RESUMO
Porcine heart xenotransplantation is a potential treatment for patients with end-stage heart failure. To understand molecular mechanisms of graft rejection after heart transplantation, we transplanted a 31-day-old alpha-1,3-galactosyltransferase knockout (GTKO) porcine heart to a five-year-old cynomolgus monkey. Histological and transcriptome analyses were conducted on xenografted cardiac tissue at rejection (nine days after transplantation). The recipient monkey's blood parameters were analyzed on days -7, -3, 1, 4, and 7. Validation was conducted by quantitative real-time PCR (qPCR) with selected genes. A non-transplanted GTKO porcine heart from an age-matched litter was used as a control. The recipient monkey showed systemic inflammatory responses, and the rejected cardiac graft indicated myocardial infarction and cardiac fibrosis. The transplanted heart exhibited a total of 3748 differentially expressed genes compared to the non-transplanted heart transcriptome, with 2443 upregulated and 1305 downregulated genes. Key biological pathways involved at the terminal stage of graft rejection were cardiomyopathies, extracellular interactions, and ion channel activities. The results of qPCR evaluation were in agreement with the transcriptome data. Transcriptome analysis of porcine cardiac tissue at graft rejection reveals dysregulation of the key molecules and signaling pathways, which play relevant roles on structural and functional integrities of the heart.
Assuntos
Rejeição de Enxerto , Transplante de Coração , Transplante Heterólogo , Animais , Biomarcadores , Biologia Computacional/métodos , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Ontologia Genética , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Haplorrinos , Transplante de Coração/efeitos adversos , Imunossupressores/farmacologia , Masculino , Anotação de Sequência Molecular , Suínos , Transcriptoma , Transplante Heterólogo/efeitos adversosRESUMO
We report a morphometric evaluation of α1,3-galactosyltransferase knockout (GTKO) pig heart and kidney (n = 9) at the end of one, three and seven months. Organs parameters gradually increased with the age (p < 0.05) and body weight (p < 0.05) of the pigs. Organs morphometries were significantly correlated to the age and body weight of the animal. We were able to conclude the average size of GTKO pig heart and kidney based on age and body weight, which could be helpful in estimating the size of these organs non-invasively for transplantation.
Assuntos
Galactosiltransferases/deficiência , Coração/anatomia & histologia , Rim/anatomia & histologia , Sus scrofa/anatomia & histologia , Animais , Feminino , Técnicas de Inativação de Genes , Masculino , Sus scrofa/genéticaRESUMO
In this study, we attempted to upgrade GT -MCP/-MCP pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT -MCP/-MCP pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT -MCP/-MCP /mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.
