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Doctoral Thesis
DOI
Document
Author
Full name
Natalia Aparecida de Paula
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Frade, Marco Andrey Cipriani (President)
Silva Neto, José Freire da
Frantz, Fabiani Gai
Moraes, Milton Ozório
Title in Portuguese
Pele humana como modelo ex vivo para manutenção do bacilo Mycobacterium leprae
Keywords in Portuguese
Cultura ex-vivo
hOSEC
Morfologia
Mycobacterium leprae
Viabilidade
Abstract in Portuguese
Mycobacterium leprae (M. leprae), descrito como causador da hanseníase em 1873, ainda hoje não há meios de cultivá-lo artificialmente in vitro, sendo que atualmente a única forma de mantêlo viável e replicando-se é a inoculação in vivo, principalmente em camundongos nude, porém trata-se de um modelo in vivo, trabalhoso e demorado. Esse trabalho propõe padronizar o cultivo do M. leprae, em modelo ex vivo, a partir de explantes de pele humana (hOSEC - human organotypic skin explant culture) tanto para ensaios laboratoriais quanto para auxílio clínico. Para isso, M. leprae, cepa Thai-53, obtido de camundongos nude, foi inoculado em fragmentos de pele humana e mantidos em placas com meio de cultura DMEM, a 5% de CO2. A manutenção, crescimento e morfologia dos bacilos nos explantes foram avaliadas após os períodos de cultura (4, 7, 14, 28 e 60 dias) por coloração de Fite-Faraco (FF) e por biologia molecular (RLEP/16s rRNA). Após 28 e 60 dias de cultura, bacilos foram recuperados dos explantes e inoculados na pata de camundongo nude para avaliar manutenção da viabilidade e infectividade. À coloração FF, bacilos foram encontrados em todos os tempos nas formas bacilar íntegra e fragmentada, isolados ou formando alguns aglomerados e raras globias. A viabilidade bacilar foi demonstrada pela positividade da RT-PCR para 16s rRNA em todos os explantes em todo o período de cultura. Dentre os animais inoculados com os bacilos previamente cultivados nos explantes por 28 e 60 dias, após manutenção por 5 meses, 28,4% apresentaram-se positivos para os bacilos, em pelo menos uma das técnicas utilizadas, FF (31,8%), Zihel-Neelsen em suspensão de macerado da pata (77,3%) e RT-PCR (45%). Suspensão de bacilos oriundas de raspados dérmicos e hansenomas de pacientes, foram inoculadas nos explantes e cultivados da mesma maneira já citada, mantendose positivas ao FF e RT-PCR após cultivo médio de 28 dias. Os bacilos de amostras clínicas também foram recuperados dos explantes e inoculados nos camundongos e também apresentaram positividade após cinco meses de manutenção in vivo. Pela primeira vez na literatura, nossos 9 resultados demonstraram com sucesso que é possível manter o M. leprae viável no modelo de pele humana ex vivo por até 60 dias, tanto a partir de amostras laboratoriais quanto clínicas, importante passo no desenvolvimento de modelos experimentais para estudos da biologia do M. leprae e suas interações, além de clínicos, como imunologia e susceptibilidade a drogas
Title in English
Human skin as an ex vivo model for maintenance of the Mycobacterium leprae bacillus
Keywords in English
Ex-vivo culture
hOSEC
Morphology
Mycobacterium leprae
Viability
Abstract in English
Mycobacterium leprae (M. leprae), described as the cause of leprosy in 1873, there is still no way to grow it artificially in vitro, and currently the only way to keep it viable and replicating is in vivo inoculation, especially in nude mice, but it is an in vivo, laborious and time-consuming model. This work proposes to standardize the cultivation of M. leprae, in an ex vivo model, from human organotypic skin explant culture (hOSEC) for both laboratory and clinical assistance. For this, M. leprae, Thai-53 strain, obtained from nude mice, was inoculated into fragments of human skin and kept in plates with DMEM culture medium, at 5% CO2. The maintenance, growth and morphology of the bacilli in the explants were evaluated after culture periods (4, 7, 14, 28 and 60 days) by Fite-Faraco staining (FF) and by molecular biology (RLEP / 16S rRNA). After 28 and 60 days of culture, bacilli were recovered from the explants and inoculated into the nude mouse paw to evaluate maintenance of viability and infectivity. At FF staining, bacilli were found at all times in whole and fragmented bacillary forms, isolated or forming some agglomerates and rare globes. Bacillary viability was demonstrated by the positivity of RT-PCR to 16S rRNA in all explants throughout the culture period. Among the animals inoculated with the bacilli previously cultured in the explants for 28 and 60 days, after maintenance for 5 months, 28.4% were positive for the bacilli in at least one of the techniques used, FF (31.8%), Zihel-Neelsen in paw maceration suspension (77.3%) and RT-PCR (45%). Bacilli's suspension of bacilli from skin smear and injury biopsy of patients were inoculated into the explants and cultured in the same manner as mentioned above, and show positivity for FF and RT-PCR after an average culture of 28 days. Clinical sample bacilli were also recovered from the explants and inoculated in the mice and also showed positivity after five months of in vivo maintenance. For the first time in the literature, our results have successfully demonstrated that it is possible to maintain M. leprae viable in the ex vivo human skin model for up to 60 days, both from laboratory and clinical samples, an 11 important step in the development of experimental models for studies the biology of M. leprae and its interactions, as well as clinical, such as immunology and drug susceptibility
 
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Publishing Date
2019-11-12
 
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