Transplante interespecífico de células germinativas-tronco em Siluriformes Neotropicais
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Data
2018-02-27
Autores
López Suárez, Lucia
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Universidade Estadual Paulista (Unesp)
Resumo
No Brasil os siluriformes neotropicais são o segundo grupo mais ameaçado de extinção com 91 espécies. Neste trabalho, foi empregada a técnica de quimerismo usando duas espécies de siluriformes neotropicais o mandi (Pimelodus maculatus) e o bagre sapo (Pseudopimelodus mangurus), visando a reconstituição da segunda espécie. Para tal foi estabelecido um protocolo de isolamento, separação, transplante e criopreservação de células germinativas tronco. As gônadas de P. mangurus foram digeridas enzimáticamente e as células germinativas tronco foram separadas por gradiente descontinuo de densidade, marcadas com PKH26 e transplantadas via papila urogenital em 32 receptores triploides de P. maculatus. Oito gônadas de P. maculatus que receberam transplantes foram analisadas por cortes histológicos e microscopia de epifluorescência para identificação das células germinativo tronco transplantadas, sendo encontradas células do doador em duas gônadas receptoras. Parte das gônadas, bem como do tecido muscular de P. maculatus e de P. mangurus foram utilizados para a extração do DNA genômico e serão utilizados para análise molecular, por a construção de primers, para confirmação do quimerismo. O protocolo estabelecido de criopreservação de células germinativas-tronco de P. mangurus permitirá transplante via intraperitoneal em larvas triploides, durante o período reprodutivo. Este trabalho é pioneiro no transplante de células germinativas-tronco em espécies de siluriformes neotropicais, usando receptores adultos triploides. Os resultados obtidos neste deste estudo poderá ser futuramente empregado em outras espécies nativas que están em perigo de extinção ou de importância na aquicultura.
In Brazil, Neotropical siluriformes are the second more endangered group with 91 species. In this work, the technique of chimerism was used with two species of Neotropical siluriformes, □mμandi□(¶ Pimelodus maculatus) and □baμgre sapo□¶□ (Pseudopimelodus mangurus), aiming the reconstitution of the second species. Therefore, was established a protocol for isolation; separation and transplantation of stem germ-cells. The gonads of P. mangurus were enzymatic digested and the stem germ-cells separated using a discontinuous density gradient, labeled with PKH26 and transplanted through the urogenital papillae in 32 triploids recipients of P. maculatus. The gonads of eight fish that have received the stem germ-cells were analyzed by histology and fluorescent microscopy for identification of the germ-cell transplanted, and cells from the donor fish were identified in the recipient gonads. For confirmation of chimerism, part of the gonads, as well the muscle tissue of P. maculatus and P. mangurus was used for extraction of genomic DNA, for primers construction and confirmation of the chimerism. The developed protocol for cryopreservation of stem germ-cells of P.mangurus will allow the transplant via urogenital papila in triploid larvae during the spawning season. This work is pioneer in the transplant of stem germ-cells in Neotropical siluriformes using adult triploids as recipients. The results of this study may be applied in future endangered native species or with importance to aquaculture.
In Brazil, Neotropical siluriformes are the second more endangered group with 91 species. In this work, the technique of chimerism was used with two species of Neotropical siluriformes, □mμandi□(¶ Pimelodus maculatus) and □baμgre sapo□¶□ (Pseudopimelodus mangurus), aiming the reconstitution of the second species. Therefore, was established a protocol for isolation; separation and transplantation of stem germ-cells. The gonads of P. mangurus were enzymatic digested and the stem germ-cells separated using a discontinuous density gradient, labeled with PKH26 and transplanted through the urogenital papillae in 32 triploids recipients of P. maculatus. The gonads of eight fish that have received the stem germ-cells were analyzed by histology and fluorescent microscopy for identification of the germ-cell transplanted, and cells from the donor fish were identified in the recipient gonads. For confirmation of chimerism, part of the gonads, as well the muscle tissue of P. maculatus and P. mangurus was used for extraction of genomic DNA, for primers construction and confirmation of the chimerism. The developed protocol for cryopreservation of stem germ-cells of P.mangurus will allow the transplant via urogenital papila in triploid larvae during the spawning season. This work is pioneer in the transplant of stem germ-cells in Neotropical siluriformes using adult triploids as recipients. The results of this study may be applied in future endangered native species or with importance to aquaculture.
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Palavras-chave
Célula germinativas-tronco, transplante célular, criopreservação