅࡿGFPᶆ㆑㓝ẕࡢྲྀࡾ㎸ࡳࠋ᫬㛫ࡣࠊྲྀࡾ㎸ࡳ

ࢆ㛤ጞࡋ࡚࠿ࡽ⤒㐣ࡋࡓ᫬㛫ࢆ⾲ࡍࠋ⣽⬊ෆࡢGFPᶆ㆑㓝ẕࡢᩘࢆࡑࢀࡒࢀ10ಶయ௨ୖ

࡟ࡘ࠸࡚ ᐃࡋࠊᖹᆒ࡜ᶆ‽೫ᕪ㸦Წ㸧ࢆ♧ࡋࡓࠋ

࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟࠾ࡅࡿ㣵ࡢྲྀࡾ㎸ࡳࡣࠊ⣽⬊እ࡟࠶ࡿ㣵ࢆ⧄ẟ㐠ື࡟ࡼ

ࡗ࡚⣽⬊ཱྀ࡬㐠ᦙࡍࡿẁ㝵࡜ࠊ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟ࡼࡗ࡚⣽⬊ཱྀ࠿ࡽ㣗⬊࡟

ྲྀࡾ㎸ࡴẁ㝵ࡢ 2 ࡘ࡟ศࡅࡽࢀࡿࠋᐃᖖᮇ࡟࠾ࡅࡿ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ࡢྲྀ

ࡾ㎸ࡳ࡟࠾࠸࡚࡝ࡕࡽࡢẁ㝵ࡀᢚไࡉࢀ࡚࠸ࡿ࠿ࢆㄪ࡭ࡿࡓࡵ࡟ࠊ࣑ࢻࣜࢰ࢘

࣒ࣜࢩࡢ㔝⏕ᰴ࡟ CongoRed ࡛ᰁⰍࡋࡓ㓝ẕࢆ୚࠼ࠊࡑࡢ⣽⬊ཱྀ࡬ࡢ㐠ᦙࢆග Ꮫ㢧ᚤ㙾࡛ほᐹࡋࡓ㸦ᅗ 8㸧ࠋࡑࡢ⤖ᯝࠊᐃᖖᮇ࡟࠾࠸࡚ࡶࠊ⧄ẟ㐠ື࡟ࡼࡿ⣽

⬊ཱྀ࡬ࡢ㣵ࡢ㐠ᦙࡣᑐᩘቑṪᮇ࡜ྠᵝ࡟ṇᖖ࡟⾜ࢃࢀ࡚࠸ࡓࠋࡇࡢࡇ࡜࠿ࡽࠊ ᐃᖖᮇ࡟࠾࠸࡚Ⓨ⌧ࡀᢚไࡉࢀࡿࢱࣥࣃࢡ㉁ࡣࠊ⣽⬊ཱྀ࡬ࡢ㐠ᦙ࡛ࡣ࡞ࡃ࢚ࣥ

ࢻࢧ࢖ࢺ࣮ࢩࢫ࡟㛵୚ࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ㧗࠸ࠋ

19

ᅗᅗ 8. 㔝⏕ᰴ⣽⬊ࡢ⣽⬊ཱྀ࡟྾࠸㎸ࡲࢀࡿCongoRedᰁⰍ㓝ẕࡢほᐹ㸦▮༳㸧ࠋᕥഃࡀᑐ

ᩘቑṪᮇ㸦ᇵ㣴4᪥┠㸧ࡢ⣽⬊ࠊྑഃࡀᐃᖖᮇ㸦ᇵ㣴14᪥┠㸧ࡢ⣽⬊ࠋ

3.5 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᾘ໬άᛶ

࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝࡟࠾ࡅࡿྲྀࡾ㎸ࢇࡔ㣵ࡢᾘ໬ࢆᐃ㔞ⓗ࡟ㄪ࡭ࡿࡓ

ࡵ࡟ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ㎸ࡲࡏࠊ⣽⬊እࡢ㓝ẕࢆ㝖ཤࡋ

࡚࠿ࡽ୍ᐃ᫬㛫⤒㐣ᚋ࡟⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿ㓝ẕࡢᩘࢆィ ࡋࡓࠋ⣽⬊እ࡟

᤼ฟࡉࢀࡓศࢆぢ✚ࡶࡿࡓࡵ࡟ࠊྠ᫬࡟⣽⬊እ࡟Ꮡᅾࡋ࡚࠸ࡿ㓝ẕࡢᩘࡶィ  ࡋࡓࠋ ᐃࡢ1౛࡜ࡋ࡚ࠊᇵ㣴㛤ጞ࠿ࡽ4᪥┠ࡢ㔝⏕ᰴ࡟GFPᶆ㆑㓝ẕࢆྲྀࡾ

㎸ࡲࡏࡓሙྜࢆᅗ9࡟♧ࡍࠋ⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿ㓝ẕࡣ᫬㛫࡜࡜ࡶ࡟ῶᑡࡋࠊ 3᫬㛫࡛࡯࡜ࢇ࡝࡞ࡃ࡞ࡗࡓࠋࡑࢀ࡟ᑐࡋ࡚ࠊ⣽⬊እ࡟Ꮡᅾࡍࡿ㓝ẕࡢᩘࡣࠊ࣑

