피부 광과민성 예측을 위한 각질세포 유래 VEGF 연구

Abstract

The needs for development of animal alternative tests have been growing based on the dual motivation of animal welfare and systems toxicology. Animal testing of cosmetics has been banned in 2013, and alternative tests for skin toxicity are currently under active development. Assessment of skin sensitization and photo-sensitization has been conducted by animal tests, such as the mouse Local Lymph Node Assay (LLNA). In this study, we tried to develop animal alternative test methods to evaluate the sensitizing/ photosensitizing potential of chemicals in vitro, using two different approaches. First, a sequential culture system of human keratinocytes (HaCaT) and human dendritic cells (THP-1) was introduced to simulate the complicated skin sensitization processes. We measured the expression level of cell surface proteins, such as CD54 and CD86, on THP-1 cells by established seven photo-sensitizers. CD54 and CD86 expression level in THP-1 were up-regulated by HaCaT conditioned-media even without chemical challenge or UV irradiation. Despite the optimization of the protocol, it was difficult to observe the significant difference between control group and sensitizer-treated groups. Second, we tried to identify a released biomarker upon activation of keratinocytes by chemicals. The level of vascular endothelial growth factor (VEGF), which plays important roles in skin inflammation, was significantly increased in HaCaT cells co-exposed to cobalt chloride (sensitizer and photo-sensitizer) and UVA. In summary, we showed that secreted mediators in HaCaT conditioned-media induce activation of THP-1 in sequential culture system, and the VEGF could be suggested as novel biomarkers to predict skin photo-sensitization in keratinocyte. The predictive capacity of VEGF needs to be further validated. We suggest that our findings may contribute to the development of in vitro sensitization or photo-sensitization assay.|시스템 독성학과 동물 복지의 인식이 증가함에 따라 동물대체시험법 개발의 필요성이 점점 증가하고 있다. 2013년 화장품에 있어 동물 실험이 금지되면서, 현재 피부 독성 평가에 대한 대체시험법 연구가 활발하게 진행되고 있다. 지금까지 피부 감작과 광감작은 동물 실험인 국소림프절시험법 (LLNA) 으로 평가되어 왔다. 본 연구에서는, 화학물질의 감작/광감작능을 평가하기 위한 동물대체시험법을 개발하기 위해 2가지 방법으로 접근하였다. 첫 번째, 생체 내 복잡한 피부 감작 과정을 구현하기 위한 사람 각질세포와 수지상세포의 연속적 배양계 구축이다. 이미 알려진 7가지 광감작물질에 의한 수지상세포의 표면단백질인 CD54와 CD86의 수준을 측정하였다. 화학물질과 자외선 노출이 없었던 각질세포 (HaCaT) 의 배지만으로도 수지상세포 (THP-1) 에서 CD54와 CD86의 수준이 증가하였다. 이를 최소화하고자 연속적 배양계를 최적화하였지만, 대조군과 감작물질 처리군에서의 유의적인 차이를 관찰하는데 어려움이 있었다. 두 번째, 감작/광감작물질에 의해 활성화된 각질세포에서 분비되는 생체지표를 관찰하였다. 코발트 염화물 (감작물질이면서 광감작물질) 과 자외선 A (UVA) 에 의해 피부 염증반응에서 중요한 역할을 하는 혈관내피세포생성인자 (VEGF) 의 수준이 유의적으로 증가하였다. 요약하여, 본 연구자는 각질세포-수지상 세포 연속배양계를 통해 각질세포 유래 물질이 수지상 세포를 활성화하는 것을 확인하였으며, 피부 광감작을 예측하는 신규 생체지표로서 혈관내피세포생성인자의 가능성을 확인하였다. 본 연구의 결과는 향후 피부 감작 및 광감작시험법의 개발에 기여할 것으로 기대된다.; The needs for development of animal alternative tests have been growing based on the dual motivation of animal welfare and systems toxicology. Animal testing of cosmetics has been banned in 2013, and alternative tests for skin toxicity are currently under active development. Assessment of skin sensitization and photo-sensitization has been conducted by animal tests, such as the mouse Local Lymph Node Assay (LLNA). In this study, we tried to develop animal alternative test methods to evaluate the sensitizing/ photosensitizing potential of chemicals in vitro, using two different approaches. First, a sequential culture system of human keratinocytes (HaCaT) and human dendritic cells (THP-1) was introduced to simulate the complicated skin sensitization processes. We measured the expression level of cell surface proteins, such as CD54 and CD86, on THP-1 cells by established seven photo-sensitizers. CD54 and CD86 expression level in THP-1 were up-regulated by HaCaT conditioned-media even without chemical challenge or UV irradiation. Despite the optimization of the protocol, it was difficult to observe the significant difference between control group and sensitizer-treated groups. Second, we tried to identify a released biomarker upon activation of keratinocytes by chemicals. The level of vascular endothelial growth factor (VEGF), which plays important roles in skin inflammation, was significantly increased in HaCaT cells co-exposed to cobalt chloride (sensitizer and photo-sensitizer) and UVA. In summary, we showed that secreted mediators in HaCaT conditioned-media induce activation of THP-1 in sequential culture system, and the VEGF could be suggested as novel biomarkers to predict skin photo-sensitization in keratinocyte. The predictive capacity of VEGF needs to be further validated. We suggest that our findings may contribute to the development of in vitro sensitization or photo-sensitization assay.