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Effect of L-Ascorbic Acid on Collagen Synthesis in 3T6 Fibroblasts and Primary Cultured Cells of Chondrocytes

3T6 세포주 및 연골 초대배양세포의 Collagen 합성에 미치는 비타민 C의 영향

  • Kim, Mi-Hyang (Dept. of Food Science and Nutrition, Silla University, and Fusion Technology Research Institute for Marine Pharmaceutical Materials)
  • 김미향 (신라대학교 식품영양학과 및 해양식의약소재융합기술연구소)
  • Published : 2006.01.01

Abstract

L-Ascorbic acid (AsA) is an essential nutrient for prevention of scurvy in humans, primates and guinea pigs that lack $L-gulono-\gamma-lactone$ oxidase which is required for the final step of AsA biosynthesis. AsA participates in various hydroxylation reactions involved in the biosynthesis of collagen. The purpose of this study is to clarify the role of AsA on collagen synthesis in 3T6 fibroblasts and primary cultured cells of chondrocytes. Cells were cultured in medium supplemented with catalase and AsA at various concentration. Supplement of AsA induced collagen synthesis in 3T6 fibroblasts and primary cultured cells of chondrocytes. The most remarkable induction of collagen synthesis by AsA was found in primary cultured chondrocytes. The content of collagen representing the amounts of extracellular matrix significantly increased in the cells of which growth was stimulated by AsA, while it decreased with increasing passage numbers of subculture in cells. It showed that the content of collagen decreased in the medium which contained AsA at the concentration higher than 5.0 mM. However, the contents of collagen to DNA were not different among various AsA concentrations. Supplementing with AsA resulted in enhancement of collagen formation and extracellular matrix. Therefore, there might be a Positive correlation between the activity of catalase and the AsA concentration. Moreover, it can be assumed that AsA stimulates the collagen synthesis by optimizing the cell-culture environment.

본 연구에서는 비타민 C의 배양세포 증식을 촉진하는 기능을 세포 간 결합조직의 주성분인 콜라겐 생합성에 초점을 맞추어 3T6 세포주 및 흰쥐에서 분리한 초대 연골세포를 이용하여, 세포 및 세포 외액 중의 콜라겐 함량의 분석과 콜라겐 생합성에 미치는 비타민 C의 영향을 각 농도별로 분석하여 콜라겐 생합성에 필요한 비타민 C의 적합한 첨가 농도를 검토하였다. 비타민 C를 1.0 mM 농도가 되도록 첨가하여 3T6 섬유아세포 및 초대연골 세포를 배양하였을때 양 세포 모두 콜라겐 양은 비타민 C를 첨가한 세포가 높은 수치를 나타내어 비타민 C에 의한 콜라겐 합성의 촉진효과가 현저하였으며 일수의 증가에 따라 그 합성량도 증가하였다. 비타민 C 무첨가의 경우 세포층의 콜라겐 합성량을 3T6 섬유아세포와 연골세포를 비교하였을 때 3T6 섬유아세포의 경우 배양일수의 경과와 더불어 콜라겐 양이 증가하였으나 연골세포의 경우 1일째부터 21일째까지 콜라겐양은 거의 변화하지 않아, 본 실험에 사용한 연골세포는 초대 배양세포로서 세포외로부터 비타민 C의 공급이 되지 않을 경우콜라겐 합성은 일어나지 않는 것으로 사료된다. 따라서 배양세포의 콜라겐 합성에 미치는 비타민 C의 영향을 검토하기 위하여 초대연골세포를 이용하여 비타민 C의 농도를 변화시켜 가면서 콜라겐 합성량을 측정하였다. 5 mM, 7 mM, 10 mM의 고농도의 비타민 C를 첨가한 경우 저 농도의 비타민 C 첨가 경우보다 plate 내의 총 콜라겐 함량은 적었다. 그러나 DNA 양에 대한콜라겐 함량을 비교하였을 때 5mM 이상의 비타민 C 첨가에서는 콜라겐 합성 증가량이 조금 낮은 경향을 보였으나, $0.1\~2mM$을 첨가하였을 때의 콜라겐 양과 최종적으로는 거의 비슷한 수치를 나타내었다. 0.5 mM 이상의 비타민 C 첨가는 콜라겐 합성을 저해한다는 기존의 보고와는 달리 본 연구에서는 세포독성을 억제할 목적으로 배지 중에 catalase 첨가하였으며, 그 결과 $0.1\~10mM$의 비타민 C 농도범위에서는 콜라겐 합성량에 차이가 크게 없는 것으로 나타나, $0.1\~10mM$의 비타민 C 농도로는 catalase를 첨가한 배지를 사용할 경우 세포내의 콜라겐 양에 대한 비타민 C 첨가농도별 차이는 크게 없는 것으로 추측된다.