ࢻࣜࢰ࣒࢘ࣜࢩ⣽⬊እࡢ㓝ẕࢆ㝖࠸࡚࠿ࡽ10 ศ࡜ 2᫬㛫࡛࡯࡜ࢇ࡝ኚࢃࡽࡎࠊ

࣑ࢻࣜࢰ࣒࢘ࣜࢩ1 ⣽⬊࠶ࡓࡾ࠾࠾ࡼࡑ 1 ಶࡢࡲࡲࡔࡗࡓࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩ ࡀྲྀࡾ㎸ࢇࡔ㓝ẕࢆᾘ໬ࡏࡎ࡟᤼ฟࡋࡓ࡜ࡍࢀࡤࠊ⣽⬊ෆ࡛ῶᑡࡋࡓศ⣽⬊እ ࡢ㓝ẕࡢᩘࡣቑຍࡍࡿࡣࡎ࡞ࡢ࡛ࠊ⣽⬊ෆࡢ㓝ẕᩘࡀῶᑡࡋࡓࡢࡣ࡯࡜ࢇ࡝ࡀ ᾘ໬࡟ࡼࡿࡶࡢ࡜ゝ࠼ࡿࠋ࡞࠾ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀྲྀࡾ㎸ࢇࡔ㣵ࢆ඲࡚ᾘ໬

ࡍࡿࡢ࡟࠿࠿ࡿ᫬㛫ࡣࠊቑṪ᫬ᮇ࡟ࡼࡽࡎ࠾࠾ࡼࡑ 3 ᫬㛫ࡔࡗࡓ㸦ࢹ࣮ࢱࡣ♧

ࡉࡎ㸧ࠋ

20

ᅗᅗ 9. 㔝⏕ᰴ㸦ᇵ㣴4᪥┠㸧࡟࠾ࡅࡿGFPᶆ㆑㓝ẕࡢᾘ໬ࠋ᫬㛫ࡣࠊ㓝ẕࢆ㝖࠸࡚࠿ࡽ

⤒㐣ࡋࡓ᫬㛫ࢆ⾲ࡍࠋ⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿGFPᶆ㆑㓝ẕࡢᩘࢆࡑࢀࡒࢀ10⣽⬊௨ୖ࡟

ࡘ࠸࡚ ᐃࡋࠊᖹᆒ࡜ᶆ‽೫ᕪ㸦Წ㸧ࢆ㉥࡛♧ࡋࡓࠋ⣽⬊እ࡟ᏑᅾࡍࡿGFPᶆ㆑㓝ẕࡢᩘ

㸦࣑ࢻࣜࢰ࣒࢘ࣜࢩ1⣽⬊࠶ࡓࡾ㸧ࡣ㟷࡛♧ࡋࡓࠋ

㔝⏕ᰴ࡜ⓑ໬ᰴࡢᾘ໬άᛶࢆẚ㍑ࡍࡿࡓࡵ࡟ࠊGFP ᶆ㆑㓝ẕࢆ⏝࠸࡚୧⪅࡟

࠾ࡅࡿᾘ໬ࢆㄪ࡭ࡓ㸦ᅗ10㸧ࠋࡓࡔࡋ3.3⠇࡛㏙࡭ࡓ㏻ࡾࠊ㔝⏕ᰴ࡜ⓑ໬ᰴ࡛ࡣ

᭱ึ࡟ྲྀࡾ㎸ࡴ㓝ẕࡢᩘࡀ␗࡞ࡿࡓࡵࠊ⣽⬊ෆ࡟ṧᏑࡍࡿ㓝ẕࡢᩘࢆࡑࡢࡲࡲ

ẚ㍑ࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡑࡇ࡛ࠊᾘ໬ࡢ඲యࡀ1ẁ㝵ࡢ཯ᛂ࡛࠶ࡿ࡜௬ᐃࡋࠊ ṧᏑࡍࡿ GFP ᶆ㆑㓝ẕࡢᩘࢆᣦᩘ㛵ᩘ࡛㏆ఝࡋ࡚ࡑࡢ୍ḟ㏿ᗘᐃᩘࢆồࡵࡓࠋ ࡑࡢ⤖ᯝࠊᾘ໬ࡢぢ࠿ࡅࡢ୍ḟ㏿ᗘᐃᩘࡣࠊ㔝⏕ᰴࡀ 0.74 (h^-1)ࠊⓑ໬ᰴࡀ

0.61 (h^-1)࡜࡞ࡾࠊ࡯࡜ࢇ࡝ኚࢃࡽ࡞࠿ࡗࡓࠋ௨ୖࡼࡾࠊ㣵ࡢᾘ໬άᛶࡣ㔝⏕ᰴ

࡜ⓑ໬ᰴ࡛࡯࡜ࢇ࡝ኚࢃࡽࡎࠊඹ⏕⸴ࡢᏑᅾࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢᾘ໬άᛶ࡟

ࡣᙳ㡪ࡋ࡞࠸ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

21

ᅗᅗ 10. 㔝⏕ᰴ࡜ⓑ໬ᰴ࡟࠾ࡅࡿGFPᶆ㆑㓝ẕࡢᾘ໬ࠋ᫬㛫ࡣࠊ㓝ẕࢆ㝖࠸࡚࠿ࡽ⤒㐣ࡋ ࡓ᫬㛫ࢆ⾲ࡍࠋ⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿGFPᶆ㆑㓝ẕࡢᩘࢆࡑࢀࡒࢀ10⣽⬊௨ୖ࡟ࡘ࠸࡚

