Padronização de cultura organóide cutânea e avaliação da resposta melanogênica no melasma ao UVB, UVA e luz visível.
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Data
2019-09-26
Autores
Alcantara, Giovana Piteri
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Universidade Estadual Paulista (Unesp)
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FUNDAMENTOS: Melasma é hipermelanose crônica, focal, adquirida decorrente de patogênese não totalmente compreendida, resultado da alteração funcional e arquitetural dos melanócitos. A predisposição genética, aspectos hormonais e exposição à radiação solar são os elementos mais associados ao desenvolvimento da doença e essenciais para entendimento da fisiopatologia. É bem estabelecida a relação entre a exposição à radiação solar e a piora do quadro, entretanto, o efeito independente do UVB, UVA, assim como a atuação da luz visível (LV) são pouco estudados. OBJETIVOS: Padronização de um modelo de cultura organoide cutâneo primário para estudo da melanogênese da pele com melasma e pele normal adjacente, à irradiação com diferentes comprimentos de onda (UVB, UVA, LV). MÉTODOS: Etapa 1: Amostras de pele da região retroauricular (punch 3mm), de 10 voluntários foram seccionados longitudinalmente, e cultivados em meio DMEM segundo protocolo estabelecido por Ayres, para padronização de viabilidade e dosimetria das radiações induzindo melanogênese. Um dos fragmentos foi irradiado e o outro mantido ao abrigo da luz por 72h. Foram avaliados aspectos morfológicos e arquiteturais da histologia (H&E e Fontana-Masson) e por rt-PCR para comparação da expressão quantitativa de gene constitucional (GAPDH) entre as peles recém-coletadas e as cultivadas. Foram padronizadas as doses de radiação e tempo de cultura que promovessem viabilidade da amostra e aumento de 10% na intensidade de pigmentação da camada basal. Etapa 2: Amostras de pele facial com melasma e normal adjacente (distância menor que 2cm), de 22 voluntários foram acondicionadas em meio de cultura DMEM: um fragmento ao abrigo da luz e a outra exposta às radiações (UVB, UVA ou LV), segundo a padronização prévia. A pigmentação melânica induzida pelas radiações foi avaliada, na camada basal, pela coloração de Fontana-Masson. Os valores das intensidades de pigmentação melânica das amostras foram comparados entre os grupos (UVB x UVA x LV), entre os procedimentos (irradiado x não-irradiado) e entre as topografias (melasma x normal adjacente) por modelo linear generalizado de efeitos mistos. RESULTADOS: No processo de padronização, as doses de UVB, UVA e LV (luz azul) indutoras de melanogênese diferencial foram: 166mJ/cm2, 1,524J/cm2 e 40J/cm2. Houve manutenção arquitetural e a expressão de GAPDH não diferiu entre a amostra de pele recém-coletada e a cultivada por 72h. Na fase de comparação, houve melanogênese no melasma e na pele normal adjacente após todas as irradiações (p<0,01), sem diferença entre as topografias (p=0.66). Após ajuste para o fototipo, houve aumento na granulação da melanina somente após a irradiação com UVA e apenas na pele com melasma (p<0,01). Entretanto, a maior proeminência dos dendritos ocorreu apenas na pele com melasma para irradiação com UVB e para LV (p<0,01), sem diferença entre UVB e LV. LIMITAÇÕES: Estudo em cultura de pele, dissociada da regulação hormonal, neural e vasomotora do organismo, e avaliação do efeito das radiações de forma independente. CONCLUSÃO: Padronizou-se cultura organoide primária de pele, com sobrevida de 72h, manutenção das propriedades melanogênicas e resposta às diferentes radiações. Explorou-se o papel melanogênico de UVB, UVA e LV no melasma comparada à pele normal adjacente, fundamental no desenvolvimento de novas terapêuticas e a otimização da fotoproteção na doença. Este modelo pode fundamentar estudos em fotobiologia, fotoproteção e regulação da melanogênese.
