Investigation of vocal fold epithelial barrier function

• Project/Area Number: 17K16909
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Otorhinolaryngology
• Research Institution: Kyoto University
• Principal Investigator: Mizuta Masanobu 京都大学, 医学研究科, 客員研究員 (20777875)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2019: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 声帯 / 上皮細胞 / 培養 / バリア機能 / 声帯上皮細胞 / 分離培養 / タイトジャンクション / 声帯上皮 / バリア

Outline of Final Research Achievements

The characteristics and function of vocal fold epithelial cells remains unclear because of the lack of in vitro experiments. In this study, we established the culture methods of the epithelial cells of rat vocal folds to establish an in vitro experimental model. The toluidine blue staining showed multi-layered epithelial structure of the cultured epithelial cell layer and immunohistochemistry revealed the expression of occludin and ZO-1 in the cultured cells. Transepithelial electrical resistance (TER) was measured for the evaluation of epithelial barrier function. Results revealed more than 1000 ohms x cm2 of TER on day 4, passage 1. After exposing the cultured cell layer to acidic-pepsin, TER values promptly decreased and recovered to the baseline within 24 hours.

Academic Significance and Societal Importance of the Research Achievements

音声障害の原因となる声帯疾患の発生には、声帯上皮の防御機構の破綻が関与すると考えられる。このため音声障害の予防のためには声帯上皮の防御機構を維持することが重要であるが、今まで有用な声帯上皮の培養システムが確立されていなかったため声帯防御機構の研究は進んでいなかった。本研究において上皮としての防御機能を有する声帯上皮をラットから培養するシステムおよび実験系が確立されたことにより、今後声帯上皮の防御機構の解明が進むことが期待される。

Research Products

• [Journal Article] Vibratory function and healing outcomes after small intestinal submucosa biomaterial implantation for chronic vocal fold scar (2018)
  • Author(s): Pitman MJ, Kurita T, Powell ME, Kimball EE, Mizuta M, Chang S, Garrett CG, Rousseau B
  • Journal Title: Laryngoscope Volume: 128 Issue: 4 Pages: 901-908
  • DOI: 10.1002/lary.26883
• [Journal Article] Process of tight junction recovery in the injured vocal fold epithelium: Morphological and paracellular permeability analysis (2017)
  • Author(s): Suzuki Ryo、Katsuno Tatsuya、Kishimoto Yo、Nakamura Ryosuke、Mizuta Masanobu、Suehiro Atsushi、Yamashita Masaru、Nakamura Tatsuo、Tateya Ichiro、Omori Koichi
  • Journal Title: The Laryngoscope Volume: 128 Issue: 4
  • DOI: 10.1002/lary.26959
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] In vivo measurement of vocal fold surface resistance (2017)
  • Author(s): Mizuta M, Kurita T, Dillon NP, Kimball EE, Garrett CG, Sivasankar MP, Webster RJ 3rd, Rousseau B
  • Journal Title: Laryngoscope Volume: 127 Issue: 10
  • DOI: 10.1002/lary.26715
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structurally and functionally characterized in vitro model of rabbit vocal fold epithelium (2017)
  • Author(s): Mizuta M, Kurita T, Kimball EE, Rousseau B
  • Journal Title: Tissue Cell Volume: 49 Issue: 3 Pages: 427-434
  • DOI: 10.1016/j.tice.2017.03.006
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Recovery of Vocal Fold Epithelium after Acute Phonotrauma (2017)
  • Author(s): Rousseau B, Kojima T, Novaleski CK, Kimball EE, Valenzuela CV, Mizuta M, Daniero JJ, Garrett CG, Sivasankar MP
  • Journal Title: Cells Tissues Organs Volume: 204 Issue: 2 Pages: 93-104
  • DOI: 10.1159/000472251
• [Journal Article] The efficacy of a novel collagen-gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar. (2016)
  • Author(s): Hiwatashi, N., Hirano, S., Mizuta, M., Kobayashi, T., Kawai, Y., Kanemaru, SI., Nakamura, T., Ito, J., Kawai, K., Suzuki, S.
  • Journal Title: J Tissue Eng Regen Med. Volume: in press Issue: 5 Pages: 0-0
  • DOI: 10.1002/term.2060
  • Peer Reviewed
• [Presentation] Development of an in vitro model of rat vocal fold epithelium (2019)
  • Author(s): Keisuke Kojima
  • Organizer: 2019 Combined Otolaryngology Spring Meetings
  • Int'l Joint Research
• [Presentation] Distribution of the tight junction proteins occluding, ZO-1, and claudins in the laryngeal glands. (2018)
  • Author(s): Ryo Suzuki, Yo Kishimoto, Shinji Kaba, Masanobu Mizuta, Atsushi Suehiro, Masaru Yamashita, Ichiro Tateya, Koichi Omori
  • Organizer: 第119回日本耳鼻咽喉科学会
• [Presentation] 塩酸ペプシン塗布によるラット声帯上皮バリアへの影響 (2018)
  • Author(s): 鈴木良、岸本曜、樋渡直、水田匡信、末廣篤、楯谷一郎、大森孝一
  • Organizer: 第31回日本喉頭科学会
• [Presentation] 声帯上皮バリア機能評価の試み (2017)
  • Author(s): 水田匡信，土師知行，佐藤進一，玉木久信
  • Organizer: 第43回日本耳鼻咽喉科学会中国地方部会
• [Presentation] in vivo環境での声帯上皮バリア機能評価の試み (2017)
  • Author(s): 水田匡信，楯谷一郎，大森孝一
  • Organizer: 第29回日本喉頭科学会
• [Presentation] Recovery process of TJ-based functional barriers in injured vocal fold epithelium (2017)
  • Author(s): Ryo Suzuki, Yo Kishimoto, Tatsuya Katsuno, Ryosuke Nakamura, Yoshitaka Kawai, Masanobu Mizuta, Ichiro Tateya, Koichi Omori
  • Organizer: The 138th Annual Meeting of the American Laryngological Association