A: Ca 2+ 結合飽和状態 B:Ca 2+ -free
VIII. 総括
本研究では，生体内環境下での顕微鏡観察が可能な膜状の骨格筋である脊柱 僧帽筋に，蛍光 Ca2+指示薬を用いたバイオイメージングを適用し，筋細胞内の Ca2+の動態を様々な条件下で検討することを目的として，以下の研究課題を遂行 した．
研究課題1. 生体内（in vivo）環境下における骨格筋細胞内Ca2+-kineticsの可視 化
in vitroでは時間経過と共に細胞内Ca2+増加が観察されたが，血流を維持した
in vivoにおいては細胞内Ca2+は安静レベルを維持していた．in vivoにおいても，
薬理学的処置によって Ca2+変化が生じたことから，脊柱僧帽筋を観察対象とし たバイオイメージングは，生体内環境をよく保存した状態での筋細胞内 Ca2+観 察が可能であることが明らかになった．
研究課題2. 筋収縮様式の違いに着目した骨格筋細胞内Ca2+-kineticsの差異
（1）様式の異なる筋収縮負荷後の筋細胞内Ca2+-kineticsの測定
アイソメトリック収縮は，収縮を繰り返すことによってわずかな細胞内 Ca2+
増加を示したが，一方でエキセントリック収縮においては安静時，およびアイ ソメトリック収縮時と比べても大きな細胞内 Ca2+の増加が，収縮回数の少ない 段階から観察された．
（2）伸張性筋収縮で生じるCa2+蓄積に寄与するCa2+増加経路の検討
SAC 阻害薬であるストレプトマイシンとガドリニウムイオン（Gd3+）はエキ セントリック収縮後の Ca2+蓄積を有意に抑制することが明らかになった．この
結果は，エキセントリック収縮後にはSACを介したCa2+蓄積が生じていること を示すものであった．
研究課題3. 骨格筋in vivoモデルを用いた細胞内Ca2+測定の発展的応用
（1）性差によって生じる筋機能の違いと細胞内Ca2+動態の関連について
細胞へのストレスに対して保護的な性差を生じるメスの骨格筋では，エキセ ントリック収縮後に生じる筋細胞内への Ca2+蓄積が大幅に抑制されていること が示された．また，この抑制は卵巣を摘出したメスラットにおいても同様に見 られたことから，エキセントリック収縮後の筋細胞内 Ca2+に対して，エストロ ゲンの関与は小さいことが示唆された．
（2）糖尿病がもたらす筋機能の低下と細胞内Ca2+動態の関連について
筋が萎縮し，循環系機能も低下していると考えられる糖尿病の骨格筋におい て，正常な筋と同様にエキセントリック収縮後に筋細胞内 Ca2+蓄積が生じた．
また，正常な筋と比較して，アイソメトリック収縮後においても高い筋細胞内 Ca2+状態を示した．このことから糖尿病状態の骨格筋では，細胞内Ca2+恒常性が 破綻しやすい状態にあると考えられた．
以上の研究項目を総括し，本研究の結論として，in vivo における脊柱僧帽筋 細胞内の Ca2+測定が，生体内環境下での細胞内イベントを観察することに非常 に有効な手法であることが示された．特に筋収縮については，細胞内 Ca2+の変 化がリアルタイムで観察可能であり，収縮様式や筋モデルの違いによって細胞 内Ca2+の恒常性に差異が生じることが明らかになった．
謝辞
本論文の作成にあたり，終始懇切丁寧な御指導，御校閲を賜りました狩野豊 准教授に心から感謝の意を表します．また，本学量子・物質工学科の丹羽治樹 教授，中村整教授，白川英樹准教授，本学知能機械工学科の山田幸生教授，カ ンザス州立大学のDr. Poole氏をはじめ，諸先生方から多くの御指導を賜りまし た．ここに深厚なる謝意を表します．最後に，実験に際し御協力頂いた狩野研 究室の皆様に御礼申し上げます．
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