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Abstract(s)
O vírus do Papiloma Humano (HPV) é o principal fator de risco para o cancro do colo do útero, uma vez que as oncoproteínas E6 e E7 comprometem a expressão ou a atividade das proteínas supressoras de tumor p53 e pRb. Muitas abordagens terapêuticas têm sido consideradas ineficazes, existindo a necessidade de desenvolver novas estratégias como a terapia génica. O DNA minicircular (mcDNA) surge como um vetor inovador, que resulta de um processo de recombinação in vivo, no qual um plasmídeo parental (PP) leva à formação de um mini-plasmídeo (mP) que contém as sequências procariotas, e um mcDNA que contém apenas a cassete de expressão eucariótica. O mcDNA apresenta uma elevada eficiência de transfecção devido ao seu tamanho reduzido, sendo também considerado uma terapia mais segura do que o DNA plasmídico. Assim, o presente trabalho tem como objetivo, numa primeira fase, produzir e purificar um vetor de mcDNA que codifica o gene p53 para aplicação em estudos in vitro. Com o intuito de isolar a isoforma superenrolada (sc) do mcDNA-p53, a partir de quantidades residuais de PP e impurezas bacterianas, foram explorados monolitos com ligandos de L-histidina e 1-benzil-L-histidina, assim como uma coluna cromatográfica de filtração em gel (Sephacryl S-1000 SF). A força iónica e o pH dos passos de ligação e eluição foram ajustados ao longo das estratégias de purificação. Os resultados indicaram que a filtração em gel foi a melhor abordagem para o isolamento da biomolécula de interesse, assegurando um grau de pureza superior e obtenção de maior quantidade de amostra. Posteriormente, foram realizados estudos de transfecção in vitro em células HeLa com os vetores PP-p53 e mcDNA-p53, tendo-se verificado a internalização celular de ambos os vetores. Para além disto, foi confirmada a transcrição do gene de interesse p53 e detetada a tradução na respetiva proteína alvo, assim como foi realizada a sua quantificação. Por último, de modo a avaliar a ação terapêutica do gene p53 codificado nestes vetores, foram realizados ensaios de proliferação celular, citotoxicidade e apoptose. De um modo geral, todos os estudos in vitro realizados sugeriram que o mcDNA-p53 apresenta resultados mais promissores, com potencial para ser utilizado numa nova estratégia terapêutica para o cancro do colo do útero. Numa segunda fase, o presente trabalho também teve como objetivo a construção de um vetor de mcDNA multigénico, com clonagem de dois genes terapêuticos no PP (gene p53 e gene pri-miRNA-375), visando uma potencial aplicação terapêutica combinada e mais eficaz no cancro do colo do útero.
Human Papillomavirus (HPV) is considered the main causative agent for the development of cervical cancer since its E6 and E7 oncoproteins compromise the expression or activity of p53 and pRb tumor suppressor proteins. Many cervical cancer therapeutic approaches have demonstrated to be ineffective, which emphasizes the need for new strategies such as gene therapy. Minicircle DNA (mcDNA) is a small eukaryotic expression vector that results from an in vivo site-specific recombination process, in which a parental plasmid (PP) leads to the formation of a miniplasmid (mP), carrying the undesired backbone sequences, and a mcDNA, carrying the therapeutic expression cassette. The mcDNA allows higher transfection efficiency due to its smaller size and it is a safer therapeutic approach than plasmid DNA. Therefore, the present work aims, in a first stage, to produce and purify a mcDNA encoding for p53 gene regarding future in vitro studies. L-histidine and 1-benzyl-L-histidine modified monoliths, as well the gel filtration column Sephacryl S-1000 SF, were explored to isolate the supercoiled (sc) mcDNA-p53 from residual amounts of parental plasmid and bacterial impurities. The ionic strength and pH of binding and elution steps were adjusted to enhance the purification performance. The chromatographic results suggest that gel filtration column presents a better approach for sc mcDNA-p53 isolation, ensuring a higher degree of purity and providing a larger quantity of recovered sample. Afterwards, HeLa cells were transfected with PP-p53 and mcDNA-p53 vectors. The cellular internalization and the transfection efficiency of both vectors were evaluated. In addition, both the transcription and translation of gene p53 was confirmed, following by the quantification of the target protein. Finally, the therapeutic effect of p53 gene encoded in both vectors was carry out through cell proliferation, cytotoxicity and apoptosis assays. Overall, these in vitro studies suggest that mcDNA-p53 is highly promising to be used as a new therapeutic strategy for cervical cancer in the future. Moreover, in a second stage, this work aims to construct a multigenic mcDNA vector encoding the p53 gene and pri-miRNA-375 gene, aiming to establish a potential combined therapeutic approach more effective for cervical cancer.
Human Papillomavirus (HPV) is considered the main causative agent for the development of cervical cancer since its E6 and E7 oncoproteins compromise the expression or activity of p53 and pRb tumor suppressor proteins. Many cervical cancer therapeutic approaches have demonstrated to be ineffective, which emphasizes the need for new strategies such as gene therapy. Minicircle DNA (mcDNA) is a small eukaryotic expression vector that results from an in vivo site-specific recombination process, in which a parental plasmid (PP) leads to the formation of a miniplasmid (mP), carrying the undesired backbone sequences, and a mcDNA, carrying the therapeutic expression cassette. The mcDNA allows higher transfection efficiency due to its smaller size and it is a safer therapeutic approach than plasmid DNA. Therefore, the present work aims, in a first stage, to produce and purify a mcDNA encoding for p53 gene regarding future in vitro studies. L-histidine and 1-benzyl-L-histidine modified monoliths, as well the gel filtration column Sephacryl S-1000 SF, were explored to isolate the supercoiled (sc) mcDNA-p53 from residual amounts of parental plasmid and bacterial impurities. The ionic strength and pH of binding and elution steps were adjusted to enhance the purification performance. The chromatographic results suggest that gel filtration column presents a better approach for sc mcDNA-p53 isolation, ensuring a higher degree of purity and providing a larger quantity of recovered sample. Afterwards, HeLa cells were transfected with PP-p53 and mcDNA-p53 vectors. The cellular internalization and the transfection efficiency of both vectors were evaluated. In addition, both the transcription and translation of gene p53 was confirmed, following by the quantification of the target protein. Finally, the therapeutic effect of p53 gene encoded in both vectors was carry out through cell proliferation, cytotoxicity and apoptosis assays. Overall, these in vitro studies suggest that mcDNA-p53 is highly promising to be used as a new therapeutic strategy for cervical cancer in the future. Moreover, in a second stage, this work aims to construct a multigenic mcDNA vector encoding the p53 gene and pri-miRNA-375 gene, aiming to establish a potential combined therapeutic approach more effective for cervical cancer.
Description
Keywords
Cancro do Colo do Útero Dna Minicircular Gene P53 Mirna-375 Plasmídeo Parental