Los tumores de mama pertenecen a un grupo de lesiones neoplásicas denominadas tumores desmoplásicos que, bajo la influencia de ciertos factores epiteliales, originan una estructura estromal rígida responsable de la consistencia dura de la masa tumoral. Este proceso fibrótico ocurre en etapas tempranas de la enfermedad, es controlado por una forma de fibroblastos conocidos como miofibroblastos, o fibroblastos activados, y sus mecanismos son pobremente comprendidos. La activación del tejido estromal es una etapa fundamental en la progresión tumoral, permitiendo tanto la adquisición de propiedades malignas en células epiteliales, como la capacidad invasiva y metastásica. En esta memoria de título estudiamos la miodiferenciación de fibroblastos mamarios no tumorales RMF-EG, frente al estímulo de TGF-β1, secretado por células tumorales y que es abundante en el microambiente tumoral. Los resultados mostraron que 5ng/mL de TGF-β1 aumentó la expresión de actina α-SMA, marcador de miofibroblastos, y de CTGF, molécula asociada a diversos desórdenes fibróticos. A través del uso del inhibidor DPI y el knock-down de NOX4 demostramos que TGF-β1 promovió un ambiente oxidativo que favoreció la miodiferenciación fibroblástica de células RMF-EG. Determinamos también que TGF-β1 activó la ruta de señalización JNK1,2 y que esta activación era fundamental para el aumento de la expresión de CTGF, NOX4 y α-SMA. Estudiamos la influencia de la activación de esta ruta alternativa junto con el aumento del tenor oxidativo, sobre la activación de la ruta canónica Smad2,3. Los resultados mostraron que el aumento en la expresión de NOX4 y la fosforilación de JNK1,2 actuaban de manera sinérgica para activar la ruta Smad2,3. En conjunto, estos resultados demuestran que TGF-β1 provoca la miodiferenciación de fibroblastos no tumorales, a través de un mecanismo que requiere de la activación de JNK1,2, el aumento temprano de la expresión de CTGF y NOX4 con un consecuente aumento de los niveles intracelulares de ROS. / Memoria para optar al título de Bioquímico / Brest tumors belong to a group of neoplastic lesions known as desmoplastics or scirrhous tumors which, under the influence of tumor cell factors, originate a fibrous structure responsible for the hard consistency of the tumor mass. This fibrotic process occurs during early stages of the disease, it is orchestrated by activated fibroblast i.e. myofibroblast and its mechanisms are poorly understood. Activation of the stromal compartment is a critical step in tumor progression, enabling the epithelial acquisition of malignant properties, such as invasive and metastatic capacities. In the present study, we investigated the myofibroblastic differentiation of normal human mammary fibroblast RMF-EG, induced by TGF-β1, a growth factor secreted by tumor cells and abundant in tumor microenvironment. Our results reveal that a 5ng/mL TGF-β1 stimulus increased the expression of myofibroblast marker α-SMA and CTGF, a molecule associated to several fibrotic disorders. Using a NOX inhibitor (DPI) and a siRNA for NOX4, we demonstrated that TGF-β1 promoted an oxidative environment that favors myofibroblastic differentiation of RMF-EG cells. We also determined that TGF-β1-dependant activation of JNK1,2 was essential for CTGF, NOX4 and α-SMA increased expression. We assessed the influence of JNK1,2 activation and NOX4 activity on canonical Smad2,3 activation. Our results reveal that the TGF-β1-dependant increase of NOX4 expression and JNK1,2 phosphorylation induced a synergical activation of the canonical TGF-β1 pathway, Smad2,3.Taken together, these results demonstrate that TGF-β1 promotes myofibroblastic differentiation of normal fibroblasts RMF-EG through a mechanism that requires JNK1,2 activation, early increase of CTGF and NOX4 expression with a consequent increase of intracellular ROS levels

Tesis presentada a la Universidad de Chile para optar al grado de Doctora en Farmacología / La fibrosis cardiaca se genera por un depósito exagerado de proteínas de matriz extracelular (MEC) producto de la activación de los fibroblastos (FC). Estas células, representan cerca de un 60 a 70% de las células del corazón, y en respuesta a un daño, son activadas por diversas citoquinas y factores de crecimiento (entre ellas, TGF-β1, Ang II, etc.), los que desencadenan en ellas un cambio estructural y funcional que las diferencia a miofibroblastos cardiacos (MFC). Los MFC tienen como principal función la de secretar proteínas de la MEC con el fin de contraer y reparar la zona dañada en el caso de un infarto al corazón. Así, el MFC juega un papel crucial en la respuesta frente a un daño en el tejido cardiaco. A pesar de ello, es muy poco lo que se sabe con respecto al comportamiento de este tipo celular frente al remodelado cardiaco. Además, se tiene un escaso