• BMB Reports
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• 제51권4호
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• pp.188-193
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• 2018

Caffeoylserotonin (CaS), one derivative of serotonin (5-HT), is a secondary metabolite produced in pepper fruits with strong antioxidant activities. In this study, we investigated the effect of CaS on proliferation and migration of human keratinocyte HaCaT cells compared to that of 5-HT. CaS enhanced keratinocyte proliferation even under serum deficient condition. This effect of CaS was mediated by serotonin 2B receptor (5-HT2BR) related to the cell proliferation effect of 5-HT. We also confirmed that both CaS and 5-HT induced G1 progression via 5-HT2BR/ERK pathway in HaCaT cells. However, Akt pathway was additionally involved in upregulated expression levels of cyclin D1 and cyclin E induced by CaS by activating 5-HT2BR. Moreover, CaS and 5-HT induced cell migration in HaCaT cells via 5-HT2BR. However, 5-HT regulated cell migration only through ERK/AP-1/MMP9 pathway while additional Akt/NF-${\kappa}B$/MMP9 pathway was involved in the cell migration effect of CaS. These results suggest that CaS can enhance keratinocyte proliferation and migration. It might have potential as a reagent beneficial for wound closing and cell regeneration.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제27권6호
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• pp.510-522
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• 2005

To assess the clinical applicability of bio-artificial mucosa which was made with autologous oral keratinocytes and human acellular dermal matrix, the formation of basement membrane and stratification of oral keratinocytes were evaluated. Six New Zealand white rabbits (around 2kg in weight) were anesthetized and its buccal mucosa was harvested (1.0 $\times$ 0.5cm size). Oral keratinicytes were extracted and cultured primarily with the feeder layer of pretreated NIH J2 3T3 fibroblast. These confluent cells were innoculated on the human acellular dermal matrix and cultured in multiple layer by air-rafting method. After 3, 5, 7, 10, 14 days of culture, each cultured bio-artificial mucosa was investigated the number of epthelial layer of by H&E stain and toluidine blue stain. The immuhohistochemical methods were used to evaluate the cell division capacity, the formation of basement membrane, and it's property of specific cells (PCNA, cytokeratin 14, laminin). Transmission electromicroscopy was used for the attachment between cells and matrix with the number of hemidesmosome. In result, the numbers of layer of stratified growth of oral keratinocyte cultured on the human acellular dermal matrix and the number of hemidesomal attachment between epithelial cells and human acellular dermal matrix were similar to the layers of normal oral mucosa after 10 days of culture. The cell division rate, basement membrane formation and proliferation rate increased as culture period increased. With these results, bio-artificial mucosa with autologous oral epithelial cells cultured on the acellular dermal matrix had clinically adaptable properties after 10 days' culture and this new bio-artificial mucosa model with relatively short culture time can be expected clinical applicability.

• 대한치과마취과학회지
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• 제14권2호
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• pp.101-106
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• 2014

