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Characterization of ROCK inhibitor-treated extracellular vesicles and analysis of their effect on osteogenesis

研究課題

研究課題/領域番号 22K17135
研究種目

若手研究

配分区分基金
審査区分 小区分57050:補綴系歯学関連
研究機関新潟大学

研究代表者

Thant Lay  新潟大学, 医歯学系, 特任助教 (10941867)

研究期間 (年度) 2022-04-01 – 2024-03-31
研究課題ステータス 中途終了 (2023年度)
配分額 *注記
4,550千円 (直接経費: 3,500千円、間接経費: 1,050千円)
2023年度: 2,210千円 (直接経費: 1,700千円、間接経費: 510千円)
2022年度: 2,340千円 (直接経費: 1,800千円、間接経費: 540千円)
キーワードExtracellular vesicles / ROCK inhibitor / Bone regeneration
研究開始時の研究の概要

First, we will elucidate the composition of ROCK inhibitor treated extracellular vesicles secreted from mesenchymal cells and its secretory control pathway. Next, we will analyze the mineralization of osteoblasts and bone regeneration by administering ROCKin'-EV to calvarial defects in rats.

研究実績の概要

For the proteomic analysis of ROCK inhibitor-treated extracellular vesicles (ROCKin'-EVs), MagCapture Exosome Isolation Kit was used to prepare Control extracellular vesicles (Control-EVs) and ROCKin'-EVs from the culture medium of MC3T3-E1 cells. Mass spectrometry was performed to elucidate the changes in the proteomic composition. We detected there were changes ROCKin'-EVs protein composition. We identified that a number of Rab GTPase (known as the key regulators of intercellular vesicle transport) were upregulated in ROCKin'-EVs.

For the analysis of osteoblastic mineralization regulated by ROCKin'-EVs, we isolated the Control-EVs and ROCKin'-EVs again from the culture medium of MC3T3-E1 cells on Day3, 6 and 9 of osteoblastic differentiation using the culture filtration device. We measured the insolated proteins in the Control-EVs and ROCKin'-EVs. The protein concentration of the ROCKin'-EVs were 5.9times more than the Control-EVs. The ROCKin'-EVs were diluted to the same concentration as the Control-EVs, and MC3TC-E1 cells were treated with dimethyl sulfoxide (DMSO) (Control), 5 μM ROCK inhibitor (ROCK), 5 μg/ml Control-EVs, and ROCKin'-EVs, with induction of osteoblast differentiation. For the evaluation of mineralization activity, ALP staining and Picrosirus Red staining were performed. ALP and picrosirus red activity were significantly increase in ROCK inhibitor treated cells (ROCK) than in Control, as expected. However, the ALP staining and Picrosirus Red staining of Control-EVs and ROCKin'-EVs treated cells didn’t show significant difference from the Control.

報告書

(2件)
  • 2023 実績報告書
  • 2022 実施状況報告書
  • 研究成果

    (9件)

すべて 2024 2023 2022

すべて 雑誌論文 (5件) (うち国際共著 2件、 査読あり 5件、 オープンアクセス 3件) 学会発表 (4件)

  • [雑誌論文] Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix2024

    • 著者名/発表者名
      Kaku Masaru、Thant Lay、Dobashi Azusa、Ono Yoshiki、Kitami Megumi、Mizukoshi Masaru、Arai Moe、Iwama Hajime、Kitami Kohei、Kakihara Yoshito、Matsumoto Masaki、Saito Isao、Uoshima Katsumi
    • 雑誌名

      Scientific Reports

      巻: 14 号: 1 ページ: 354-354

    • DOI

      10.1038/s41598-023-51054-8

    • 関連する報告書
      2023 実績報告書
    • 査読あり / オープンアクセス
  • [雑誌論文] Effect of Sparc knockout on the extracellular matrix of mouse periodontal ligament cells2024

    • 著者名/発表者名
      Arai Moe、Kaku Masaru、Thant Lay、Kitami Megumi、Ono Yoshiki、Dobashi Azusa、Iwama Hajime、Mizukoshi Masaru、Kitami Kohei、Matsumoto Masaki、Saito Isao、Uoshima Katsumi
    • 雑誌名