 ᐃࡋࠊᖹᆒ࡜ᶆ‽೫ᕪ㸦Წ㸧ࢆ♧ࡋࡓࠋ᭤⥺ࡣᣦᩘ㛵ᩘ࡟ࡼࡿ㏆ఝࠋ

3.6 ࢰ࣒࢘ࣜࢩࡢ㣗స⏝

࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡜ྠᒓ࡛࠶ࡿࡀඹ⏕⸴ࢆࡶࡓ࡞࠸ࢰ࣒࢘ࣜࢩ㸦Paramecium caudatum^{㸧࡟ࡘ࠸࡚ࡶࠊ}GFPᶆ㆑㓝ẕࢆ⏝࠸࡚㣗స⏝ࢆㄪ࡭ࡓ㸦ᅗ11㸧ࠋࢰ࢘

࣒ࣜࢩࡢ⣽⬊ࡢࢧ࢖ࢬࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡼࡾࡶ኱ࡁ࠸࡟ࡶ㛵ࢃࡽࡎࠊ1⣽⬊࠶

ࡓࡾྲྀࡾ㎸ࡴ㓝ẕࡢᩘࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡼࡾࡶᑡ࡞࠿ࡗࡓࠋࡲࡓ⣽⬊ࡈ࡜ࡢ ᕪࡶ኱ࡁ࠿ࡗࡓࠋࡑࡇ࡛ࠊ㓝ẕࢆ㣵࡜ࡋ࡚ࢰ࣒࢘ࣜࢩࢆᇵ㣴ࡋࠊቑṪࢆㄪ࡭ࡓ 㸦ᅗ12㸧ࠋKlebsiellaࢆຍ࠼ࡓࣞࢱࢫᾐฟᾮ࡛ࢰ࣒࢘ࣜࢩࢆᇵ㣴ࡋࡓሙྜࡣࠊ⣙

1500 cells/mlࡲ࡛ቑṪࡋࠊࡑࡢᚋ2㐌㛫௨ୖ⏕Ꮡࡋ⥆ࡅࡓࡢ࡟ᑐࡋࠊ㓝ẕࢆ㣵

࡜ࡋ࡚ᇵ㣴ࡋࡓሙྜቑṪࡣ㐜ࡃࠊ9᪥┠࡛඲⁛࡟⮳ࡗࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊࢰ࢘

࣒ࣜࢩࡣ㓝ẕࢆ࠶ࡲࡾᦤྲྀ࣭฼⏝࡛ࡁ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

22

ᅗᅗ 11. ࢰ࣒࢘ࣜࢩHC3ᰴ࡟࠾ࡅࡿGFPᶆ㆑㓝ẕࡢᾘ໬ࠋ᫬㛫ࡣࠊ㓝ẕࢆ㝖࠸࡚࠿ࡽ⤒㐣 ࡋࡓ᫬㛫ࢆ⾲ࡍࠋ⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿGFPᶆ㆑㓝ẕࡢᩘࢆࡑࢀࡒࢀ10⣽⬊௨ୖ࡟ࡘ࠸

࡚ ᐃࡋࠊᖹᆒ࡜ᶆ‽೫ᕪ㸦Წ㸧ࢆ㉥࡛♧ࡋࡓࠋ⣽⬊እ࡟ᏑᅾࡍࡿGFPᶆ㆑㓝ẕࡢᩘ㸦࣑

ࢻࣜࢰ࣒࢘ࣜࢩ1⣽⬊࠶ࡓࡾ㸧ࡣ㟷࡛♧ࡋࡓࠋHC3ᰴࡣࠊ2012ᖺ࡟ᙜ◊✲ᐊ࡟ࡼࡗ࡚ᘯ ๓ᕷࡔࢇࡪࡾụࡼࡾ᥇㞟ࡉࢀࡓࠋ

ᅗ 12. Klebsiellaࢆຍ࠼ࡓࣞࢱࢫᾐฟᾮࡲࡓࡣฟⱆ㓝ẕࢆ㣵࡜ࡋ࡚ᇵ㣴ࡋࡓሙྜࡢࢰ࢘ࣜ

࣒ࢩHC3ᰴࡢቑṪ᭤⥺ࠋ

23 4 ⪃ᐹ

4.1 GFPᶆ㆑㓝ẕࢆ⏝࠸ࡓ⧄ẟ⹸ࡢ㣗స⏝ࡢ ᐃ

GFPᶆ㆑㓝ẕࢆ⏝࠸࡚࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࢆᐃ㔞ⓗ࡟ ᐃࡍࡿࡓࡵ࡟

ࡣࠊ㓝ẕࡀ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵࡜ࡋ࡚㐺ࡋ࡚࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸ࠋࡑࡇ࡛࣑

ࢻࣜࢰ࣒࢘ࣜࢩ࡟㣵࡜ࡋ࡚ฟⱆ㓝ẕࢆ୚࠼࡚ᇵ㣴ࡋࡓ࡜ࡇࢁࠊ᪤Ꮡࡢ㣵࡜㐯Ⰽ

࡞࠸ቑṪࢆ♧ࡋࡓࡇ࡜࠿ࡽࠊ㓝ẕࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵࡜ࡋ࡚㐺ࡋ࡚࠸ࡿࡇ

࡜ࡀศ࠿ࡗࡓࠋ୍᪉࡛ࢰ࣒࢘ࣜࢩ㸦Paramecium caudatum^{㸧࡟㓝ẕࢆ୚࠼࡚ᇵ} 㣴ࡋ࡚ࡶቑṪࡀ㐜ࡃࠊṚ⁛࡟⮳ࡿࡢࡶ᪩࠿ࡗࡓࡇ࡜࠿ࡽࠊࢰ࣒࢘ࣜࢩࡣ㓝ẕࢆ