Background: Remifentanil, an ultra-short-acting mu-opioid receptor agonist, is unique from other opioids because of its esterase-based metabolism, minimal accumulation, and very rapid onset and offset of clinical action. Remifentanil can prevent the inflammatory response and can suppress inducible nitric oxide synthase expression in a septic mouse model. However, the effects of remifentanil on human keratinocyte and autophagy have yet to be fully elucidated during hypoxia-reoxygenation. Here we investigated whether remifentanil confers protective effect against hypoxia-reoxygenation in human keratinocyte and, if so, whether autophagy mediates this effect. Methods: The human keratinocytes were cultured under 1% oxygen tension. The cells were gassed with 94% $N_2$, and 5% $CO_2$ and incubated for 24 h at $37^{\circ}C$. To determine whether the administration of affects human keratinocytes hypoxia-reoxygenation injury, cells were then exposed to various concentrations of remifentanil (0.01, 0.1, 0.5 and 1 ng/ml) for 2 h. After remifentanil treatment, to simulate reoxygenation and recovery, the cells were reoxygenated for 12 h at $37^{\circ}C$. Control group did not receive remifentanil treatment. Normoxia group did not receive hypoxia and remifentanil treatment for 36 h. 3-MA group was treated 3-methyladenine (3-MA) for 1h before remifentanil treatment. Cell viability was measured using a quantitative colorimetric assay with MTT, showing the mitochondrial activity of living cells. Cells were stained with fluorescence and analyzed with Western blot analysis to find out any relations with activation of autophagy. Results: Prominent accumulation of autophagic specific staining MDC was observed around the nuclei in RPT group HaCaT cells. Similarly, AO staining, red fluorescent spots appeared in RPT group HaCaT cells, while the Normoxia, control and 3-MA groups showed mainly green cytoplasmic fluorescence. We here examined activation of autophagy related protein under H/R-induced cells by Western blotting analysis. Atg5, Beclin-1, LC3-II (microtubule-associated protein 1 light chain 3 form II) and p62 was elevated in RPT group cells. But they were decreased when autophagy was suppressed by 3-MA (Fig. 5). Conclusions: Although the findings of this study are limited to an in vitro interpretation, we suggest that remifentanil may have a beneficial effect in the recovery of wound from hypoxia-reoxygenation injury.

• 대한치과보철학회지
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• 제37권6호
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• pp.717-729
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• 1999

Although many studies on the cytotoxicity of the dental cast base metal alloys and their components have been carried out, the results are rather conflicting because of the different type of cells used and the various experimental procedures taken. Recently a number of scientists have claimed that it would be preferable to focus on the use of cells from relevant specific location of the human bodies. Consequently, the primary cultured oral keratinocyte derived from oral mucous along with nickel chloride and several of widely used dental cast base metal alloys(two Ni-Cr alloys and one Co-Cr alloy)in domestic were selected for this study, from which 1) The amounts of released metal ions were determined using atomic absorption spectrometry, 2) The cytotoxicity of nickel chloride and dental cast base metal alloys was evaluated via MTT assay, and finally, 3) The amounts of released metal ions and the cytotoxicity of nickel chloride were correlated with the cytotoxicity of dental cast base metal alloys And, the results were summarized as follows; 1. Nickel ion from Ni-Cr alloys and Cobalt ion from Co-Cr alloys resulted in maximum releasing rate during first 2h hours, followed by a decrease in releasing rate with time. Chromium ion were found to be minimal in all alloys. 2. In cytotoxic test. with $40{\mu}M,\;80{\mu}M$ of nickel chloride, there were observed an increase in the relative cell number compared to control samples after 24 hours. With $160{\mu}M$, there was found to be no difference in the relative cell number with control, except that 48 hour showed a increase in relative cell number. With $320{\mu}M$, the relative cell number remained constant and decreased after 48 hours, and with $640{\mu}M$, a continuing decrease in relative cell number was observed throughout test period. 3 The sensitivity of primary cultured oral epithelium to nickel was lower compared to the cells used in other studies. 4. CB-80 Soft and Regalloy showed no cytotoxicity to primary cultured oral epithelium and New crown resulted in a slight cytotoxicity. In conclusion, it was shown that the primary cultured oral keratinocytes could be applied successfully as testing cells in cytotoxicity test. Futhermore, the dental cast base metal alloys used in this study were found to be biocompatible.

