Journal of the Korea Academia-Industrial cooperation Society (한국산학기술학회논문지)

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
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Establishment and characterization of porcine mammary gland epithelial cell line using three dimensional culture system

3차원 배양 시스템을 이용한 돼지 유선 상피 세포 주 특성과 설정

  • Chung, Hak-Jae (Swine Science Division, National Institute of Animal Science, Rural Development Administration)
  • 정학재 (농촌진흥청 국립축산과학원 양돈과)
  • Received : 2017.09.20
  • Accepted : 2017.10.13
  • Published : 2017.10.31
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Abstract

To study and validate tissue-specific promoters and vectors, it is important to develop cell culture systems that retain the tissue and species specificity. Such systems are attractive alternatives to transgenic animal models. This study established a line of porcine mammary gland epithelial cells (PMECs) from a primary culture based on the cellular morphology and mRNA levels of porcine beta-casein (CSN2). The selected PMECs were stained with the cytokeratin antibody, and were shown to express milk protein genes (CSN2, lactoferrin, and whey acidic protein). In addition, to confirm the acini structure of PMEC932-7 in 3D culture, live cells were stained with SYTO-13 dye, which binds to nucleic acid. The acini of these PMECs on matrigel were formed by the aggregation of peripheral cells and featured a hollow lumens. The system was demonstrated by testing the effects of the culture conditions to cell culture including cell density and matrigel methods of the PMECs. These results suggest that PMECs possess the genetic and structural features of mammary epithelial cells.

조직특이적 프로모터 및 벡터를 연구하고 검증하기 위해서는 조직 및 종의 특이성을 유지하는 세포 시스템을 개발하는 것이 바람직하다. 이러한 시스템은 형질전환동물 모델에 대한 효과적인 대안이다. 우리는 베타 카제인 (CSN2)의 세포 형태와 mRNA 수준에 기초하여 일차 배양으로부터 돼지 유선 상피 세포 주 (PMECs)를 확립하였다. 선택된 PMECs는 cytokeratin 항체에 의해 염색되었으며, 유선 상피 세포에 존재한다고 생각되어지는 유즙 단백질 유전자 (CSN2, 락토페린 및 유청 단백질)를 발현하는 것으로 나타났다. 또한, 3D 배양에서 PMECs932-7의 acini 구조를 확인하기 위해 살아있는 세포를 핵산에 결합하는 SYTO-13으로 염색하였다. 우리는 마트리겔 (matrigel)에 있는 PMECs의 acini가 말초 세포의 응집에 의해 형성되고 공간의 lumen을 특징으로 한다는 것이 관찰하였다. 우리는 PMECs의 matrigel 사용법과 세포 밀도를 포함한 세포 배양 조건의 영향을 시험함으로서 시스템을 시연했다. 이러한 결과는 PMCEs의 유선 상피 세포는 유전적 또는 구조적 특징을 갖고 있음을 시사하고 있다.

