Proposta de modelo in vitro de Doença Hepática Gordurosa Não-Alcoólica (DHGNA): Silenciamento do fator de crescimento TGF-a em células AML-12.
Data
2023-07-10
Autores
Tamashiro, Juliana Almeida 
Tipo
Trabalho de conclusão de curso
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ISSN da Revista
Título de Volume
Resumo
Introdução: A Doença Hepática Gordurosa Não Alcoolica é uma doença que acomete cerca de 25% da população mundial, caracterizada pelo acúmulo de triacilglicerídeos (TAG) no citoplasma dos hepatócitos, podendo evoluir a esteatohepatite, cirrose e hepatocarcinoma. Uma das vias que contribui para o acúmulo lipídico é a de novo lipogenesis (DNL), estimulada pelo alto consumo de frutose de alimentos ultraprocessados. A linhagem celular AML-12, de hepatócito de camundongo, é utilizado para o estudo da doença. No entanto, resultados prévios do nosso grupo, ainda não publicados, demonstram que ao invés da expressão esperada de Glicoquinase (GCK), a isoenzima expressa é a Hexoquinase II (HK2), impactando diretamente no metabolismo de glicose e consequentemente, na de novo lipogenesis. Materiais e Métodos: Foi construído através de clonagem, um vetor plasmidial contendo um short hairpin RNA (shRNA) direcionado ao RNA mensageiro (mRNA) do Transforming Growth Factor alpha (TGF-ɑ) a fim de realizar um silenciamento por interferência por RNA deste gene, responsável pela imortalização da linhagem AML-12. O estabelecimento do protocolo foi testado através do ensaio de beta-galactosidase. Resultados: A partir do seqüenciamento pelo método de Sanger, foi possível confirmar a obtenção correta do plasmídio contendo o inserto de shRNA para o gene do TGFα. Conclusão: É possível concluir que as modificações propostas no protocolo do fabricante foram decisivas para a obtenção do plasmídio de silenciamento.
Introduction: Non-Alcoholic Fatty Liver Disease is a disease that affects about 25% of the world population, characterized by the accumulation of triacylglycerides in the cytoplasm of hepatocytes, which can progress to steatohepatitis, cirrhosis and hepatocellular carcinoma. One of the pathways that contribute to lipid accumulation is de novo lipogenesis, stimulated by high consumption of fructose from ultra-processed foods. The mouse hepatocyte cell line AML-12 is used to study this disease. However, previous results from our group, not yet published, demonstrated that, considering the expected expression of Glucokinase, the expressed isoenzyme is Hexokinase II, directly impacting glucose metabolism and, consequently, de novo lipogenesis. Materials and Methods: A plasmid vector containing a short hairpin RNA (shRNA) directed to the Transforming Growth Factor alpha (TGF-ɑ) mRNA was constructed through cloning in order to carry out silencing by RNA interference of this gene, responsible for immortalization of the AML-12 lineage. The establishment of the protocol was tested through the beta-galactosidase assay. Results: From the sequencing by the Sanger method, it was possible to confirm the correct obtaining of the plasmid containing the shRNA insert for the TGFα gene. Conclusion: It is possible to conclude that the proposed modifications in the manufacturer's protocol were supported for obtaining the silencing plasmid.
Introduction: Non-Alcoholic Fatty Liver Disease is a disease that affects about 25% of the world population, characterized by the accumulation of triacylglycerides in the cytoplasm of hepatocytes, which can progress to steatohepatitis, cirrhosis and hepatocellular carcinoma. One of the pathways that contribute to lipid accumulation is de novo lipogenesis, stimulated by high consumption of fructose from ultra-processed foods. The mouse hepatocyte cell line AML-12 is used to study this disease. However, previous results from our group, not yet published, demonstrated that, considering the expected expression of Glucokinase, the expressed isoenzyme is Hexokinase II, directly impacting glucose metabolism and, consequently, de novo lipogenesis. Materials and Methods: A plasmid vector containing a short hairpin RNA (shRNA) directed to the Transforming Growth Factor alpha (TGF-ɑ) mRNA was constructed through cloning in order to carry out silencing by RNA interference of this gene, responsible for immortalization of the AML-12 lineage. The establishment of the protocol was tested through the beta-galactosidase assay. Results: From the sequencing by the Sanger method, it was possible to confirm the correct obtaining of the plasmid containing the shRNA insert for the TGFα gene. Conclusion: It is possible to conclude that the proposed modifications in the manufacturer's protocol were supported for obtaining the silencing plasmid.
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Citação
TAMASHIRO, Juliana Almeida. Proposta de modelo in vitro de Doença Hepática Gordurosa Não-Alcoólica (DHGNA): Silenciamento do fator de crescimento TGF-a em células AML-12. 2023. 63 f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) – Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2023.