인체 두피 모낭의 장기간 배양을 위한 기관 배양 배지의 개발

Development of Organ Culture Medium for Long Term Culture of Human Hair Follicle

  • 유보영 (동국대학교 공과대학 생명화학공학과) ;
  • 윤희훈 (동국대학교 공과대학 생명화학공학과) ;
  • 신연호 (동국대학교 공과대학 생명화학공학과) ;
  • 서영권 (동국대학교 공과대학 생명화학공학과) ;
  • 이두훈 (동국대학교 공과대학 생명화학공학과) ;
  • 송계용 (중앙대학교 의과대학 병리학교실) ;
  • 황성주 (황성주 털털피부과) ;
  • 박정극 (동국대학교 공과대학 생명화학공학과)
  • Yoo, Bo-Young (Department of Chemical and Biochemical Engineering, Dongguk University) ;
  • Yoon, Hee-Hoon (Department of Chemical and Biochemical Engineering, Dongguk University) ;
  • Shin, Yeon-Ho (Department of Chemical and Biochemical Engineering, Dongguk University) ;
  • Seo, Young-Kwon (Department of Chemical and Biochemical Engineering, Dongguk University) ;
  • Lee, Doo-Hoon (Department of Chemical and Biochemical Engineering, Dongguk University) ;
  • Song, Kye-Yong (Department of Pathology, Chung-Ang University) ;
  • Hwang, Sung-Joo (SJ Hair Hair Clinic) ;
  • Park, Jung-Keug (Department of Chemical and Biochemical Engineering, Dongguk University)
  • 투고 : 2005.12.10
  • 심사 : 2006.01.20
  • 발행 : 2006.02.28

초록

본 연구에서는 인체 두피조직에서 미세수술법을 이용하여 모낭을 성공적으로 분리하였으며 다양한 조건으로 액침기관배양을 수행하였다. 우태아 혈청첨가시 모낭의 길이 성장이 저해되는 것으로 확인되어 무혈청 배지 조성을 시도하였다. 무혈청 배지로는 모낭 기관배양에 널리 이용되는 Williams'E medium을 기본으로 하는 Philpott medium과 자체 개발한 고농도 아미노산과 비타민(B군) 조성의 DHGM(Dongguk hair growth medium)을 이용하였다. 그리고 IMDM은 DHGM의 비교 대조군으로 이용하였다. 연구 결과 Philpott medium과 IMDM으로 배양한 모낭은 구조상으로는 길이 성장이 각각 9일과 12일 정도에 멈추며, 낮은 알카라인 포스파테이즈 발현, CK19 발현이 거의 없는 것으로 보아 세포사멸에 의한 퇴화(regression)가 빠르게 일어나는 것으로 판단되었다. 반면, DHGM으로 배양한 모낭은 상대적으로 긴 기간 동안 성장기의 구조를 보이며 25일까지 지속적인 길이 증가 및 3배 높은 알카라인 포스파테이즈 발현, 전반적인 CK19 발현을 나타내었다. 따라서 고농도의 아미노산 및 비타민 배지 조성이 생체 외에서 모낭을 장기간 배양하는데 중요한 역할을 하는 것으로 판단된다. 이러한 배양 방법은 장기간 검사를 필요로 하는 모낭에 대한 기초 생물학적 연구뿐만 아니라 새로운 탈모치료제의 효능 평가 분야에 이용될 수 있을 것이다.

We successfully isolated human anagen hair follicles from human scalp skin by microdissection and tried to culture them under various conditions. First we confirmed negative effect of serum on human hair follicle organ culture. As a next step serum-free medium compositions, Philpott medium, IMDM, and DHGM (Dongguk hair growth medium) were tried. Philpott medium is a general medium for hair organ culture based on Williams' E medium and DHGM is a special self-developed medium containing high amino acids and vitamins (B group) composition. As results, hair follicle in Philpott medium and IMDM showed anagen phase morphological structure, but rapid loss of hair elongation, low alkaline phosphatase expression, and very low expression of CK19. It is thought these hair follicles rapidly regressed from apoptosis. However, hair follicles in DHGM showed long term anagen phase morphological structure, continuous hair elongation, high alkaline phosphatase, and CK19 expression. These results demonstrate that high amino acids and vitamins (B group) composition are essential to in vitro long term human hair follicle organ culture and this culture medium will be useful in basic study of hair biology or application study to the development of alopecia treatment drugs.

