Células tronco mesenquimais caninas cultivadas com desferroxamine (dfo) e interferon gamma, mimetizando condições de hipóxia e inflamação
Carregando...
Data
2021-07-01
Autores
Ocampo Ortiz, Pablo Eduardo
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
As células tronco mesenquimais (MSC) tem se destacado como candidatas ao uso em terapias regenerativas. Porém, foi demonstrado que nem todas as condições clínicas podem ser favorecidas pela presença das MSCs, já que suas propriedades são influenciadas pelas condições do microambiente onde serão aplicadas, que normalmente estão acompanhadas da presença de fatores inflamatórios e isquêmicos. Devido a isto, este estudo teve por objetivo avaliar os efeitos in vitro da adição de DFO e/ou ao IFN- no meio de cultivo de células tronco mesenquimais (MSCs do inglês Mesenchymal Stem Cells) de tecido adiposo canino (cAT-MSCs). Foram utilizadas MSCs originadas de 6 cadelas e pertencentes a um banco celular em terceira passagem. As amostras foram divididas em 4 grupos, controle (MSCs em condições normais de cultivo em meio de manutenção), grupo DFO (50 µmol de DFO no meio de manutenção), grupo IFN- (50 ng/mL de IFN- no meio de manutenção) e grupo IFN-/DFO (50 µmol de DFO e 50 ng/mL IFN- no meio de manutenção). O teste de proliferação foi feito por 144 horas de cultivo e, viabilidade, apoptose e expressão gênica foram avaliadas após 48 horas de cultivo. Na expressão genica foram analisados transcritos relacionados com apoptose (BCL2, CASP8 e CASP9), sobrevivência celular (DKC1, HDAC1 e DNMT1), imunomodulação (HGF, IDO, PGE2 e IL-6), proliferação e diferenciação (FGF2), angiogênese (MMP2 e VEGFA), reguladores do ciclo celular (Cyclin-D1 e TP53) e regulador celular de resposta adaptativa a hipóxia (HIF1-). As cAT-MSCs mostraram durante e ao final do cultivo morfologia fibroblastóide e aderência ao plástico. No entanto, os grupos tratados apresentaram maior distanciamento entre as células aderidas. Após 144 horas de cultivo, todos os grupos tiveram comportamento similar quanto a curva de proliferação celular, com diminuição da concentração nas primeiras 48 horas, seguido por uma fase de manutenção e finalizando com um ligeiro crescimento. A expressão gênica revelou que as cAT-MSCs tratadas com IFN- apresentaram um incremento na expressão do gene pró-apoptótico Casp9 quando comparado com o grupo IFN-/DFO; o gene FGF2 importante para os processos de proliferação e diferenciação celular foi aumentado no grupo IFN- quando comparado aos outros grupos tratados; os genes DKC1 e TP53 envolvidos com a sobrevida celular e supressão tumoral respectivamente, tiveram um incremento no grupo IFN- quando comparado com o grupo DFO; a expressão do gene relacionado com os processos de angiogênese, o VEGFA, foi maior nos grupos onde o DFO foi usado. Concluímos que condicionar as cAT-MSCs com a citocina pro-inflamatória IFN- estimulou um aumento na sobrevivência, proliferação celular e angiogênese mediada por MMP2. Já a mimetização de hipóxia, levou a uma diminuição da expressão de genes relacionados a estas propriedades associadas a um estímulo para a angiogênese dependente de VEGF. Apesar disso, os genes relacionados a imunomodulação não foram influenciados pelas condições de cultivo. Os resultados obtidos indicam que as propriedades de indução da regeneração tecidual foram mais afetadas pelas condições de cultivo, em comparação às propriedades imunomodulados das cAT-MSC.