Keywords

References

  1. Van Cott, K. E, W. H. Velander, "Transgenic animals as drug factories: a new source of recombinant protein therapeutics", Expert Opin. Investig. Drugs, vol. 7, no. 10, pp. 1683-1690, 1998. DOI: http://dx.doi.org/10.1517/13543784.7.10.1683
  2. Janne, J, L. Alhonen, J. M. Hyttinen, T. Peura, M. Tolvanen, V. P. Korhonen, "Transgenic bioreactors", Biotechnol. Annu. Rev. vol. 4, pp. 55-74. 1998. DOI: https://doi.org/10.1016/S1387-2656(08)70067-X
  3. Rudolph, N. S, "Biopharmaceutical production in transgenic livestock", Trends Biotechnol, vol. 17, no. 9, pp. 367-374, 1999. DOI: http://dx.doi.org/10.1016/S0167-7799(99)01341-4
  4. Wheeler, M. B, E. M. Walters, "Transgenic technology and applications in swine", Theriogenology, vol. 56, no. 8, pp. 1345-1369, 2001. DOI: https://doi.org/10.1016/S0093-691X(01)00635-5
  5. Pipe, S. W, "The promise and challenges of bioengineered recombinant clotting factors", J. Thromb. Haemost, vol. 3, no. 8, pp. 1692-1701, 2005. https://doi.org/10.1111/j.1538-7836.2005.01367.x
  6. Velander, W. H, J. L. Johnson, R. L. Page, C. G. Russell, A. Subramanian, T. D. Wilkins, F. C. Gwazdauskas, C. Pittius, W. N. Drohan, "High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C", Proc. Natl. Acad. Sci, vol. 89, no. 24, pp. 12003-12007. 1992. https://doi.org/10.1073/pnas.89.24.12003
  7. Paleyanda, R. K. W. H. Velander, T. K. Lee, D. H. Scandella, F. C. Gwazdauskas, J. W. Knight, L. W. Hoyer, W. N. Drohan, H. Lubon, "Transgenic pigs produce functional human factor VIII in milk", Nat. Biotechnol, vol. 15, no. 10, pp. 971-975, 1997. DOI: https://doi.org/doi:10.1038/nbt1097-971
  8. Park, J. K. Y. K. Lee, P. Lee, H. J. Chung, S. Kim, H. G. Lee, M. K. Seo, J. H. Han, C. G. Park, H. T. Kim, T. K. Kim, K. S. Min, J. H. Kim, H. T. Lee, and W. K. Chang, "Recombinant human erythropoietin produced in milk of transgenic pigs", J. Biotechnol, vol. 122, no. 3, pp. 362-371, 2006. DOI: https://doi.org/10.1016/j.jbiotec.2005.11.021
  9. Sharp, J. A. K. N. Cane, S. L. Mailer, W. H. Oosthuizen, J. P. Amould, K. R. Nicholas, "Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)", Matrix. Biol. vol. 25, no. 7, pp. 430-442, 2006. DOI: https://doi.org/10.1016/j.matbio.2006.05.003
  10. Fata, J. E. Z. Werb, and M. J. Bissell, "Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes", Breast Cancer Res. vol. 6, no. 1, pp. 1-11, 2003. DOI: https://doi.org/10.1186/bcr634
  11. Bissell, M. J. A Rizki, and I. S. Mian, "Tissue architecture: the ultimate regulator of breast epithelial function", Curr. Opin. Cell. Biol. vol. 15, no. 6, pp. 753-762, 2003. DOI: https://doi.org/doi:10.1016/j.ceb.2003.10.016
  12. Bissell, M. J, "Modelling molecular mechanisms of breast cancer and invasion: lessons from the normal gland", Biochem. Soc. Trans, vol. 35, no. 1, pp. 18-22, 2007. DOI: https://doi.org/10.1042/BST0350018
  13. Lee, G. Y, P. A. Kenny, E. H. Lee, M. J. Bissell, "Three-dimensional culture models of normal and malignant breast epithelial cells", Nat. Methods, vol. 4, no. 4, pp. 359-365, 2007. DOI: https://doi.org/10.1038/nmeth1015
  14. Kumura, H, A. Tanaka, Y. Abo, S. Yui, K. Shimazaki, E. Kobayashi, K. Satama, "Primary culture of porcine mammary epithelial cells as a model system for evaluation of milk protein expression", Biosci. Biotechnol. Biochem. vol. 65, no. 9, pp. 2098-2101. 2001. DOI: https://doi.org/10.1271/bbb.65.2098
  15. Sun, Y. L, C. S. Lin, Y. C. Chou, "Gene transfection and expression in a primary culture of mammary epithelial cells isolated from lactating sows", Cell. Biol. Int, vol. 29, no. 7, pp. 576-582, 2005 DOI: https://doi.org/10.1016/j.cellbi.2005.03.021
  16. Sun, Y. L, C. S. Lin, Y. C. Chou, "Establishment and characterization of a spontaneously immortalized porcine mammary epithelial cell line", Cell. Biol. Int, vol. 30, no. 12, pp. 970-976, 2006. DOI: https://doi.org/10.1016/j.cellbi.2006.06.023
  17. Zucchi, I. and R. Dulbecco, "Proteomic dissection of dome formation in a mammary cell line", J. Mammary Gland Biol. Neoplasia, vol. 7, no. 4, pp. 373-384, 2002. DOI: https://doi.org/10.1023/A:1024081914634
  18. Debnath, J, S. K. Muthuswamy, J. S. Brugge, "Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures", Methods, vol. 30, no. 3, pp. 256-268, 2003. DOI: https://doi.org/10.1016/S1046-2023(03)00032-X
  19. Murtagh, J, E. McArdle, E. Gilligan, L. Thornton, F. Furlong, F. Martin, "Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling", J. Cell. Biol. vol. 166, no. 1, pp. 133-143, 2004. DOI: https://doi.org/10.1083/jcb.200403020
  20. Danielson, K. G, C. J. Oborn, E. M. Durban, J. S. Butel, D. Medina, "Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro", Proc. Natl. Acad. Sci. vol. 81: pp. 3756-3760, 1984. DOI: https://doi.org/10.1073/pnas.81.12.3756
  21. Gordon, K. E, B. Binas, R. S. Chapman, K. M. Kurian, R. W. E. Clarkson, A. J. Clark, E. B. Lane, J. Watson Christine, "A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland", Breast Cancer Res, vol. 2: no. 3, pp. 222-235, 2000. DOI: https://doi.org/10.1186/bcr57