Navegando por Palavras-chave "Integrase"

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    Engenharia genética de um sistema de expressão duradoura e seletiva do VEGF para condição isquêmica baseado no sistema integrase C31 e HRE
    (Universidade Federal de São Paulo (UNIFESP), 2010-05-26) Yasumura, Eduardo Gallatti [UNIFESP]; Han, Sang Won [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Peripheral arterial disease is characterized by insufficient blood flow to the extremities and affects more than 10 million people worldwide and can occur strong pain, non-healing ulcers and loss of limb. With the aim to help these patients, gene therapy has been presented new studies to stimulate the process of neoangiogenesis by injecting genes that encode growth factors. Among the problems used in gene therapy for ischemic diseases, lack of expression of transgenes is the main. Most used vectors, because of the lack of expression levels, can lead to pathological processes of angiogenesis. The ideal vector for this condition would be one who possessed a mechanism of recognition of the ischemic condition to regulate the expression of the transgene and at the same time be integrated into the host cell genome perpetuating their action. Thus, the duration of gene expression would be long enough for the recovery of ischemic tissue and its level would be adjusted as the change in local oxygen concentration. Therefore, this thesis aims to develop a vector that satisfies these conditions using the gene for human vascular endothelial growth factor (hVEGF). To transfer the gene a vector was constructed using the C31 integrase system, which has been shown to mediate a robust and stable integration of the transgene into the genome of the cells. Along with this system, were also inserted the 9 copies of the consensus sequence of hypoxia response element (HRE) in order to control the expression according to oxygen concentration. ELISA, PCR, necrosis degree, determination of muscle force, muscle weigth and immunohistochemistry were performed to evaluate the behavior of the vector and its efficacy in the treatment of ischemia. The results demonstrated the integrative capacity and lasting expression of the vector both in vitro and in vivo. The presence of 9 copies of the HRE sequence, under hypoxia condition, allowed the modulation of gene expression with increased concentration of the protein. These characteristics have resulted in greatly improved in visual appearance of the ischemic limb and became evident the action of hVEGF in terms of increased strength and muscle weigth, decrease of fibrous tissue and increased number of vessels. This study suggests that the joint action of integrase C31 system and HRE may prolong and modulate gene expression in addition to enabling an improvement in treatment for a murine model of limb ischemia.