FUNDAMENTALS: Melasma is chronic hypermelanosis, focal, acquired due to pathogenesis not fully understood, resulting from the functional and architectural alteration of melanocytes. Genetic predisposition, hormonal aspects and exposure to solar radiation are the elements most associated with the development of the disease and essential for understanding the pathophysiology. The relationship between exposure to solar radiation and worsening of the condition is well established, however, the independent effect of UVB, UVA, as well as the action of visible light (VL) are poorly studied. OBJECTIVES: Standardization of a primary cutaneous organoid culture model to study melanogenesis of skin with melasma and adjacent normal skin to irradiation with different wavelengths (UVB, UVA, VL). METHODS: Step 1: Skin samples from the retroauricular region (punch 3mm) from 10 volunteers were longitudinally sectioned and cultured in DMEM medium according to the protocol established by Ayres, for viability standardization and radiation dosimetry inducing melanogenesis. One of the fragments was irradiated and the other kept in the dark for 72h. Morphological and architectural aspects of histology (H&E and Fontana-Masson) and rt-PCR were evaluated for comparison of quantitative constitutional gene expression (GAPDH) between newly collected and cultured skins. Radiation doses and culture time that promoted sample viability and a 10% increase in basal layer pigmentation intensity were standardized. Step 2: Samples of melasma and adjacent normal facial skin (distance less than 2cm) from 22 volunteers were placed in DMEM culture medium, one fragment protected from light and the other exposed to radiation (UVB, UVA or VL), according to previous standardization. Radiation-induced melanic pigmentation was evaluated in the basal layer by Fontana-Masson staining. The values of the melanin pigmentation intensities of the samples were compared among the groups (UVB x UVA x VL), between the procedures (irradiated x non-irradiated) and between the topographies (melasma x adjacent normal) by generalized linear mixed effects model. RESULTS: In the standardization process, the doses of UVB, UVA and VL (blue light) inducing differential melanogenesis were: 166mJ / cm², 1,524J / cm² and 40J / cm². There was architectural preservation, and GAPDH expression did not differ between the newly collected skin sample and the one cultivated for 72h. In the comparison phase, there was melanogenesis in the melasma and adjacent normal skin after all irradiations (p <0.01), with no difference between topographies (p=0.66). After phototype adjustment, melanin granulation increased only after UVA irradiation and only in melasma skin (p <0.01). However, the highest prominence of dendrites occurred only in skin with melasma for UVB and VL irradiation (p <0.01), with no difference between UVB and VL. LIMITATIONS: Study in skin culture, dissociated from the hormonal, neural and vasomotorl regulation of the body, and evaluation of the effect of radiation independently. CONCLUSION: Primary organoid skin culture was standardized, with 72h survival, maintenance of melanogenic properties and response to different radiation. It was explored the melanogenic role of UVB, UVA and VL in melasma compared to adjacent normal skin, fundamental in the development of new therapies and the optimization of photoprotection in the disease. This model can substantiate studies in photobiology, photoprotection and regulation of melanogenesis.
FUNDAMENTALS: Melasma is chronic hypermelanosis, focal, acquired due to pathogenesis not fully understood, resulting from the functional and architectural alteration of melanocytes. Genetic predisposition, hormonal aspects and exposure to solar radiation are the elements most associated with the development of the disease and essential for understanding the pathophysiology. The relationship between exposure to solar radiation and worsening of the condition is well established, however, the independent effect of UVB, UVA, as well as the action of visible light (VL) are poorly studied. OBJECTIVES: Standardization of a primary cutaneous organoid culture model to study melanogenesis of skin with melasma and adjacent normal skin to irradiation with different wavelengths (UVB, UVA, VL). METHODS: Step 1: Skin samples from the retroauricular region (punch 3mm) from 10 volunteers were longitudinally sectioned and cultured in DMEM medium according to the protocol established by Ayres, for viability standardization and radiation dosimetry inducing melanogenesis. One of the fragments was irradiated and the other kept in the dark for 72h. Morphological and architectural aspects of histology (H&E and Fontana-Masson) and rt-PCR were evaluated for comparison of quantitative constitutional gene expression (GAPDH) between newly collected and cultured skins. Radiation doses and culture time that promoted sample viability and a 10% increase in basal layer pigmentation intensity were standardized. Step 2: Samples of melasma and adjacent normal facial skin (distance less than 2cm) from 22 volunteers were placed in DMEM culture medium, one fragment protected from light and the other exposed to radiation (UVB, UVA or VL), according to previous standardization. Radiation-induced melanic pigmentation was evaluated in the basal layer by Fontana-Masson staining. The values of the melanin pigmentation intensities of the samples were compared among the groups (UVB x UVA x VL), between the procedures (irradiated x non-irradiated) and between the topographies (melasma x adjacent normal) by generalized linear mixed effects model. RESULTS: In the standardization process, the doses of UVB, UVA and VL (blue light) inducing differential melanogenesis were: 166mJ / cm², 1,524J / cm² and 40J / cm². There was architectural preservation, and GAPDH expression did not differ between the newly collected skin sample and the one cultivated for 72h. In the comparison phase, there was melanogenesis in the melasma and adjacent normal skin after all irradiations (p <0.01), with no difference between topographies (p=0.66). After phototype adjustment, melanin granulation increased only after UVA irradiation and only in melasma skin (p <0.01). However, the highest prominence of dendrites occurred only in skin with melasma for UVB and VL irradiation (p <0.01), with no difference between UVB and VL. LIMITATIONS: Study in skin culture, dissociated from the hormonal, neural and vasomotorl regulation of the body, and evaluation of the effect of radiation independently. CONCLUSION: Primary organoid skin culture was standardized, with 72h survival, maintenance of melanogenic properties and response to different radiation. It was explored the melanogenic role of UVB, UVA and VL in melasma compared to adjacent normal skin, fundamental in the development of new therapies and the optimization of photoprotection in the disease. This model can substantiate studies in photobiology, photoprotection and regulation of melanogenesis.
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Palavras-chave
Fotobiologia, Melasma, Cultura organóide, Ultravioleta, Luz visível, Photobiology, , Organoid Culture, Ultraviolet, Visible Light