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KMID : 0350519930460020583
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Journal of Catholic Medical College
1993 Volume.46 No. 2 p.583 ~ p.598
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The Effects of Collagenase and Trypsin on Cell Growth and Protein Synthesis in Human Skin Fibroblast Culture
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Abstract
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Fibroblasts are mesenchymal cells in most organs, which play a crucial role in repair processes but are also involed in development of fibrosis. Although there were many reports about fibroblasts, their growth kinetics and multiple functions such
as
migration, chemotaxis and protein synthesis have recently been investigated. This study was performed to investigate the effects of extracellular matrix and tissue proteolytic enzymes on growth of human skin fibroblasts.
Collagen, collagenase and trypsin were added to cultured fibroblasts for investigating the effects on cell growth and protein synthesis. For cell growth determination, DNA and protein synthesis were measured by [3H]-thymidine, [3H]-leucine and
[3H]-proline incorporation. We also observed the effects of trypsin in various doses and fetal calf serum on growthm DNA and protein synthesis of fibroblast using the same method. Trypsin was treated with heat(56℃ or 85℃) for inactivation of
cytolytic
activity. The synthetic polypeptide, milk casein and gelatin agar were used as the substrate for the enzyme activity assay to determine proteolytic activites in conditioned media and cultured supernatant.
@ES The results were as follows :
@EN 1) There was a significant decrease of DNA and protein synthesis of cultured fibroblasis in the presence of collagen matri compared with in control group (P<0.01).
2) . DNA and proein synthesis of cultured fibroblasts were ecreased by below 20% in collagenase or trypsin treated group compared with in control group (P<0.001).
3) DNA and protein synthesis of cultured fibroblasts in 56℃-trypsin-treated group were lower than those in 85℃-trypsin-treated group, but no significant difference between the two groups was found in the amont of DNA.
4) Proteolytic activity in conditioned media of 56℃-trypsin-treated group was higher than that of 85℃-trypsin-treated group, but no significant difference between the two groups of cultured fibroblasts was found in the amount of DNA.
5) There was a significant decrease of DNA and protein synthesis of cultured fibroblasts in serum free media compared with in serum containing media (P<0.001).
6) When fibroblasts were cultured in serum free media, the growth rates showed no significant difference depending upon various concentrations of trypsin.
In conclusion, collagen matrix may affect the down modulation of fibroblast growth and protein synthesis as dose dependent manner, and the low dose of proteolytic enzyme without cytolytic activity may inhibit fibroblast growth, DAN and protein
synthesis
in culture.
With above results, we suppose that modulation of fibrosis kinetics by non-specific proteolytic enzymes, such as trypsin may be helpful to the treatment or prevention of pathologic fibrosis.
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KEYWORD
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