࠶ࡲࡾᦤྲྀ࣭฼⏝࡛ࡁ࡞࠸࡜⪃࠼ࡽࢀࡿࠋࡇࡢࡼ࠺࡟ࢰ࣒࢘ࣜࢩ࡜࣑ࢻࣜࢰ࢘

࣒ࣜࢩ࡛㓝ẕ࡟ᑐࡍࡿ㣗ᛶࡀ␗࡞ࡿ࡜࠸࠺⤖ᯝࡣࠊࢰ࣒࢘ࣜࢩࡀ⣽⳦㣗⪅࡛࠶

ࡿࡢ࡟ᑐࡋ࡚ࠊඹ⏕⸴ࢆࡶࡘ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀ⸴㣗⪅࡛࠶ࡿࡇ࡜ࢆ⾲ࡋ࡚࠸

ࡿࡢ࠿ࡶࡋࢀ࡞࠸ࠋฟⱆ㓝ẕࡣ⥳⸴㢮ࡢࢡࣟࣞࣛ࡜ྠࡌࡃ┿᰾⏕≀࡛࠶ࡾࠊ⣽

⬊ࡢ኱ࡁࡉ࡞࡝ࡶẚ㍑ⓗ㏆࠸ࡓࡵࠊ⸴㣗⪅࡟࡜ࡗ࡚ࡣࡼ࠸㣵࡟࡞ࡿࡢ࠿ࡶࡋࢀ

࡞࠸ࠋ

⣽⬊ෆ࡟ඹ⏕⏕≀ࢆࡶࡘ⏕≀ࡢ㣗స⏝ࢆほᐹࡍࡿሙྜࡣࠊྲྀࡾ㎸ࢇࡔ㣵࡜ඹ

⏕⏕≀ࢆ࡝ࡢࡼ࠺࡟ࡋ࡚༊ูࡍࡿ࠿ࡀၥ㢟࡜࡞ࡿࠋᮏ◊✲࡛ࡣࠊ⥳Ⰽ⺯ගࢱࣥ

ࣃࢡ㉁GFPࢆ㐣๫Ⓨ⌧ࡋ࡚࠸ࡿ㓝ẕࡀᙉ࠸⥳Ⰽࡢ⺯ගࢆฟࡍࡢ࡟ᑐࡋ࡚ࠊඹ⏕

⸴ࡣගྜᡂⰍ⣲ࡢࢡࣟࣟࣇ࢕ࣝ࡟⏤᮶ࡍࡿ㉥Ⰽࡢ⮬ᐙ⺯ගࢆฟࡍࡇ࡜ࢆ฼⏝ࡋ

࡚ࠊ⺯ග㢧ᚤ㙾࡜ 2 ✀㢮ࡢࣇ࢕ࣝࢱ࣮ࢭࢵࢺࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ࠊ୧⪅ࢆࡑ

ࢀࡒࢀูࠎࡢ⺯ගീ࡜ࡋ᳨࡚ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡇࡢࡼ࠺࡟ࠊGFP ᶆ㆑㓝ẕ

࠾ࡼࡧ⺯ග㢧ᚤ㙾ࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ࠊඹ⏕⸴࡜㣗స⏝࡟ࡼࡗ࡚⣽⬊ෆ࡟ྲྀ

ࡾ㎸ࡲࢀࡓ㣵ࢆ༊ูࡍࡿࡇ࡜ࡀྍ⬟࡟࡞ࡗࡓࠋ

㣗స⏝ࢆᐃ㔞ⓗ࡟ㄪ࡭ࡿࡓࡵ࡟ࡣࠊྲྀࡾ㎸ࢇࡔ㣵ࡢᾘ໬࡜᤼ฟࡀ༊ู࡛ࡁࡿ

ࡇ࡜ࡶ㔜せ࡛࠶ࡿࠋGFPࢆ⣽⬊ෆ࡟㐣๫Ⓨ⌧ࡋ࡚࠸ࡿ㓝ẕࡣࠊ⣽⬊ࡀ⏕ࡁ࡚࠸

ࡿ㝈ࡾ⺯ගࢆฟࡋࠊᾘ໬࡟ࡼࡗ࡚⣽⬊ቨ࡜⭷ࡀኻࢃࢀࡿ࡜⺯ගࡀ኱ࡁࡃῶᑡࡍ

ࡿࡇ࡜ࡀண᝿ࡉࢀࡿࡓࡵࠊGFPᶆ㆑㓝ẕࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚ᾘ໬ࡢ᳨ฟࡀྍ

⬟࡟࡞ࡿࡇ࡜ࡀᮇᚅࡉࢀࡿࠋࡲࡎ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࠿ࡽᮍᾘ໬ࡢ㓝ẕࡀ⣽⬊እ

࡟᤼ฟࡉࢀࡿᵝᏊࢆほᐹࡋࡓ࡜ࡇࢁࠊ᤼ฟࡉࢀࡓGFPᶆ㆑㓝ẕࡣ᤼ฟᚋࡶ㢧ᚤ 㙾࡟ࡼࡗ᳨࡚ฟࡍࡿࡇ࡜ࡀ༑ศ࡟ྍ⬟࡛࠶ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ୍᪉GFPᶆ㆑㓝