      Biochemical and Biophysical Research Communications

      巻: 692 ページ: 149364-149364

    • DOI

      10.1016/j.bbrc.2023.149364

    • 関連する報告書
      2023 実績報告書
    • 査読あり
  • [雑誌論文] Acellular Extrinsic Fiber Cementum Is Invariably Present in the Superficial Layer of Apical Cementum in Mouse Molar2024

    • 著者名/発表者名
      Iwama Hajime、Kaku Masaru、Thant Lay、Mizukoshi Masaru、Arai Moe、Ono Yoshiki、Kitami Kohei、Saito Isao、Uoshima Katsumi
    • 雑誌名

      Journal of Histochemistry & Cytochemistry

      巻: 72 号: 2 ページ: 109-120

    • DOI

      10.1369/00221554241229130

    • 関連する報告書
      2023 実績報告書
    • 査読あり
  • [雑誌論文] Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress2022

    • 著者名/発表者名
      Thant Lay、Kakihara Yoshito、Kaku Masaru、Kitami Megumi、Kitami Kohei、Mizukoshi Masaru、Maeda Takeyasu、Saito Isao、Saeki Makio
    • 雑誌名

      Biochemical and Biophysical Research Communications

      巻: 624 ページ: 16-22

    • DOI

      10.1016/j.bbrc.2022.07.061

    • 関連する報告書
      2022 実施状況報告書
    • 査読あり / オープンアクセス / 国際共著
  • [雑誌論文] Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress2022

    • 著者名/発表者名
      Thant Lay、Kaku Masaru、Kakihara Yoshito、Mizukoshi Masaru、Kitami Megumi、Arai Moe、Kitami Kohei、Kobayashi Daiki、Yoshida Yutaka、Maeda Takeyasu、Saito Isao、Uoshima Katsumi、Saeki Makio
    • 雑誌名

      Frontiers in Physiology

      巻: 13 ページ: 899699-899699

    • DOI

      10.3389/fphys.2022.899699

    • 関連する報告書
      2022 実施状況報告書
    • 査読あり / オープンアクセス / 国際共著
  • [学会発表] Chemical digestion-assisted extracellular matrix profiling of differentiating osteoblastic cells2023

    • 著者名/発表者名
      Lay Thant, Masaru Kaku, Hlaing Pwint Phyu, Azusa Dobashi, Yoshito Kakihara, Isao Saito, Katsumi Uoshima
    • 学会等名
      Australia and New Zealand Bone and Mineral Society Annual Scientific Meeting
    • 関連する報告書
      2023 実績報告書
  • [学会発表] Chemical digestion-assisted extracellular matrix profiling of human periodontal ligament2023

    • 著者名/発表者名
      Lay Thant, Masaru Kaku, Azusa Dobashi, Yoshito Kakihara, Isao Saito, Katsumi Uoshima
    • 学会等名
      21st Annual Meeting of Japanese Proteome Society
    • 関連する報告書
      2023 実績報告書
  • [学会発表] Alteration of the ECM composition and fiber organization in periodontal ligament under mechanical loading2022

    • 著者名/発表者名
      Lay Thant, Masaru Kaku, Yoshito Kakihara, Masaru Mizukoshi, Megumi Kitami, Kohei Kitami, Daiki Kobayashi, Yutaka Yoshida, Takeyasu Maeda, Katsumi Uoshima, Isao Saito, Makio Saeki
    • 学会等名
      第54回日本結合組織学会学術大会
    • 関連する報告書
      2022 実施状況報告書
  • [学会発表] Extracellular matrix profiling of cultured PDL cells by chemical digestion-assisted proteomics2022

    • 著者名/発表者名
      Lay Thant, Masaru Kaku, Azusa Dobashi, Yoshito Kakihara, Isao Saito, Katsumi Uoshima
    • 学会等名
      第22回日本再生医療学会総会
    • 関連する報告書
      2022 実施状況報告書

URL: 

公開日: 2022-04-19   更新日: 2024-12-25  

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