• 한국융합학회논문지
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• 제10권4호
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• pp.81-89
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• 2019

이 연구는 HPV 16 E6/E7 도입 불멸화 구강상피세포의 배양 미세환경과 세포 분화간의 관계를 분석하였다. 배양환경을 변화시켜서 IHOK-EF 세포와 IHOK-EFKGM 세포를 얻었고, 이들 세포의 특성변화를 세포증식분석, 면역형광분석 및 마이크로어레이와 실시간 정량 PCR분석으로 알아보았다. IHOK-EF 세포는 상피세포의 특성을 상실하고 간엽세포의 특성을 획득하였고, 마이크로어레이 분석결과, 분화억제 유전자인 ID2, IL6, TWIST1이 과발현 되었다. 이러한 변화는 초기의 배양환경으로 회복되었을 때, 특별히, ID2와 IL6에서 유전자발현의 복귀를 나타내면서 세포의 특성이 부분적으로 회복되었다. 이 연구는 세포의 특성을 결정하는 연구에서 배양 미세환경의 변화에 따른 세포의 생존을 위한 적응양상을 이해하는데 공헌할 것이며, 향후, 암세포의 미세환경변화에 따른 생존연구에 적용하여 질병에 대한 치료적 접근을 가능하게 할 것이다.

• International Journal of Oral Biology
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• 제39권2호
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• pp.97-105
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• 2014

The aim of this study was to determine the beneficial effect of propofol on human keratinocytes that have undergone hypoxia reoxygenation (H/R) injury and to investigate whether autophagy is associated with the protective mechanism. Thus, we evaluated how propofol influences the intracellular autophagy and apoptosis during the H/R process in the HaCaT cells. The cultured human keratinocyte cells were exposed to 24 h of hypoxia (5% $CO_2$, 1% $O_2$, 94% $N_2$) followed by 12 h of reoxygenation (5% $CO_2$, 21% $O_2$, 74% $N_2$). The experiment was divided into 4 groups: (1) Control=Normoxia ; (2) H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=Propofol Preconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+ H/R=3-MA-Methyladenine+Propofol Preconditioning+ Hypoxia Reoxygenation. In addition, Western blot analysis was performed to identify the expression of apoptotic pathway parameters, including Bcl-2, Bax, and caspase 3 involved in mitochondrial-dependent pathway. Autophagy was determined by fluorescence microscopy, MDC staining, AO staining, and western blot. The H/R produced dramatic injuries in keratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. The treatment with 25, 50, and $100{\mu}M$ Propofol in H/R induced HaCaT cells enhanced cell viability in a dose-dependent manner and $100{\mu}M$ was the most effective dose. The Atg5, Becline-1, LC3-II, and p62 were elevated in PPC group cells, but H/R-induced group showed significant reduction in HaCaT cells. The Atg5 were increased when autophagy was induced by Propofol, and they were decreased when autophagy was suppressed by 3-MA. These data provided evidence that propofol preconditioning induced autophagy and reduced apoptotic cell death in an H/R model of HaCaT cells, which was in agreement with autophagy playing a very important role in cell protection.

• Imaging Science in Dentistry
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• 제33권2호
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• pp.97-105
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• 2003

Purpose : To evaluate the effect of all-trans-retinoic acid (ATRA) on the radiosensitivity of normal human oral keratinocyte (NHOK). Materials and methods: Relative cell survival fraction including SF2 (survival fraction at 2 Gy) was calculated on the basis of colony formation assay. Data were fitted to the linear-quadratic model to establish the survival curve and calculate α and β values. Using flow cytometry at 1, 2, 3, 4, and 5 days after exposure to 2 and 10 Gy irradiation, cell cycle arrest and apoptosis were analysed. To understand the molecular mechanism of the radiosensitization of ATRA on NHOK, proteins related with apoptosis and cell cycle arrest were investigated by Western blot analysis. Results: Treatment with ATRA resulted in a significant decrease of SF2 value for NHOK from 0.63 to 0.27, and increased α and β value, indicating that ATRA increased radiosensitivity of NHOK. ATRA increased LDH significantly, but increasing irradiation dose decreased LDH, suggesting that the radiosensitizing effect of ATRA is not directly related with increasing cell necrosis by ATRA. ATRA did not induce appotosis but increased G2 arrest after 10 Gy irradiation, implying that the increased radiosensitivity of NHOK may be due to a decrease in mitosis casued by increasing G2 arrest. ATRA inhibited the reduction of p53 at 3 days after l0Gy irradiation and increased p21 at 1 day after 10 Gy irradiation. Further study is required to determine the precise relationship between this effect and the radiosensitizing effect of A TRA. Conclusion: These results suggested that ATRA increase radiosensitivity by inhibiting mitosis caused by increasing G2 arrest.