24

ẕࢆ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟ྲྀࡾ㎸ࡲࡏ࡚࠿ࡽእࡢ㓝ẕࢆ㝖ࡃ࡜ࠊ⣽⬊ෆ࡟ṧᏑࡋ

࡚࠸ࡿ㓝ẕࡢᩘࡣ᫬㛫ࡀ⤒ࡘ࡟ࡘࢀ࡚ῶᑡࡋࡓࡢ࡟ᑐࡋࠊ⣽⬊እ࡟Ꮡᅾࡍࡿ㓝 ẕࡢᩘࡣࠊ࡯࡜ࢇ࡝ኚ໬ࡏࡎ⣽⬊ෆࡢ㓝ẕࡼࡾࡶࡎࡗ࡜ᑡ࡞࠸ࡲࡲࡔࡗࡓࠋ࣑

ࢻࣜࢰ࣒࢘ࣜࢩࡀ㓝ẕࢆᾘ໬ࡏࡎ࡟᤼ฟࡋ࡚࠸ࡿ࡜ࡍࢀࡤࠊ⣽⬊እ࡟Ꮡᅾࡍࡿ

㓝ẕࡢᩘࡣቑຍࡍࡿࡣࡎ࡞ࡢ࡛ࠊ⣽⬊ෆ࡟ṧᏑࡋ࡚࠸ࡿ㓝ẕࡀῶᑡࡋࡓࡢࡣ࡯

࡜ࢇ࡝ࡀᾘ໬࡟ࡼࡿ࡜⤖ㄽ࡛ࡁࡿࠋࡼࡗ࡚ࠊGFP ᶆ㆑㓝ẕࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ

࡚ࠊྲྀࡾ㎸ࢇࡔ㓝ẕࡢᾘ໬࡜᤼ฟࢆ༊ูࡍࡿࡇ࡜ࡀྍ⬟࡟࡞ࡗࡓ࡜ゝ࠼ࡿࠋ௨

ୖࡢ⤖ᯝࢆࡲ࡜ࡵࡿ࡜ࠊᑡ࡞ࡃ࡜ࡶ୍㒊ࡢ⧄ẟ⹸࡟ࡘ࠸࡚ࡣࠊGFPᶆ㆑㓝ẕࡣࠊ 㣗స⏝ࢆᐃ㔞ⓗ࡟ ᐃࡍࡿࡢ࡟᭷⏝࡛࠶ࡿ࡜⤖ㄽࡉࢀࡿࠋ

4.2 ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊ෆඹ⏕࡜㣗స⏝

GFPᶆ㆑㓝ẕࢆ⏝࠸࡚ࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣗స⏝ࢆᐃ㔞ⓗ࡟ ᐃࡋࡓࠋ㣗 స⏝ࡣࠊ㣵ࡢ⣽⬊ෆ࡬ࡢྲྀࡾ㎸ࡳ࡜ྲྀࡾ㎸ࢇࡔ㣵ࡢᾘ໬ࡢ2ࡘࡢ㐣⛬࠿ࡽ࡞ࡿࠋ

ࡲࡎྲྀࡾ㎸ࢇࡔ㓝ẕࡢᾘ໬࡟ࡘ࠸࡚ࡣࠊ㔝⏕ᰴ࡜ⓑ໬ᰴ࡛࡯࡜ࢇ࡝ኚࢃࡽ࡞࠿

ࡗࡓࠋࡇࡢ⤖ᯝ࠿ࡽࠊ⣽⬊ෆ࡟ඹ⏕⸴ࢆᣢࡘ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟࠾࠸࡚ࠊඹ⏕

⸴ࡀศゎࡉࢀࡿࡢࢆ㜵ࡄࡓࡵ࡟ࠊ⣽⬊඲యࡢᾘ໬άᛶࡀᢚไࡉࢀࡿࡇ࡜ࡣ≉࡟

࡞࠸ࡇ࡜ࡀศ࠿ࡗࡓࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡟࠾࠸࡚ࡣࠊඹ⏕⸴ࡣඹ⏕⬊࡟ໟࡲࢀ

࡚࠾ࡾ㣵࡞࡝ࢆศゎࡍࡿ㣗⬊࡜ࡣ᏶඲࡟ศ㞳ࡉࢀ࡚࠸ࡿࡓࡵ㸦1㸧ࠊඹ⏕⸴ࡢᏑ ᅾࡀ㣗⬊ࡢᾘ໬άᛶ࡟ᙳ㡪ࡍࡿࡇ࡜ࡣ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

୍᪉ࠊྲྀࡾ㎸ࡴ㓝ẕࡢᩘ࡟ࡘ࠸࡚ࡣࠊⓑ໬ᰴࡢ᪉ࡀ㔝⏕ᰴࡼࡾࡶከ࠿ࡗࡓࠋ ࡇࡢࡇ࡜࠿ࡽࠊඹ⏕⸴ࡢᏑᅾࡣ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶࢆపୗࡉࡏ

ࡿࡇ࡜ࡀศ࠿ࡗࡓࠋ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀ㓝ẕࢆྲྀࡾ㎸ࡴ㝿࡟⣽⬊ෆࡢඹ⏕⸴ᩘ

