Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Primary Cultures of Drosophila melanogaster Gut Cells for Studies of Intestinal Stem Cell Regulation

장줄기세포 조절 연구를 위한 초파리 장세포의 일차배양

  • Yoon, Young-Il (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Hwang, Jae-Sam (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Goo, Tae-Won (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Han, Myung-Sae (Department of Bio-Fibers and Materials Science, Kyungpook National University) ;
  • Ahn, Mi-Young (Department of Agricultural Biology, National Academy of Agricultural Science, RDA) ;
  • Yun, Eun-Young (Department of Agricultural Biology, National Academy of Agricultural Science, RDA)
  • 윤영일 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 황재삼 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 구태원 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 한명세 (경북대학교 바이오섬유소재학과) ;
  • 안미영 (농촌진흥청 국립농업과학원 농업생물부) ;
  • 윤은영 (농촌진흥청 국립농업과학원 농업생물부)
  • Received : 2012.02.14
  • Accepted : 2012.05.07
  • Published : 2012.05.30
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Abstract

$Drosophila$ $melanogaster$ has been used as a useful model to study development and disease. In this study, we established the primary culture method of $Drosophila$ in the intestine to understand how intestinal stem cells (ISCs) mediate tissue repair during infection and disease. To obtain intestinal cells, we separated intestines from adult flies and isolated single cells by enzymatic treatment. The survival of cultured cells was measured using MTS-analysis. The maximum growth rate of the cells was observed on the 9th day after seeding. In addition, the presence of ISCs and enteroendocrine cells was confirmed by delta and prospero staining. Accordingly, we supposed that $Drosophila$ $melanogaster$ gut cells established in this study are probably useful in studies about intestinal stem cell regulation and various diseases occurring in the intestine.

초파리는 발생과 질병연구를 위한 모델 곤충으로 널리 이용되어 왔다. 본 연구에서도 초파리를 모델곤충으로 한 장질환 연구의 일환으로 다양한 병원균 감염 및 장질환 유발시 어떻게 장줄기세포가 작용하는지를 이해하기 위해 초파리 장세포의 일차배양 방법을 확립하였다. 초파리 성충으로부터 장을 해부하고 다양한 효소를 처리하여 장세포를 분리한 후 배양하였다. 배양세포의 생존여부는 현미경 검경 및 MTS assay에 의해 확인한 결과 배양 후 9일째 최대 증식되었고 14일까지 생존함을 확인할 수 있었다. 또한 장줄기세포 및 장내분비세포의 존재도 immunostaining에 의해 확인하였다. 따라서 본 연구에서 구축된 초파리 일차배양 장세포는 다양한 유전자에 의한 장줄기 세포 조절연구뿐만 아니라 장에서 발생하는 다양한 질병을 연구하는 도구로 매우 유용할 것으로 추측된다.

