Efecto de bacterias lácticas de la microbiota de salmónidos, sobre genes efectores del sistema inmune innato en la línea celular SHK-1
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2015
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es
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Universidad Andrés Bello
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La microbiota del tracto gastrointestinal ha sido ampliamente estudiada en peces por los potenciales beneficios que presenta, uno de ellos es la estimulación del sistema inmune innato. El sistema inmune innato es la primera barrera de defensa frente a los patógenos, y en peces una cantidad importante de genes relacionados a esta respuesta han sido identificados. Dentro de ellos se encuentran las citoquinas Il-1b, TNF-a e IL-8, óxido nítrico sintasa (iNOS), hepcidina (hep) y lisozima (lys), la proteína del suero amiloide A (SAA) y la ciclooxigenasa 2 (COX-2. En ésta tesis se busca determinar si bacterias de origen láctico, de un set de bacterias provenientes de la microbiota de salmónidos, son capaces de estimular ciertos efectores asociados al sistema inmune innato en la línea celular SHK-1. Las bacterias utilizadas correspondieron a cepas generalmente presentes en la microbiota de salmónidos, tales como, Aeromonas sp (Aehp), Shewanella sp (I8), Pseudomonas sp (P1), Carnobacterium sp (39L y 2A5), Lactococcus sp (B0P1-8) y Pediococcus sp (B022), más dos probióticos comerciales (Bactocell® y Biolactus®). Primero se midió el efecto sobre la integridad de las células SHK-1, donde Aeromonas sp provocó un total desprendimiento de la monocapa a las 48 horas de exposición (hpe), siendo la única bacteria en producir tal daño. Luego, las bacterias fueron clasificadas según su capacidad de proliferación y tiempo de duplicación y de ésta forma normalizar los inóculos iniciales. Se dividió en dos grupos: el grupo 1 correspondió a las bacterias que presentaron un crecimiento más acelerado y fue conformado por las bacterias Aehp, I8, P1 más el probiótico para humanos. El grupo 2 lo forman las bacterias que presentaron un crecimiento más retrasado, que correspondieron a las bacterias B0p1-8, 39L, 2A5, B022 más el probiótico para peces. Para los ensayos de expresión, las bacterias se incorporaron a una MOI 0,1 para el grupo 1 y una MOI 1 para el grupo 2 (inóculo inicial). Para todos los genes, existió un perfil de expresión que se adelantó por parte de las bacterias del grupo 1.En el caso de IL-1b y IL-8, la curva de expresión presenta un pick y luego un descenso paulatino en la mayoría de las bacterias, no así para TNF-a que tras el pick la expresión cae drásticamente en todos los casos. Para el gen iNOS, la expresión es baja para todas las bacterias, siendo para el grupo 2 prácticamente imperceptible. El probiótico para peces, no presenta expresión significativa para ninguno de los genes en estudio, lo que sugiere que éste probiótico no otorga beneficios asociados a la estimulación del sistema inmune innato en peces.
Los resultados indican que las bacterias provenientes de la microbiota de salmónidos, son capaces de estimular genes efectores del sistema inmune innato en células SHK-1, siendo candidatas para futuros estudios de probióticos en la industria acuícola. A su vez, el probiótico para humanos resultó ser más eficaz que el probiótico para peces, ya que presenta una mayor estimulación de los genes, sin que ésta respuesta genere una daño a la célula. Sin embargo se necesita un mayor número de ensayos a nivel de organismo completo, para definir si es un mejor probiótico en este caso para peces.