࡜ྲྀࡾ㎸ࡴ㓝ẕࡢᩘ࡟┦㛵ࡀ࠶ࡿ࠿ㄪ࡭ࡓ࡜ࡇࢁࠊᙅ࠸ṇࡢ┦㛵ࡣ࠶ࡿࡶࡢࡢࠊ

≉࡟㈇ࡢ┦㛵ࡣ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊ⣽⬊ෆࡢඹ⏕⸴ࡀᑡ࡞ࡅࢀࡤከࡃࡢ㓝ẕࢆ

ྲྀࡾ㎸ࡴࢃࡅ࡛ࡣ࡞࠸ࠋࡼࡗ࡚ࠊⓑ໬ᰴࡢ᪉ࡀ㣵ࡢྲྀࡾ㎸ࡳάᛶࡀ㧗࠸ࡢࡣࠊ ༢࡟ඹ⏕⸴ࡀᏑᅾࡋ࡞࠸ࡓࡵ㣵ࢆྲྀࡾ㎸ࡴ✵㛫ࡀከ࠸࠿ࡽ࡜࠸࠺ࢃࡅ࡛ࡣ࡞࠸

࡜ゝ࠼ࡿࠋᙅ࠸ṇࡢ┦㛵ࡣࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊ࡀ኱ࡁ࠸࡯࡝ࠊྲྀࡾ㎸ࡴ

㓝ẕࡢᩘ࡜ඹ⏕⸴ࡢ୧᪉ࡀከࡃ࡞ࡿࡇ࡜࡟ࡼࡿࡶࡢ࠿ࡶࡋࢀ࡞࠸ࠋ௨ୖࡼࡾࠊ ඹ⏕⸴ࡢᏑᅾ࡟ࡼࡗ࡚࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ྲྀࡾ㎸ࡳάᛶࡀపୗࡍࡿࡢࡣࠊ༢

࡟✵㛫ⓗ࡞ၥ㢟࡛ࡣ࡞ࡃࠊᐟ୺ࡢ⏕⌮ⓗᛶ㉁࡟ࡼࡿ࡜⪃࠼ࡽࢀࡿࠋ

25

㔝⏕ᰴࡢሙྜࠊቑṪ࡟࠾ࡅࡿ᫬ᮇ࡟ࡼࡗ࡚ࡶྲྀࡾ㎸ࡴ㓝ẕࡢᩘ࡟ᕪࡀࡳࡽࢀࠊ ᐃᖖᮇ࡟ධࡗ࡚᫬㛫ࡀ⤒ࡘ࡯࡝ῶᑡࡋࡓࠋ ࡞࠾ࢰ࣒࢘ࣜࢩ㸦Paramecium

caudatum㸧࡟࠾࠸࡚ࡶࠊᐃᖖᮇ࡟ධࡿ࡜㣵ࡢྲྀࡾ㎸ࡳࡀῶᑡࡍࡿࡇ࡜ࡀศ࠿ࡗ

࡚࠸ࡿ㸦14㸧ࠋࡇࡢᐃᖖᮇࡢ⣽⬊࡟㓝ẕࢆ୚࠼ࡿ࡜ࠊ2᪥࡛ྲྀࡾ㎸ࡳάᛶࡀᑐᩘ

ቑṪᮇ࡜ྠ⛬ᗘ࡟ᡠࡗࡓࠋࡋ࠿ࡋࠊ㓝ẕࢆ୚࠼ࡿ࡜ྠ᫬࡟ࢱࣥࣃࢡ㉁ྜᡂ㜼ᐖ

๣ࡶຍ࠼ࡿ࡜ྲྀࡾ㎸ࡳάᛶࡣᡠࡽ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡣ࿘ᅖ

࡟㣵ࡀ࠶ࡗ࡚ቑṪࢆ㛤ጞࡍࡿ࡜㣵ࢆྲྀࡾ㎸ࡴࡢ࡟ᚲせ࡞ࢱࣥࣃࢡ㉁ࢆከࡃⓎ⌧

ࡋࠊᐃᖖᮇ࡛ࡣࡑࡢࡼ࠺࡞ࢱࣥࣃࢡ㉁ࡢⓎ⌧ࡀᢚไࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ ᐃᖖᮇ࡟࠾࠸࡚ࡶ⧄ẟ㐠ື࡟ࡼࡿ⣽⬊ཱྀ࡬ࡢ㣵ࡢ㐠ᦙࡣṇᖖ࡟⾜ࢃࢀ࡚࠸ࡓࡢ

࡛ࠊᐃᖖᮇ࡟࠾࠸࡚Ⓨ⌧ࡀᢚไࡉࢀࡿࢱࣥࣃࢡ㉁ࡣࠊ㣵ࡢ⣽⬊ཱྀ࡬ࡢ㐠ᦙ࡛࡞

ࡃ࢚ࣥࢻࢧ࢖ࢺ࣮ࢩࢫ࡟㛵୚ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ

௨ୖࡢࡼ࠺࡟࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ㣵ࡢྲྀࡾ㎸ࡳάᛶࡀࡉࡲࡊࡲ࡞≧ἣ࡟ᛂࡌ

࡚ኚ໬ࡍࡿࡇ࡜ࡣࠊΰྜᰤ㣴⏕≀࡛࠶ࡿ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀࠊ⏕ᏑࡸቑṪࡢຠ

⋡ࢆ㧗ࡵࡿࡓࡵ࡟㝈ࡾ࠶ࡿ⣽⬊ෆࡢ㈨※㸦ࢱࣥࣃࢡ㉁㸧ࢆඹ⏕⸴ࡢቑṪࡸගྜ

ᡂ࡜⮬㌟ࡢ㣗స⏝ࡢ࡝ࡕࡽ࡟᣺ࡾศࡅࡿࡢࡀ᭷฼࠿࡜࠸࠺ࠊ㈨※ࡢ᭱㐺࡞ศ㓄 ᡓ␎࡟ࡼࡗ࡚ㄝ᫂ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ㔝⏕ᰴࡢ᪉ࡀⓑ໬ᰴࡼࡾࡶ㣵ࡢྲྀࡾ㎸ࡳ

