A legislação brasileira para cosméticos exige que os apelos mercadológicos desses produtos sejam comprovados. Os testes in vivo utilizando animais para avaliação desta categoria de produtos ou os princípios ativos nela contidos são, atualmente, bastante criticados. O presente trabalho teve como objetivo desenvolver um modelo de cultura de pele humana e avaliar a viabilidade dos melanócitos durante o período de 7 dias. A manutenção do tecido foi avaliada pela observação microscópica após coloração com HE e Masson, os melanócitos ativos pela reação de DOPA e a melanina pela coloração Fontana-Masson. Fragmentos de pele com 2mm2 mantidos em meio de Leibovitz à temperatura ambiente e atmosfera com 95% de ar e 5% de CO2, sofreram menos alterações morfológicas comparados aos mantidos em DMEM à temperatura de 37°C, 5% de CO2, 40% de O2 e 55% de N2. Fragmentos com 20mm2 mantidos em Leibovitz apresentaram alterações semelhantes aos de 2mm2. Células em divisão foram observadas em amostras de pele mantidas em Leibovitz enriquecido com SFB e ácido retinóico. A presença de melanina foi verificada durante todo o período de cultura, bem como a dos melanócitos, que se mostraram DOPA reativos. A radiação UVA/UVB, empregada com a finalidade de verificar se os melanócitos sofriam alguma alteração na atividade e morfologia, não provocou qualquer mudança nestas células, por outro lado induziu uma redistribuição da melanina nos queratinócitos dos fragmentos irradiados. Os resultados obtidos mostraram que é possível manter pele humana em cultura por 7dias, bem como a viabilidade dos melanócitos e sugere ser possível a aplicação do modelo estudado em futuros ensaios de eficácia e segurança de produtos tópicos. / The Brazilian law for cosmetics demands that the marketing of these products should be proofed. The tests in vivo utilizing animals for evaluation of this category of products or the active components in it, are very criticized nowadays. The present work had as an objective to develop a model of culture of human skin, and evaluate the viability of the melanocytes during a period of 7 days. The maintenance of the tissue was evaluated by the microscopic observation after coloring with HE and Masson, the active melanocytes by the DOPA reaction and the melanin by coloring of Fontana-Masson. Fragments of skin with 2mm2 kept in Leibovitz at room temperature and atmosphere with 95 % of air and 5 % of CO2, presented less morphologic alterations than the ones kept in DMEM at the temperature of 37, 5% of CO2, 40% of O2 and 55% of N2. Fragments with 20mm2 kept in Leibovitz presented similar alterations to the 2mm2. Mitotic cells were observed in samples of skin kept in enriched Leibovitz with FBS and retinoic acid. The presence of melanin was verified through all of the period of culture as well as the melanocytes that were DOPA-reactive. The radiation UVA/UVB, used with the aim of verifying if the melanocytes presented any alteration in the activity and morphology, didn\'t provoke any changes in the cells, on the other hand it induced a redistribution of the melanin in the keratinocytes of the irradiated fragments. The results obtained showed that it is possible to keep the human skin in culture for 7 days as well as the viability of melanocytes, and suggests the possibility of application of the studied model in future research on the efficacy and the safety of topic products.

A legislação brasileira para cosméticos exige que os apelos mercadológicos desses produtos sejam comprovados. Os testes in vivo utilizando animais para avaliação desta categoria de produtos ou os princípios ativos nela contidos são, atualmente, bastante criticados. O presente trabalho teve como objetivo desenvolver um modelo de cultura de pele humana e avaliar a viabilidade dos melanócitos durante o período de 7 dias. A manutenção do tecido foi avaliada pela observação microscópica após coloração com HE e Masson, os melanócitos ativos pela reação de DOPA e a melanina pela coloração Fontana-Masson. Fragmentos de pele com 2mm2 mantidos em meio de Leibovitz à temperatura ambiente e atmosfera com 95% de ar e 5% de CO2, sofreram menos alterações morfológicas comparados aos mantidos em DMEM à temperatura de 37°C, 5% de CO2, 40% de O2 e 55% de N2. Fragmentos com 20mm2 mantidos em Leibovitz apresentaram alterações semelhantes aos de 2mm2. Células em divisão foram observadas em amostras de pele mantidas em Leibovitz enriquecido com SFB e ácido retinóico. A presença de melanina foi verificada durante todo o período de cultura, bem como a dos melanócitos, que se mostraram DOPA reativos. A radiação UVA/UVB, empregada com a finalidade de verificar se os melanócitos sofriam alguma alteração na atividade e morfologia, não provocou qualquer mudança nestas células, por outro lado induziu uma redistribuição da melanina nos queratinócitos dos fragmentos irradiados. Os resultados obtidos mostraram que é possível manter pele humana em cultura por 7dias, bem como a viabilidade dos melanócitos e sugere ser possível a aplicação do modelo estudado em futuros ensaios de eficácia e segurança de produtos tópicos. / The Brazilian law for cosmetics demands that the marketing of these products should be proofed. The tests in vivo utilizing animals for evaluation of this category of products or the active components in it, are very criticized nowadays. The present work had as an objective to develop a model of culture of human skin, and evaluate the viability of the melanocytes during a period of 7 days. The maintenance of the tissue was evaluated by the microscopic observation after coloring with HE and Masson, the active melanocytes by the DOPA reaction and the melanin by coloring of Fontana-Masson. Fragments of skin with 2mm2 kept in Leibovitz at room temperature and atmosphere with 95 % of air and 5 % of CO2, presented less morphologic alterations than the ones kept in DMEM at the temperature of 37, 5% of CO2, 40% of O2 and 55% of N2. Fragments with 20mm2 kept in Leibovitz presented similar alterations to the 2mm2. Mitotic cells were observed in samples of skin kept in enriched Leibovitz with FBS and retinoic acid. The presence of melanin was verified through all of the period of culture as well as the melanocytes that were DOPA-reactive. The radiation UVA/UVB, used with the aim of verifying if the melanocytes presented any alteration in the activity and morphology, didn\'t provoke any changes in the cells, on the other hand it induced a redistribution of the melanin in the keratinocytes of the irradiated fragments. The results obtained showed that it is possible to keep the human skin in culture for 7 days as well as the viability of melanocytes, and suggests the possibility of application of the studied model in future research on the efficacy and the safety of topic products.

