송아지 혈청 농도가 인체의 각막상피세포에 미치는 영향
Effects of Calf Serum on Human Corneal Epithelial Cells in Vitro
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목적: 송아지 혈청 농도에 따른 인체의 각막상피세포의 증식력 및 독성에 관한 효과를 알아보고자 하였다. 대상과 방법: 인체각막상피세포를 일차 배양하여 송아지 혈청 10, 20, 30% 농도로 1, 4, 12, 24시간 동안 접촉시킨 후, MTT분석법을이용하여 세포대사력을 비교하고, 각막상피세포에서 분비되는 IL-1α, TGF-β1, MMP-9 등의 생성량을 비교하였다. Lactate dehydrogenase (LDH) assay를 이용하여 세포독성을 비교하고, 전자현미경을 이용하여 형태학적 변화를 관찰하였다. 결과: MTT상 혈청의 농도가 높을수록 세포 대사활성도가 감소하였고, LDH 역가는 접촉 시간이 길수록, 농도가 높을수록 증가하였다. IL-1α와 TGF-β1, MMP-9의 경우 모든 농도에서 대조군에 비해 생성량이 감소되었다. 세포의 손상 정도는 핵막주변부의 핵질 농축과세포질 내 구조물의 확대소견 등이 농도가 높을수록 뚜렷하게 나타났다. 결론: 송아지 혈청은 10% 농도에서 20%, 30%보다 각막상피세포의 대사력이 좋으면서 세포 독성이 적고 염증성 사이토카인의 생성에미치는 영향력도 효과적이어서 임상 적용에 유리할 것으로 생각한다.
Abstract
Purpose: To investigate the biologic effects of topical calf serum on corneal epithelial cells in vitro. Methods: The effects of calf serum on the corneal epithelial cells were evaluated using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and the concentration of IL-1α, TGF-β1 and MMP-9 in the cells was measured. Cell damage was determined using lactate dehydrogenase (LDH), and cellular morphologies were examined by transmission electromicroscopy. Results: Metabolic activity of the corneal epithelial cells decreased at higher concentrations and longer exposure durations. IL-1α, TGF-β1 and MMP-9 titers were lower in calf serum-treated cells than in the control. LDH and cellular damage to the corneal epithelial cells, such as chromatin margination and cytoplasmic organelle swelling, were prominent in cells treated with 30% calf serum. Conclusions: Cellular metabolic activity was higher and cellular toxicity was lower in cells treated with 10% calf serum compared to those treated with the 20% and 30% concentrations. Furthermore, inflammatory cytokines were sufficiently inhibited in cells treated with the 10% solution. These results indicate that 10% calf serum could be used clinically.
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