• 한국치위생학회지
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• 제15권4호
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• pp.719-725
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• 2015

Objectives: The purpose of this study was to determine the toxic effects of sodium lauryl sulfate(SLS) in human keratinocyte HaCaT cells and mouse fibroblast NIH-3T3 cells. Methods: The effect of sodium lauryl sulfate(SLS) cell viability and proliferation were determined by WST-1 assay and changes shape of nucleus were evaluated by Hoechst staining under fluorescence microscopy. Additionally, observation of cell morphological changes under light microscopy. Results: SLS induced cytotoxicity and a marked apoptosis in both HaCaT and NIH-3T3 cell lines. With the result of the WST-1 assay, SLS induced the cytotoxicity of 0.005% and 0.0075%, 0.01% SLS for 24 h after HaCaT and NIH-3T3 cells in time and dose-dependent manner(p<0.005). SLS inhibited cell growth and caused apoptosis as evidenced by nuclear fragmentation and condensation. Thus, determination of the morphological changes to define apoptosis was visualized using inverted phase contrast microscopy. Conclusions: SLS had toxicity of the human keratinocyte cells and mouse fibroblast cells and this study will provide the basic data for the development of proper SLS concentration in dentifrice.

• 한국콘텐츠학회논문지
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• 제22권3호
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• pp.586-592
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• 2022

본 연구는 기존의 치면열구전색재 ConciseTM에 세륨옥사이드 나노입자(Cerium oxide nano particles; CNPs)를 0-4.0 wt%를 첨가하여 새로운 치면열구전색재를 제조하여 물리적 성질과 세포독성을 평가하였다. 물리적 성질은 중합깊이, 물흡수도와 용해도를 측정하였고 세포독성평가는 불멸화된 구강점막세포(Immortalized human oral keratinocyte (IHOK))를 이용하여 MTT assay법으로 평가하였다. 실험 결과 중합 깊이는 CNPs 첨가량이 증가할수록 감소하였고, 용해도는 CNPs를 2.0 wt% 첨가된 실험군에서 가장 낮은 값을 보였으며 물흡수도는 각 실험군별 유의한 차이는 나타나지 않았다(p>0.05). 세포독성 실험 결과는 모든 실험군에서 높은 세포 생존율을 보였다. 이는 CNPs가 물리적 성질을 크게 약화 시키지 않으며 세포독성을 나타내지 않았으므로 생체 적합성을 입증하였다. CNPs의 특성을 고려하여 향후 CNPs의 효율적인 분산 기술에 대한 추가 연구가 필요할 것으로 사료된다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제23권3호
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• pp.258-262
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• 2001

Distraction osteogenesis(DO) can be performed in the bony defect associated with trauma, anomaly, and various kinds of disease. The gap generated by DO is filled with growing callus : during the period of distraction, the osteogenesis is continued. However, there have been few reports about expression pattern of growth factors in newly formed bone during the consolidation periods. We performed DO in the mandibular defect case and studied the expressed pattern of growth factors. Its pattern was compared to that of the same patient. BMP-2 and -4 were strongly expressed in the DO site. Particularly, BMP-4 was not expressed in the normal mature bone, but expressed in new bone in DO. However, there was no difference in the FGF-7 expression between the sites. Therefore, strong expression of BMP-4 are related to new bone formation in DO and they may not be related to the normal homeostasis in human bone. Though FGF-7 is related to the growth of keratinocyte, it may have minimal role in the DO and normal mature bone.