키워드

과제정보

연구 과제 주관 기관 : 산업자원부

참고문헌

  1. Maria, I. M. and Marjana, T. C., 'Epidermal Stem Cells: the Cradle of Epidermal Determination, Differentiation and Wound Healing,' Biol. Cell., 97(3), 173-183(2005) https://doi.org/10.1042/BC20040098
  2. Jahoda, C. A. and Reynolds, A. J., 'Dermal-Epidermal Interactions: Adult Follicle-derived Cell Populations and Hair Growth,' Dermatol. Clin., 14(4), 573-583(1996) https://doi.org/10.1016/S0733-8635(05)70385-5
  3. Eicheler, W., Happle, R. and Hoffmann, R., '$5\alpha$-Reductase Activity in the Human Hair Follicle Concentrates in Dermal Papilla,' Arch. Dermatol. Res., 290, 126-132(1998) https://doi.org/10.1007/s004030050277
  4. Messenger, A. G. and Rundegren, J., 'Minoxidil: Mechanism of Action on Hair Follicle,' Br. J. Dermatol., 150, 186-194(2004) https://doi.org/10.1111/j.1365-2133.2004.05785.x
  5. Philpott, M. P., Green, M. R. and Kealey, T., 'Human Hair Growth in vitro,' J. Cell. Sci., 97, 463-471(1990)
  6. Westgate, G. E., Gibson, W. T., Kealey, T. and Philpott, M. P., 'Prolonged Maintenance of Human Hair Follicles in vitro in a Serum-free Medium,' Br. J. Dermatol., 129, 372-379(1993) https://doi.org/10.1111/j.1365-2133.1993.tb03160.x
  7. Childs, C. B., Proper, J. A., Tucker, R. F. and Moses, H. L., 'Serum Contains a Platelet-derived Transforming Growth Factor,' Proc. Natl. Acad. Sci. USA., 79, 5312-5316(1982)
  8. Sica, D. A., 'Minoxidil: an Underused Vasodilator for Resistant or Severe Hypertension,' J. Clin. Hypertens., 6(5), 283-287(2004) https://doi.org/10.1111/j.1524-6175.2004.03585.x
  9. Vjayalakshhmi, B., Sesikeran, B., Udaykumar, P., Kalyanasundaram, S. and Raghunath, M., 'Effects of Vitamin Restriction and Supplementation on Rat Intestinal Epithelial Cell Apoptosis,' Free. Radic. Biol. Med., 38(12), 1614-1624(2005) https://doi.org/10.1016/j.freeradbiomed.2005.02.029
  10. Uno, H. and Kurata, S., 'Chemical Agents and Peptides Affect Hair Growth,' J. Invest. Dermatol., 101(1 Suppl), 143-147(1993) https://doi.org/10.1111/1523-1747.ep12363275
  11. Thomas, C. E., 'The Influence of Medium Components on Cu(2+)-Dependent Oxidation of Low-density Lipoproteins and its Sensitivity to Superoxide Dismutase,' Biochim. Biophys. Acta., 1128(1), 50-57(1992) https://doi.org/10.1016/0005-2760(92)90256-U
  12. Trachy, R. E., Fors, T. D., Pickart, L. and Uno, H., 'The Hair Follicle-stimulating Properties of Peptide Copper Complexes. Results in C3H Mice.,' Ann. N. Y. Acad. Sci., 26(642), 468-469(1991)
  13. Lewin, B. and Genes, V., Oxford University Press Inc., New York(1994)
  14. Paus, R. and Pecker, S., 'Biology of Hair and Nail,' In Bolognia, J. L., Jorizzo, J. L., Rapini, R. P., eds Dermatology. Mosby-Wolfe, London, 1007-1032(2003)
  15. Sheng, Y., Pero, R. W., Olsson, A. R., Bryngelsson, C. and Hua, J., 'DNA Repair Enhancement by a Combined Supplement of Carotenoids, Nicotinamide, and Zinc,' Cancer. Detect. Prev., 22, 284-292(1998) https://doi.org/10.1046/j.1525-1500.1998.CDOA37.x
  16. Marini, M. and Musiani, D., 'Micromolar Zinc Affects Endonucleolytic Activity in Hydrogen Peroxide-mediated Apoptosis,' Exp. Cell. Res., 239(2), 393-398(1998) https://doi.org/10.1006/excr.1997.3909
  17. Handjiski, B. K., Eichmuller, S., Hofmann, U., Czarnetzki, B. M. and Paus, R., 'Alkaline Phosphatase Activity and Localization During the Murine Hair Cycle,' Br. J. Dermatol., 131(3), 303- 310(1994) https://doi.org/10.1111/j.1365-2133.1994.tb08515.x
  18. Theresa, J., Enrico, A., Anil, H. and Sreedhar, K., 'Glycine Prevents the Induction of Apoptosis Attributed to Mesenteric Ischemia/ reperfusion Injury in a Rat Model,' Surgery, 134, 457-466(2003) https://doi.org/10.1067/S0039-6060(03)00164-8
  19. Uno, H., Cappas, A. and Schlagel, C., 'Cyclic Dynamics of Hair Follicles and the Effect of Minoxidil on the Bald Scalps of Stumptailed Macaques,' Am. J. Dermatopathol., 7(3), 283-297(1985) https://doi.org/10.1097/00000372-198506000-00012
  20. Philpott, M. P., Green, M. R. and Kealey, T., 'Rat Hair Follicle Growth in vitro.,' Br. J. Dermatol., 127(6), 600-607(1992) https://doi.org/10.1111/j.1365-2133.1992.tb14873.x
  21. Commo, S., Gaillard, O. and Bernard, B. A., 'The Human Hair Follicle Contains two Distinct K19 Positive Compartments in the Outer Root Sheath: a Unifying Hypothesis for Stem Cell Reservoir?,' Differentiation, 66(4-5), 157-164(2000) https://doi.org/10.1046/j.1432-0436.2000.660401.x
  22. Michel, M., Torok, N., Godbout, M. J., Lussier, M., Gaudreau, P., Royal, A. and Germain, L., 'Keratin 19 as a Biochemical Marker of Skin Stem Cells in vivo and in vitro: Keratin 19 Expressing Cells are Differentially Localized in Function of Anatomic Sites, and Their Number Varies with Donor Age and Culture Stage.,' J. Cell. Sci., 109, 1017-1028(1996)
  23. Yang, L., Zhang, B., Toku, K., Maeda, N., Sakanaka, M. and Tanaka, J., 'Improvement of the Viability of Cultured Rat Neurons by the Non-essential Amino Acids L-serine and Glycine that Upregulates Expression of the Anti-apoptotic Gene Product Bcl-w.,' Neurosci. Lett., 295(3), 97-100(2000) https://doi.org/10.1016/S0304-3940(00)01597-4