Keywords

References

  1. Peterkofsky B. 1972. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured of cultured fibroblast. Arch Biochem Biophys 152: 318-328 https://doi.org/10.1016/0003-9861(72)90221-4
  2. Murad S, Sivarajah A, Pinnel SR. 1981. Regulation of prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts by ascorbic acid. Biochem Biophys Res Commun 101: 868-875 https://doi.org/10.1016/0006-291X(81)91830-1
  3. Yu R, Kurata T, Kim M, Arakawa N. 1991. The behavior of L-ascorbic acid in the healing process of dorsal wounds in guinea pigs. J Nutr Sci Vitaminol 37: 207-211 https://doi.org/10.3177/jnsv.37.207
  4. Kim M, Otsuka M, Yu R, Kurata T, Arakawa N. 1994. The distribution of ascorbic acid and dehydroascorbic acid during tissue regeneration in wounded dorsal skin of guinea pigs. Internat J Vit Nutr Res 64: 56-59
  5. 藤本大三郞 1990. 細胞外マトリックスのバイオサイエンスとバイオテクノロジー. アイピーシー, 東京. p 32
  6. Robins SP, Baily AJ. 1997. The chemistry of the collagen cross-links. Biochem J 163: 339-346
  7. Murad S, Tajima S, Johnson GR, Sivarajah A, Pinnell SR. 1981. Collagen synthesis in cultured human skin fibrlblasts: effect of ascorbic and its analogs. J Intest Dermotol 81: 158-162
  8. Peterkofsky B. 1972. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch Biochem Biophys 152: 318-328 https://doi.org/10.1016/0003-9861(72)90221-4
  9. Murata A. 1990. Diverse functions and functional mechanism of vitamin C. Nippon Nogeikagaku Kaishi 64: 1843-1845 https://doi.org/10.1271/nogeikagaku1924.64.1843
  10. Samuni A, Aronovitch J, Godinger D, Chevion M, Czapski G. 1983. On the cytotoxicity of vitamin C and metal irons. A site-specific fenton mechanism. Eur J Biochem 137: 119-125 https://doi.org/10.1111/j.1432-1033.1983.tb07804.x
  11. Kageyama K, Yamada R, Otani S, Hasuma T, Yoshimata T, Seto C, Takeda Y, Yamaguchi Y, Kogawa H, Miwa N. 1999. Abnormal cell morphology and cytotoxic effect are induced by 6-o-palmitol-ascorbate-2-0-phosphate but not by ascorbic acid or hyperthermia alone. Anticancer Res 19: 4321-4326
  12. Jampel HD. 1990. Ascorbic acid is cytotoxic to dividing human tenon's capsule fibroblasts: a possible contributing factor in glaucoma filtration surgery success. Arch Oph-thalmol 108: 1323-1329 https://doi.org/10.1001/archopht.1990.01070110139038
  13. Yue BYJT, Niedra R, Baum JL. 1989. Human corneal endo-thellal cell culture. Invertig Ophthalmo Vit Sci 30: 248-254
  14. 野田政樹. 1998. 骨のバイオロジー. 羊士社, 東京. p 56
  15. Benton HP, Tyler JA. 1998. Inhibition of cartilage proteo-glycan synthesis by interleukin I. Biochem Biophys Res Commun 154: 421-428
  16. Berg RA, Steinmann B, Rennard SI, Crystal RG. 1983. Ascorbate deficiency results in decreased collagen pro-duction: Under hydroxylation of proline leads to increased intracellular degradation. Arch Biochem Biophys 262: 681- 686
  17. Pacifici M. 1990. Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment. Biochem J 172: 193-199
  18. Yu R, Kurata T, Kim M, Arakawa N. 1991. The behavior of L-ascorbic acid in the healing process of dorsal wounds in guinea pigs. J Nutr Sci Vitaminol 37: 207-211 https://doi.org/10.3177/jnsv.37.207
  19. Kim M, Otsuka M, Yu R, Kurata T, Arakawa N. 1992. The distribution of ascorbic acid and dehydroascrobic acid during tissue regeneration in wounded dorsal skin of guinea pigs. c 64: 56-59
  20. Kim M, Otsuka M, Shimamura E, Arakawa N. 1998. The effect of L-ascorbic acid on age-related changes of pyri-dinoline in cartilage collagen of guinea pigs. J Nutr Sci Vitaminol 44: 217-224 https://doi.org/10.3177/jnsv.44.217
  21. Woessner JF. 1961. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 93: 440-447 https://doi.org/10.1016/0003-9861(61)90291-0
  22. Kim M. 2004. The effect of high concentration of ascorbic acid on the growth of primary cultured colls of chon-dro-cytes. J Korean Soc Food Sci Nutr 33: 797-802 https://doi.org/10.3746/jkfn.2004.33.5.797
  23. Hata R. 1998. Regulation of collagen gene expression by ascorbic acid 2-phosphate, a long-acting vitamin C deriv-ative. Nippon Nogeikagakukaishi 72: 1191-1194 https://doi.org/10.1271/nogeikagaku1924.72.1191

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