Establecimiento de un modelo in vitro de la falla hepática aguda sobre crónica, para el posterior estudio del secretoma derivado de células madre mesenquimáticas
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2022
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
La insuficiencia hepática aguda sobre crónica (ACLF) es un síndrome complejo, caracterizado por un deterioro
agudo de la función hepática, una respuesta inflamatoria local y sistémica exacerbada de alta mortalidad.
Durante la ACLF, se produce la pérdida del ambiente tolerogénico hepático, mediante la activación de las
células de Kupffer que adquieren un perfil pro-inflamatorio, caracterizado por la secreción de citoquinas y
factores quimiotácticos que llevan al infiltrado y activación de los monocitos en circulación, generando una
falla sistémica.
Actualmente no existen tratamientos específicos para la ACLF, teniendo como únicas opciones el soporte
hepático y el trasplante hepático como alternativa terapéutica cuando se desencadena la falla hepática.
El secretoma de células madre mesenquimáticas (MSC-S) está compuesto por un amplio espectro de moléculas
con actividad trófica, pro-angiogénica y anti-inflamatoria, por lo que han sido propuestos como una nueva
alternativa terapéutica para esta patología. En nuestro laboratorio, resultados preliminares en un modelo
murino de ACLF indican que la administración de MSC-S disminuye la respuesta inflamatoria y la lesión
hepática, aumentando la sobrevida de los animales.
En el presente trabajo nos propusimos estandarizar un modelo in vitro de co-cultivo monocito/hepatocito y
macrófago/hepatocito, para determinar de manera más rápida y económica, que en el modelo animal, que
molécula(s) presente(s) en el MSC-S podrían dar cuenta de su efecto terapéutico sobre la ACLF, por tal motivo
planteamos como hipótesis que la administración del MSC-S precondicionado induce una respuesta
hepatoprotectora y anti-inflamatoria.
En primer lugar, montamos un sistema de co-cultivo transwell monocitos/hepatocitos, en el cual se pudo
inducir una respuesta inflamatoria por LPS, lo que afectó la viabilidad de los hepatocitos presentes en el
sistema. Al co-incubar estas células con MSC-S, se observó una mayor viabilidad de los hepatocitos, indicando
que el sistema podría ser una buena herramienta para estudiar los componentes presentes en el MSC-S con
actividad terapéutica.
En segundo lugar, establecimos el co-cultivo transwell entre macrófagos/hepatocitos. En este caso, se
estandarizaron protocolos de diferenciación a partir de monocitos, así como de estímulos. Aunque los
estímulos evaluados (LPS y LPS-IFNγ) indujeron una respuesta inflamatoria en los macrófagos, no se vio
afectada la viabilidad de los hepatocitos. Por este motivo, hasta el momento, el sistema de co- cultivo
macrófago/hepatocito, que establecimos, no sería un buen modelo para estudiar los componentes terapéuticos
presentes en el MSC-S.
Acute-on-Chronic Liver Failure (ACFL) is a complex síndrome, characterized by acute deterioration of liver function, an exacerbated local and systemic inflammatory response, and high mortality. During ACLF, the los of the hepatic tolerogenic environment occurs, through the activation of Kupffer cells that acquire a pro-inflamatory profile, characterized by the secretion of cytokines and chemotactic factors that lead to the infiltration and activation of circulating monocytes, generating a systemic failure. Currently, there are no specific treatments for ACLF, although liver transplantation is the therapeutic alternative when liver failure is triggered. The mesenchymal stem cell secretome (MSC-S) is composed of a wide spectrum of molecules with trophic, pro-angiogenic and anti-inflammatory activity, which is why they have been proposed as a new therapeutic alternative for this pathology. In our laboratory, preliminary results in a murine modelo f ACLF indicate that the administration of MSC-S decreases the inflammatory responde and liver injuty, increasing the survival of the animals. In the present work we set out to standardize an in vitro model of monocyte/hepatocyte and macrophage/hepatocyte co-culture, to determine in a faster and cheaper way, than in the animal model, which molecule or molecule are present in the MSC-S and could account for its therapeutic effect on ACLF. For this reason, we hypothesize that the administration of preconditioned MSC-S induces a hepatoprotective and antiinflammatory response. First, we set up a monocyte/hepatocyte transwell co-culture system, in which an inflammatory response could be induced by LPS, which affected the viability of the hepatocytes present in the system. By co-incubating these cells with MSC-S, a greater viability of hepatocytes was observed, indicating that the system could be a Good tool to study the components present in MSC-S with therapeutic activity. Finally, we established transwell co-culture between macrophages/hepatocytes. In this case, differentiation protocols were standardized from monocytes, as well as from stimuli. Although the stimuli tested (LPS and LPS-IFNγ) induced an inflammatory response in macrophages, hepatocytes viability was not affected. For this reason, until now, the macrophage/hepatocyte co-culture system that we established would not be a Good model to study the therapeutic components present in MSC-S.
Acute-on-Chronic Liver Failure (ACFL) is a complex síndrome, characterized by acute deterioration of liver function, an exacerbated local and systemic inflammatory response, and high mortality. During ACLF, the los of the hepatic tolerogenic environment occurs, through the activation of Kupffer cells that acquire a pro-inflamatory profile, characterized by the secretion of cytokines and chemotactic factors that lead to the infiltration and activation of circulating monocytes, generating a systemic failure. Currently, there are no specific treatments for ACLF, although liver transplantation is the therapeutic alternative when liver failure is triggered. The mesenchymal stem cell secretome (MSC-S) is composed of a wide spectrum of molecules with trophic, pro-angiogenic and anti-inflammatory activity, which is why they have been proposed as a new therapeutic alternative for this pathology. In our laboratory, preliminary results in a murine modelo f ACLF indicate that the administration of MSC-S decreases the inflammatory responde and liver injuty, increasing the survival of the animals. In the present work we set out to standardize an in vitro model of monocyte/hepatocyte and macrophage/hepatocyte co-culture, to determine in a faster and cheaper way, than in the animal model, which molecule or molecule are present in the MSC-S and could account for its therapeutic effect on ACLF. For this reason, we hypothesize that the administration of preconditioned MSC-S induces a hepatoprotective and antiinflammatory response. First, we set up a monocyte/hepatocyte transwell co-culture system, in which an inflammatory response could be induced by LPS, which affected the viability of the hepatocytes present in the system. By co-incubating these cells with MSC-S, a greater viability of hepatocytes was observed, indicating that the system could be a Good tool to study the components present in MSC-S with therapeutic activity. Finally, we established transwell co-culture between macrophages/hepatocytes. In this case, differentiation protocols were standardized from monocytes, as well as from stimuli. Although the stimuli tested (LPS and LPS-IFNγ) induced an inflammatory response in macrophages, hepatocytes viability was not affected. For this reason, until now, the macrophage/hepatocyte co-culture system that we established would not be a Good model to study the therapeutic components present in MSC-S.
Notas
Memoria de título (Ingeniero en Biotecnología)
Financiamiento de Fondecyt Regular 1200308.
Financiamiento de Fondecyt Regular 1200308.
Palabras clave
Insuficiencia Hepática, Células Madre Mesenquimales