제품화된 무방부제 힐론 점안제가 각막상피세포에 미치는 영향
Effect of Preservative-Free Healon Eye Drop on Human Corneal Epithelial Cell in Vitro
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목적: 농도가 다른 세 가지 제품화된 무방부제 힐론 점안제가 각막상피세포에 미치는 영향을 비교해 보고자 한다. 대상과 방법: 인체의 각막상피세포를 일차배양하여 3-4차례 배양 분주한 후 배지에 sodium hyaluronate 0.1% (카이닉스Ⓡ, Alcon,Seoul, Korea), sodium hyaluronate 0.18% (카이닉스2Ⓡ, Alcon, Seoul, Korea), sodium hyaluronate 0.3% (히알유니점안액 0.3%Ⓡ,Taejoon, Seoul, Korea) 점안약제를 1, 3, 6, 12, 24, 36시간 노출한 후 각막상피세포에 미치는 영향을 비교하였다. 각막 상피세포의증식력을 비교하기 위해 MTT 검사와 Ki67 검사, 각막 상피세포의 이주력을 비교하기 위해 CD44 검사와 Scratch wound assay, 각막상피세포에 미치는 세포독성을 비교하기 위해 LDH 검사와 전자현미경 및 도립위상차현미경을 이용한 형태학적 관찰을 시행하였다. 결과: 세 가지 약제에서 각 시간별 농도에 따른 MTT 결과치는 유의한 차이가 없었고(p＞0.05), Ki-67 결과치는 히알유니점안액 0.3%Ⓡ가 카이닉스Ⓡ, 카이닉스2Ⓡ보다 높았으나 유의한 차이를 보이지 않았다(p＞0.05). CD44 결과 유의한 차이는 없으나 히알유니점안액0.3%Ⓡ가 높은 수치를 보였고(p＞0.05), 48시간 후 Scratch wound assay는 히알유니점안액 0.3%Ⓡ, 카이닉스2Ⓡ, 카이닉스Ⓡ 순으로세포 이주가 증가되었다. LDH 결과치는 각 시간별 약제의 농도가 올라갈수록 증가되었지만(p＞0.05), 형태학적 관찰에서는 세포질내변화는 큰 차이를 보이지 않았다. 결론: 시중에 판매되는 제품화된 히알유니점안액 0.3%Ⓡ가 카이닉스2Ⓡ, 카이닉스Ⓡ에 비해 각막상피세포에 대한 세포의 이주능력이다소 빨리 진행되며, 세포손상에서는 약제의 농도가 높을수록 더 심하게 나타나지만 세 약제 간 유의한 차이는 없었다.
Abstract
Purpose: To investigate the biologic effects of preservative-free artificial tear drops on cultured human corneal epithelial cells invitro. Methods: The effects of preservative-free artificial tear drops (sodium hyaluronate 0.1% (KynexⓇ, Alcon, Seoul, Korea), sodiumhyaluronate 0.18% (Kynex2Ⓡ, Alcon, Seoul, Korea), and sodium hyaluronate 0.3% (Hyaluni eye drops 0.3%Ⓡ, Taejoon, Seoul,Korea)) on human corneal epithelial cells were evaluated. Cellular proliferation was determined using MTT (3- (4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) and Ki-67 assays. Cellular migration was determined using CD44 and scratch woundassays. Cell damage was determined using the lactate dehydrogenase (LDH) assay and cellular morphologies using electronicmicroscopy and inverted microscopy. Results: Proliferation of corneal epithelial cells, as determined by the MTT and Ki-67 assays, was not significantly different betweenthe different eye drops (p > 0.05). The measured value of cellular migration after exposure of the sodium hyaluronate0.3%, as determined by mean CD44 percentage and scratch wound assay, was higher than that of the sodium hyaluronate 0.1%and sodium hyaluronate 0.18%, but the CD44 value was not significantly different (p > 0.05). The LDH titer tended to increase asthe concentration of sodium hyaluronate increased (p > 0.05), but influence on cytoplasm, as determined by electronic microscopy,was not different. Conclusions: Among 3 preservative-free artificial tear drops, sodium hyaluronate 0.3% demonstrated increased migration of cornealepithelial cells. As the concentration of sodium hyaluronate in eye drops increased, the corneal cytotoxicity of corneal epithelialcells also increased. However, there was no significant difference among the 3 tear drops.
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