Keywords

References

  1. Amcheslavsky, A., Jiang, J. and Ip, Y. T. 2009. Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell 4, 49-61. https://doi.org/10.1016/j.stem.2008.10.016
  2. Apidianakis, Y. and Rahme, L. G. 2011. Drosophila melanogaster as a model for human intestinal infection and pathology. Dis. Model Mech. 4, 21-30. https://doi.org/10.1242/dmm.003970
  3. Benjamin, O. and Allan, S. 2005. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470-474.
  4. Cha, S. J. and Han, S. S. 1999. Establishment of a novel cell line from Plutella xylostella fat bodies. Korean J. Entomol. 29, 121-126.
  5. Davis, T. R., Trotter, K. M., Granados, R. R. and Wood, H. A. 1992. Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation, and secretion. Biotechnology 10, 1148-1150. https://doi.org/10.1038/nbt1092-1148
  6. Evans, G. S., Flint, N. and Potten, C. S. 1994. Primary cultures for studies of cell regulation and physiology in intestinal epithelium. Annu. Rev. Physiol. 56, 399-417. https://doi.org/10.1146/annurev.ph.56.030194.002151
  7. Grace, T. D. 1962. Establishment of four strains of cells from insect tissues grown in vitro. Nature 195, 788-789. https://doi.org/10.1038/195788a0
  8. Granados, R. R., Guoxun, L., Derksen, A. C. G. and McKenna, K. A. 1994. A new insect cell line from Trichoplusia ni (BTI-Tn-5B1-4) susceptible to Trichoplusia ni single enveloped nuclear polyhedrosis virus. J. Invertebr. Pathol. 64, 260-266. https://doi.org/10.1016/S0022-2011(94)90400-6
  9. Juan, J. G., Guoxun, L., Ping, W., Jiang, Z. and Robert, R. G. 2001. Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae). In Vitro Cell Dev. Biol. Anim. 37, 353-359.
  10. Kapuscinski, J. 1995. DAPI: a DNA-specific fluorescent probe. Biotech. Histochem. 70, 220-233. https://doi.org/10.3109/10520299509108199
  11. Lanzoni, G., Roda, G., Belluzzi, A., Roda, E. and Bagnara, G. P. 2008. Inflammatory bowel disease: Moving toward a stem cell-based therapy. World J. Gastroenterol. 14, 4616-4626. https://doi.org/10.3748/wjg.14.4616
  12. Luer, K. and Technau, G. M. 1992. Primary culture of single ectodermal precursors of Drosophila reveals a dorsoventral prepattern of intrinsic neurogenic and epidermogenic capabilities at the early gastrula stage. Development 116, 377-385.
  13. Noguchi, T. and Miller, K. G. 2003. A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster. Development 130, 1805-1816. https://doi.org/10.1242/dev.00406
  14. O'Reilly, D. R., Miller, L. K. and Luckow, V. A. 1992. Baculovirus Expression Vectors: A Laboratory Manual (New York, N. Y.: Freeman, W. H. and Company).
  15. Palli, S. R., Caputo, G. F., Brownwright, A. J. and Sofi, S. S. 1997. Studies on apoptosis in a continuous midgut cell line, CF-203, of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae). In: Maramorosch. K., and Mitsuhashi, J. (eds.), pp. 43-51, Invertebrate cell culture. Novel directions and biotechnology applications. Science, Enfield.
  16. Pan, C., Kumar, C., Bohl, S., Klingmueller, U. and Mann, M. 2009. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol. Cell Proteomics 8, 443-450. https://doi.org/10.1074/mcp.M800258-MCP200
  17. Raziel, S. H., Silvia, C., Marcia, L., and Guy, S. 2009. Primary culture of insect midgut cells. In Vitro Cell Dev. Biol. Anim. 45, 106-110. https://doi.org/10.1007/s11626-009-9176-7
  18. Ren, F., Wang, B., Yue, T., Yun, E. Y., Ip, Y. T. and Jiang, J. 2010. Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways. Proc. Natl. Acad. Sci. USA 107, 21064-21069. https://doi.org/10.1073/pnas.1012759107
  19. Roda, G., Sartini, A., Zambon, E., Calafiore, A., Marocchi, M., Caponi, A., Belluzzi, A. and Roda, E. 2010. Intestinal epithelial cells in inflammatory bowel diseases. World J. Gastroenterol. 16, 4264-4271. https://doi.org/10.3748/wjg.v16.i34.4264
  20. Sicaeros, B., Campusano, J. M. and O'Dowd, D. K. 2007. Primary neuronal cultures from the brains of late stage Drosophila pupae. J. Vis. Exp. 4, 200.
  21. Siegmund, B. and Zeitz, M. 2011. Innate and adaptive immunity in inflammatory bowel disease. World J. Gastroenterol. 17, 3178-3183.
  22. Soin, T., Swevers, L., Mosallanejad, H., Efrose, R., Labropoulou, V., Iatrou, K. and Smagghe, G. 2008. Juvenile hormone analogs do not affect directly the activity of the ecdysteroid receptor complex in insect culture cell lines. J. Insect Physiol. 54, 429-438. https://doi.org/10.1016/j.jinsphys.2007.11.001
  23. Vaughn, J. L., Goodwin, R. H., Tompkins, G. J. and McCawley, P. 1977. The Establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213-217. https://doi.org/10.1007/BF02615077
  24. Wickham, T. J. and Nemerow, G. R. 1993. Optimization of growth methods and recombinant protein production in BTI Tn-5B1-4 insect cells using the baculovirus expression vector. Biotechnol. Prog. 9, 25-30. https://doi.org/10.1021/bp00019a004
  25. Wickham, T. J., Davis, T., Granados, R. R., Shuler, M. L. and Wood, H. A. 1992. Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol. Prog. 8, 391-396. https://doi.org/10.1021/bp00017a003
  26. Wirtz, S. and. Neurath, M. F. 2007. Mouse models of inflammatory bowel disease. Adv. Drug Deliv. Rev. 59, 1073-1083. https://doi.org/10.1016/j.addr.2007.07.003