The microbiota of the gastrointestinal tract has been studied extensively in fish by the potential benefits that it presents, one of them is the stimulation of the innate immune system. The innate system is the first defense barrier to invasion by pathogens, and in fish an important numbers of genes related to this has been identified, like cytokines IL-1β, TNFα and IL-8, inducible nitric oxide synthase (iNOS), hepcidina (hep), lisozima (lys), amiloid serum protein A (SAA) and the ciclooxigenasa 2 (COX-2). The aim of this study was to establish, if lactic bacteria of a set of bacteria from the salmonid microbiota, are capable of stimulating efector genes associated with the innate immune system in the cell line SHK-1.The set of bacteria are usually present at the salmónids microbiota, such as, Aeromonas sp (Aehp), Shewanella sp (I8), Pseudomonas sp (P1), Carnobacterium sp (39L and 2A5), Lactococcus sp (B0P1-8) and Pediococcus sp (B022), and two commercials probiotics (Bactocell ® and Biolactus ®). First, the citophatic effect was measured. Aeromonas sp produced an entire release of the cell monolayer at 48 hours post incubation (hpi), being the only bacteria in produced such damage. Then, the bacteria were classified according to its capacity of proliferation and time of duplication to normalize the initial concentration. They separated in two groups: the group 1 corresponded to the bacteria that presented a more intensive growth and it was formed by the bacteria Aehp, I8, P1 and the human probiotic. The group 2 it form the bacteria that presented a more slower growth, which they corresponded to the bacteria B0p1-8, 39L, 2A5, B022 and fish probiotic. For the gene expression, the group 1 bacteria was grown to reach a MOI 0,1 and a MOI 1 for the group 2 for use as initial inocula. For all the genes, there existed a profile of expression estimated at first for the bacteria of the group 1. In case of IL-1β and IL-8, the expression curve presents a pick and then a gradual decline in most of the bacteria, as this was not for TNFα that after the pick the expression falls drastically in all the cases. For the gene iNOS, the expression is low for all the bacteria, being for the group practically imperceptible 2.The fish probiotic no exhibit any significant expression for all genes in this study, suggesting that this probiotic not show benefits associated with stimulation of the innate immune system in fish. The results indicate that the bacteria originated from the microbiota of salmonids, are capable of stimulating certain genes of the innate immune system in cells SHK-1 depending on the genus to which they belong, being candidates for future probiotics studies in the aquatic industry. In addition, the human probiotic was more effective than the fish probiotic, since it presents a major stimulation of the genes, without causing cell damage. Nevertheless a major number of assays in vivo are needed, to define if it is a better probiotic in this case for fish.
The microbiota of the gastrointestinal tract has been studied extensively in fish by the potential benefits that it presents, one of them is the stimulation of the innate immune system. The innate system is the first defense barrier to invasion by pathogens, and in fish an important numbers of genes related to this has been identified, like cytokines IL-1β, TNFα and IL-8, inducible nitric oxide synthase (iNOS), hepcidina (hep), lisozima (lys), amiloid serum protein A (SAA) and the ciclooxigenasa 2 (COX-2). The aim of this study was to establish, if lactic bacteria of a set of bacteria from the salmonid microbiota, are capable of stimulating efector genes associated with the innate immune system in the cell line SHK-1.The set of bacteria are usually present at the salmónids microbiota, such as, Aeromonas sp (Aehp), Shewanella sp (I8), Pseudomonas sp (P1), Carnobacterium sp (39L and 2A5), Lactococcus sp (B0P1-8) and Pediococcus sp (B022), and two commercials probiotics (Bactocell ® and Biolactus ®). First, the citophatic effect was measured. Aeromonas sp produced an entire release of the cell monolayer at 48 hours post incubation (hpi), being the only bacteria in produced such damage. Then, the bacteria were classified according to its capacity of proliferation and time of duplication to normalize the initial concentration. They separated in two groups: the group 1 corresponded to the bacteria that presented a more intensive growth and it was formed by the bacteria Aehp, I8, P1 and the human probiotic. The group 2 it form the bacteria that presented a more slower growth, which they corresponded to the bacteria B0p1-8, 39L, 2A5, B022 and fish probiotic. For the gene expression, the group 1 bacteria was grown to reach a MOI 0,1 and a MOI 1 for the group 2 for use as initial inocula. For all the genes, there existed a profile of expression estimated at first for the bacteria of the group 1. In case of IL-1β and IL-8, the expression curve presents a pick and then a gradual decline in most of the bacteria, as this was not for TNFα that after the pick the expression falls drastically in all the cases. For the gene iNOS, the expression is low for all the bacteria, being for the group practically imperceptible 2.The fish probiotic no exhibit any significant expression for all genes in this study, suggesting that this probiotic not show benefits associated with stimulation of the innate immune system in fish. The results indicate that the bacteria originated from the microbiota of salmonids, are capable of stimulating certain genes of the innate immune system in cells SHK-1 depending on the genus to which they belong, being candidates for future probiotics studies in the aquatic industry. In addition, the human probiotic was more effective than the fish probiotic, since it presents a major stimulation of the genes, without causing cell damage. Nevertheless a major number of assays in vivo are needed, to define if it is a better probiotic in this case for fish.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Salmónidos, Inmunología, Bacterias