άᛶࡀప࠿ࡗࡓࡢࡣࠊඹ⏕⸴ࡀ࠸ࡿ㔝⏕ᰴࡢሙྜࠊ㈨※ࢆ࠶ࡿ⛬ᗘඹ⏕⸴ࡢቑ Ṫࡸගྜᡂ࡟๭࠿࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࡓࡵࠊ㣗స⏝࡬ࡢ᣺ࡾศࡅࡀࡑࡢศῶࡿ࡜

⪃࠼ࡽࢀࡿࠋྠࡌ㔝⏕ᰴ࡛ࡶᐃᖖᮇ࡟ධࡿ࡜ྲྀࡾ㎸ࡳάᛶࡀపୗࡍࡿࡢࡣࠊ㣵 ࡀ඲ࡃᯤῬࡋ࡚࠸ࡿ≧ἣ࡛ࡣࠊ㣵ࡢྲྀࡾ㎸ࡳ࡟㈨※ࢆ๭ࡃࡢࡣ↓㥏࡟࡞ࡿྍ⬟

ᛶࡀ㧗࠸ࡓࡵ࡜⪃࠼ࡽࢀࡿࠋࡑࡢࡼ࠺࡞≧ἣ࡛ࡶࠊከᩘࡢඹ⏕⸴ࢆ⥔ᣢࡋ࡚࠸

ࢀࡤࠊඹ⏕⸴ࡢ౪⤥ࡍࡿගྜᡂ⏘≀࡟ࡼࡗ࡚ቑṪࡣฟ᮶࡞࠸ࡲ࡛ࡶ⏕࿨άືࠊ

≉࡟㐟Ὃ㐠ືࢆ⥔ᣢࡍࡿࡇ࡜ࡀ࡛ࡁࠊ㣵ࡢ࠶ࡿ⎔ቃ࡟⾜ࡁ╔ࡃྍ⬟ᛶࡀ࠶ࡿࠋ

ࡼࡗ࡚ࠊᐃᖖᮇ࡛ࡣ㣗స⏝ࡼࡾࡶඹ⏕⸴ࡢ⥔ᣢࡸගྜᡂ࡟㈨※ࢆ᣺ࡾศࡅࡿ᪉ ࡀ᭷฼࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋᮏ◊✲࡟ࡼࡗ࡚ࠊΰྜᰤ㣴⏕≀ࠊ≉࡟⣽⬊ෆ࡟⸴

㢮ࢆඹ⏕ࡉࡏ࡚࠸ࡿ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡀࠊ≧ἣ࡟ᛂࡌ࡚⮬㌟ࡢ㣗స⏝࡜ඹ⏕⸴

࡬ࡢ㈨※ࡢศ㓄ࢆኚ࠼࡚࠾ࡾࠊࡑࢀ࡟ࡼࡗ࡚㣵ࡢྲྀࡾ㎸ࡳάᛶࢆኚ໬ࡉࡏ࡚࠸

ࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ

26 ཧ⪃ᩥ⊩

1. ඣ⋢᭷⣖ࠊ⸨ᓥᨻ༤(2008) ༢⣽⬊ື≀࣑ࢻࣜࢰ࣒࢘ࣜࢩ࡜⥳⸴ࢡࣟࣞࣛ࡜

ࡢ⣽⬊ෆඹ⏕ᡂ❧ᶵᵓ ཎ⏕ື≀Ꮫ㞧ㄅ 41(2):117-132.

2. ⏣ᮧ⌶㑻 (2013) ࣑ࢻࣜࢰ࣒࢘ࣜࢩࡢ⣽⬊ෆ࡟࠾ࡅࡿඹ⏕⸴ࡢቑṪࡢゎᯒ ᖹᡂ25ᖺᗘ༞ᴗ◊✲ㄽᩥ

3. Esteban, G. F., Fenchel, T., and Finlay, B. J. (2010) Mixotrophy in Ciliates.

Protist 161: 621-641.

4. Bronmark, C., Hansson, L.A. ༨㒊ᇛኴ㑻┘ヂ㸦2007㸧 †࡜ụࡢ⏕≀Ꮫ̿

⏕≀ࡢ㐺ᛂ࠿ࡽ⩌㞟⌮ㄽ࣭ಖ඲ࡲ࡛㸦ඹ❧ฟ∧ᰴᘧ఍♫㸧

5. ୸ᒸ⚞(2004) ᩍᮦ࡜ࡋ࡚ࡢཎ⏕ື≀(2)㸫ࢰ࣒࢘ࣜࢩϨ ཎ⏕ື≀Ꮫ㞧ㄅ

37(1):19-30.

6. Berk, S.G., Parks, L.H., and Ting, R.S. (1991) Photoadaptation alters the ingestion rates of Paramecium bursaria,a mixotrophic ciliate. Applied and Environmental Microbiology 53: 2312-2316.

7. Ghafari, P., St-Denis, C. H., Power, M. E., Jin, X., Tsou, V., Mandal, H. S., Bols, N. C., and Tang, X. (2008) Impact of carbon nanotubes on the

ingestion and digestion of bacteria by ciliated protozoa. Nature Nanotechnology 3: 347-351.