Mesenchymal stem cells (MSC) have been highlighted as candidates for use in regenerative therapies. However, it has been shown that not all clinical conditions can be favored by the presence of MSCs, as their properties are influenced by the conditions of the microenvironment where it will be applied, conditions that are usually accompanied by the presence of inflammatory and ischemic factors. So, this study aimed to evaluate the in vitro effects of the addition of DFO and/or IFN- in the culture medium of mesenchymal stem cells (MSCs Mesenchymal Stem Cells) from canine adipose tissue (cAT-MSCs). MSCs from 6 bitches and belonging to a cell bank in the third passage were used. Samples were divided in 4 groups: Control (MSCs under standard culture conditions in maintenance medium); DFO group (50 µM of DFO in maintenance medium), IFN- Group (50 ng/mL of IFN- in maintenance medium) and IFN-/DFO group (50 µM of DFO and 50 ng/mL IFN- in maintenance medium). Proliferation test was performed for 144 hours of culture and viability, apoptosis and gene expression were evaluated after 48 hours of culture. In gene expression, transcripts related to apoptosis (BCL2, CASP8 and CASP9), cell survival (DKC1, HDAC1 and DNMT1), immunomodulation (HGF, IDO, PGE2 and IL-6), proliferation and differentiation (FGF2), angiogenesis (MMP2 and VEGFA), cell cycle regulators (Cyclin-D1 and TP53) and cellular regulator of adaptive response to hypoxia (HIF1-) were analyzed. The cAT-MSCs showed fibroblastoid morphology and plastic adherence during and at the end of the culture. However, the treated groups showed greater distance between adhered cells. After 144 hours of culture, all groups had a similar behavior regarding the cell proliferation curve, with a decrease in concentration in the first 48 hours, followed by a maintenance phase and ending with a slight growth. Gene expression revealed that cAT-MSCs treated with IFN- showed an increase in the expression of the pro-apoptotic Casp9 gene when compared to the IFN-/DFO group; the FGF2 gene important for cell proliferation and differentiation processes was increased in the IFN- group when compared to the other treated groups; the DKC1 and TP53 genes involved with cell survival and tumor suppression respectively, had an increase in the IFN- group when compared to the DFO group; the expression of the gene related to the angiogenesis processes, VEGFA, was higher in the groups where DFO was used. We conclude that conditioning cAT-MSCs with the pro-inflammatory cytokine IFN- stimulated an increase in survival, cell proliferation and MMP2-mediated angiogenesis. The mimicry of hypoxia, on the other hand, led to a decrease in the expression of genes related to these properties associated with a stimulus for VEGF-dependent angiogenesis. Despite this, genes related to immunomodulation were not influenced by culture conditions. The results obtained indicate that tissue regeneration induction properties were more affected by culture conditions, compared to the immunomodulated properties of cAT-MSC.
Mesenchymal stem cells (MSC) have been highlighted as candidates for use in regenerative therapies. However, it has been shown that not all clinical conditions can be favored by the presence of MSCs, as their properties are influenced by the conditions of the microenvironment where it will be applied, conditions that are usually accompanied by the presence of inflammatory and ischemic factors. So, this study aimed to evaluate the in vitro effects of the addition of DFO and/or IFN- in the culture medium of mesenchymal stem cells (MSCs Mesenchymal Stem Cells) from canine adipose tissue (cAT-MSCs). MSCs from 6 bitches and belonging to a cell bank in the third passage were used. Samples were divided in 4 groups: Control (MSCs under standard culture conditions in maintenance medium); DFO group (50 µM of DFO in maintenance medium), IFN- Group (50 ng/mL of IFN- in maintenance medium) and IFN-/DFO group (50 µM of DFO and 50 ng/mL IFN- in maintenance medium). Proliferation test was performed for 144 hours of culture and viability, apoptosis and gene expression were evaluated after 48 hours of culture. In gene expression, transcripts related to apoptosis (BCL2, CASP8 and CASP9), cell survival (DKC1, HDAC1 and DNMT1), immunomodulation (HGF, IDO, PGE2 and IL-6), proliferation and differentiation (FGF2), angiogenesis (MMP2 and VEGFA), cell cycle regulators (Cyclin-D1 and TP53) and cellular regulator of adaptive response to hypoxia (HIF1-) were analyzed. The cAT-MSCs showed fibroblastoid morphology and plastic adherence during and at the end of the culture. However, the treated groups showed greater distance between adhered cells. After 144 hours of culture, all groups had a similar behavior regarding the cell proliferation curve, with a decrease in concentration in the first 48 hours, followed by a maintenance phase and ending with a slight growth. Gene expression revealed that cAT-MSCs treated with IFN- showed an increase in the expression of the pro-apoptotic Casp9 gene when compared to the IFN-/DFO group; the FGF2 gene important for cell proliferation and differentiation processes was increased in the IFN- group when compared to the other treated groups; the DKC1 and TP53 genes involved with cell survival and tumor suppression respectively, had an increase in the IFN- group when compared to the DFO group; the expression of the gene related to the angiogenesis processes, VEGFA, was higher in the groups where DFO was used. We conclude that conditioning cAT-MSCs with the pro-inflammatory cytokine IFN- stimulated an increase in survival, cell proliferation and MMP2-mediated angiogenesis. The mimicry of hypoxia, on the other hand, led to a decrease in the expression of genes related to these properties associated with a stimulus for VEGF-dependent angiogenesis. Despite this, genes related to immunomodulation were not influenced by culture conditions. The results obtained indicate that tissue regeneration induction properties were more affected by culture conditions, compared to the immunomodulated properties of cAT-MSC.
Descrição
Palavras-chave
Imunomodulação, Inflamação, Expressão genica, Immunomodulation, Inflammation, Gene expression