In den letzen Jahren gab es durch epidemiologische und molekularbiologische Studien vermehrt Hinweise, dass kutane humane Papillomviren (HPV) ursächlich an der Entstehung nicht-melanozytärer Hauttumore (engl. NMSC) beteiligt sind. Ziel der vorliegenden Arbeit war die Identifizierung molekularer Mechanismen der viralen Proteine E6 und E7 kutaner HPV-Typen. Die E6 oder E7 Gene der verschiedenen HPV-Typen 1, 4, 5, 8, 20, 38 und RTRX7 wurden untersucht. Natürliche Wirtszellen dieser Viren, humane primäre Keratinozyten (HPK) der Haut, wurden mit rekombinanten, für E6 oder E7 kodierenden Retroviren infiziert. Die Analysen erfolgten in Monolayer-Kultur (undifferenzierte Keratinozyten) oder in organotypischen Hautmodellen (Induktion der Keratinozytendifferenzierung). Die Expression von E6 oder E7 führte in Monolayer-HPK zu einer Verlängerung der Lebensspanne und zu einer deutlich erhöhten Verdoppelungsrate. Eine Telomeraseaktivierung, die charakteristisch für immortale Zellen ist, wurde nur in HPV 8 E6 positiven HPK nachgewiesen. In organotypischen Hautmodellen induzierte das E7 Protein von HPV 1, 4 und 38 starke Veränderungen in der Differenzierung sowie eine Zunahme der Proliferation. Weiterhin wurde eine Aufhebung der normalen Zellzykluskontrolle in suprabasalen HPV 5 E7 oder HPV 8 E7 beobachtet. Hinweise auf ein starkes invasives Potential von E7-infizierten HPK wurden für HPV 8 E7 bestätigt und für HPV 4 E7, HPV 38 E7 und RTRX7 E7 erweitert. Molekulare Mechanismen der viralen Gene E6 und E7 kutaner HPV unterscheiden sich von mukosalen Typen. Das Mehrstufenmodell der Karzinogenese beinhaltet eine Reihe fundamentaler Zelltransformationen, die für eine Tumorgenese nötig sind. In dieser Arbeit beschriebene Mechanismen der Modulation der Zelldifferenzierung und Zellproliferation durch die kutanen HPV-Typen 4, 5, 8 und 38 können unter Umständen zur Induktion und Progression früher Stadien von Plattenepithelkarzinomen (SCC) beitragen. / In the last years epidemiologic and molecular biological studies accumulated increasing evidence that cutaneous human papillomaviruses are etiologically involved in the formation of non-melanoma skin cancer (NMSC). The presented work aims to identify the underlying molecular mechanisms of the viral proteins E6 and E7 of cutaneous HPV types. The E6 and E7 genes of the different HPV types 1, 4, 5, 8, 20, and RTRX7, which are in vivo associated with cutaneous benign or malignant lesions, were studied. Natural host cells of these viruses, human primary keratinocyts (HPK) of the skin, were infected with recombinant E6 and E7 encoding retroviruses. The following analyses were performed in monolayer culture (non-differentiated keratinocytes) or in organotypic skin culture (induction of keratinocyte differentiation). The expression of E6 and E7 elongated the life span of monolayer HPK and significantly increased the doubling rate. An activation of the telomerase, characteristic for immortalized cells, was only detected in HPV 8 E6 positive cells. In organotypic skin cultures E7 of HPV 1, 4 and 38 induced drastic changes in differentiation and proliferation. Additionally an impairment of the normal cell cycle control in suprabasale HPV 5 E7 and 8 E7 cultures was seen. Hints for a strong invasive potential of E7 infected HPK were proven for HPV 8 E7 and expanded to HPV 4 E7, HPV 38 E7 and RTRX E7. The viral E6 and E7 genes of cutaneous and mucosal HPV types exhibit different molecular mechanisms. The multistep model of carcinogenesis includes a series of fundamental cell transformations necessary for tumorigenesis. Mechanisms for the modulation of cell differentiation and proliferation by cutaneous HPV types 4, 5, 8 and 38 described in this work could potentially contribute to the induction and progression of early stages of squamous cell carcinoma.