8. ᐑ⬥ᩔྐ(2000) GFP࡜ࣂ࢖࢜࢖࣓࣮ࢪࣥࢢ ⺯ගࢱࣥࣃࢡ㉁ࡢⓎ⌧࡜᳨

ฟࡢᇶᮏ࠿ࡽ⏕యᶵ⬟ࡢྍど໬ࡲ࡛㸦⨺ᅵ♫㸧

9. Mitra, B.N., Yasuda, T., Kobayashi, S., Saito-Nakano, Y. and Nozaki, T.

(2005) Differences in morphology of Phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar. Cell Motility and the Cytoskeleton 62: 84-99.

10. Kodama, Y., Fujishima, M. (2008) Cycloheximide induces synchronous swelling of perialgal vacuoles enclosing symbiotic Chlorella vulgaris and digestion of the algae in the ciliate Paramecium bursaria. Protist 159(3) :

27 483-494.

11. Omura, G., Ishida, M., Arikawa, M., Khan, S.M.M.K., Suetomo, Y.,

Kakuta, S., Yoshimura, C. and Suzaki, T. (2004) A bacteria-free monoxenic culture of Paramecium bursaria: its growth characteristics and the

re-establishment of symbiosis with Chlorella in bacteria-free conditions.

Jpn J. Protozool 37: 119-130.

12. Karakashian, S. J. (1963) Growth of paramecium bursaria as influenced by the presence of algal symbionts. Physiological Zoology 36(1): 52-68.

13. Ayala, A., Weis, D.S. (1987) The effect of Puromycin and Cycloheximide on the infection of algae-free Paramecium bursaria by symbiotic

Chlorellae. J. Protozool 34(4): 377-381.

14. Fok, A.K., Allen, R.D., Kaneshiro, E.S. (1981) Axenic Paramecium caudatum. Ϫ. Biochemical and physiological changes with culture age.

European Journal of Cell Biology 25: 193-201.

15. ಖ⛉ுࠊ㙊ᡞఙ୍㑻ࠊຍ⸨㇏ࠊ௒ᮧಙᏕ(2006) ᪥ᮏ⏘࣑ࢻࣜࢰ࣒࢘ࣜࢩ⣽

⬊ෆඹ⏕⸴࡟ࡘ࠸࡚ ཎ⏕ື≀Ꮫ㞧ㄅ 39(2):173-188.

16. ஭ୖ໏(2011) ⸴㢮30൨ᖺࡢ⮬↛ྐ➨஧∧⸴㢮࠿ࡽࡳࡿ⏕≀㐍໬࣭ᆅ

⌫࣭⎔ቃ㸦ᮾᾏ኱Ꮫฟ∧఍㸧

ㅰ㎡

ᮏ◊✲ࢆ⾜࠺࡟࠶ࡓࡾࠊᣦᑟᩍဨ࡛࠶ࡿᒾ஭ⲡ௓෸ᩍᤵ㸦ᘯ๓኱Ꮫᩍ⫱Ꮫ㒊㸧

࡟ࡣ◊✲඲⯡࡟࠾ࡅࡿ᠓ษ࡞ࡿᚚᣦᑟࢆ㈷ࡾࡲࡋࡓࡇ࡜ࢆ῝ࡃᚚ♩⏦ࡋୖࡆࡲ

ࡍࠋᏲᒇኸᮁ෸ᩍᤵ㸦ᒸᒣ኱Ꮫ␗ศ㔝⼥ྜඛ➃◊✲ࢥ࢔㸧࡟ࡣࠊᮏ◊✲࡟ᚲせ

୙ྍḞ࡛࠶ࡿGFPⓎ⌧ฟⱆ㓝ẕ pTOWѸpTEF1::GFP/BY4741ᰴࢆᥦ౪࠸ࡓࡔ ࡁࡲࡋࡓࠋ⸨ᓥᨻ༤ᩍᤵ㸦ᒣཱྀ኱Ꮫ኱Ꮫ㝔⌮ᕤᏛ◊✲⛉㸧࡟ࡣࠊᮏ◊✲ᐊ࡬ࢹ

࢕ࣉࣞࢵࢩࣙࣥࢫࣛ࢖ࢻࢆᥦ౪࠸ࡓࡔ࠸ࡓࡔࡅ࡛࡞ࡃࠊᮏ◊✲඲⯡࡟㛵ࡋ࡚ບ

ࡲࡋࡢゝⴥࢆ࠸ࡓࡔࡁࡲࡋࡓࠋⓙࠎᵝ᪉࡟ࠊᨵࡵ࡚ᚚ♩⏦ࡋୖࡆࡲࡍࠋ᭱ᚋ࡟ࠊ

⤒῭ⓗ࡟ⱞປࢆ࠿ࡅ࡞ࡀࡽࡶ኱Ꮫ࡬ࡢ㐍Ꮫࢆチࡋ࡚ࡃࢀࡓ୧ぶ࡟ឤㅰࡋ࡚ࠊ⥾

ࡵᣓࡾ࡜ࡉࡏ࡚࠸ࡓࡔࡁࡲࡍࠋ

ᖹᡂ28ᖺ1᭶24᪥

ᘯ๓኱Ꮫ኱Ꮫ㝔 ᩍ⫱Ꮫ◊✲⛉

ᩍ⛉ᩍ⫱ᑓᨷ ⌮⛉ᩍ⫱ᑓಟ ⏕≀Ꮫศ㔝

13GP211 ୕ᾆ ㈗ኈ