conocimiento acerca de los receptores celulares que presenta este tipo celular, pudiendo ser piezas claves en la regulación de la secreción exacerbada de MEC y por tanto, de la generación de fibrosis del tejido. En la fibrosis cardiaca, adquiere gran importancia la participación del sistema calicreina-cinina. Se ha propuesto que la ausencia de los receptores de cininas es deletérea a nivel del tejido cardiaco, especialmente en la respuesta frente a una injuria. Dentro de este sistema, se encuentran bradicinina (BK) que actúa sobre el receptor B2 de cininas (B2R) y lis-des-Arg-BK (DAKD) que ejercen su acción sobre el receptor B1 (B1R). En FC se ha demostrado que, BK produce liberación de NO y de prostaglandinas, además de reducir la secreción de colágeno. Sin embargo, en MFC se desconocen que subtipos de receptores están presentes, así como las vías transduccionales que se activan tras la estimulación de ambos subtipos de receptores. En la presente tesis y de acuerdo con el contexto, se realizaron diversos experimentos para demostrar la existencia y funcionalidad de los receptores de cininas tanto en FC como en MFC. Por western blot (WB) se evidenció la presencia de ambos receptores tanto en FC como en MFC, no obstante, la expresión de B1R es mayor en MFC que FC; mientras que B2R permanece constante. La expresión de B1R aumentó tras el tratamiento de FC con TGF-β1, principal diferenciador a MFC. Además mediante las técnicas de inmunofluorescencia y ensayos de radioligando, se determinó que en FC el B1R se encuentra localizado principalmente a nivel intracelular, mientras que en MFC se localiza marcadamente en las membranas plasmáticas. Por otro lado, mediante el análisis de los movimientos de calcio intracelular, se demostró que B2R es funcional en ambos tipos celulares, mientras que B1R es funcional en MFC y no así en FC. Sin embargo, en FC la preincubación con el agonista de B1R (DAKD), y la posterior estimulación (30 min) con el mismo agonista, mostró que estos receptores son también funcionales. Mediante inmunofluorescencia se evidenció que tras la preincubación con el agonista existe una relocalización de B1R hacia las membranas plasmáticas, dónde son capaces de generar movimientos de calcio intracelular, demostrando que también son receptores activos. Además, en MFC se demostró que la activación de B1R y B2R por sus respectivos agonistas induce una reducción en la expresión de colágeno I (Col I). En FC, la activación de B2R induce la disminución en los niveles de Col I, mientras que la activación de B1R que provoca la reducción en la expresión de Col I, requiere de un estímulo previo que redistribuya este receptor hacia la membrana plasmática. Finalmente, el uso de distintos inhibidores permitió demostrar que en MFC, tanto la activación de B1R como B2R, conducen a la disminución en la expresión de Col I a través de la activación de una vía transduccional que es compartida por ambos receptores, y que corresponde a: PKC/PLA2/COX-2/PGI2/IPR. En resumen, los resultados de esta tesis sostienen que en MFC la activación de B1R y B2R conduce a la disminución de la expresión de Col I, lo cual se relaciona a los efectos antifibróticos de las cininas en el progreso de patologías como la fibrosis cardiaca / Cardiac fibrosis is generated by an excessive deposit of extracellular matrix proteins (ECM) product of fibroblast (CF) activation. These cells account for about 60 to 70% of the cells in the heart. In response to injury, fibroblasts are activated by various cytokines and growth factors (including TGF-β1, Ang II, etc.), which trigger in them structural and functional changes, differentiating to cardiac myofibroblasts (CMF). The main function of CMF is to secrete ECM proteins to repair the damaged area in the case of a heart attack. Thus CMF play a crucial role in the response to injury in the heart tissue. However, very little is known about the behavior of this cell type versus cardiac remodeling. Furthermore, the knowledge on the receptors present on this cell type is scarce, and they could be key elements in the regulation of the exacerbated secretion of MEC and therefore the generation of tissue fibrosis. In cardiac fibrosis, the participation of the kallikrein-kinin system is very important. It has been proposed that the absence of kinin receptors is deleterious for cardiac tissue, especially in response to an injury. Within this system are bradykinin (BK) which acts on the B2 kinin receptor (B2R) and lis-des-Arg-BK (DAKD) that exerts its action on the B1 receptor (B1R). In CF, it has been shown that BK induces NO release and prostaglandins production, which reduce collagen secretion. However, in CMF it is not known which kinin receptor subtype is present and what signal transduction pathways are activated by these receptors. In this thesis and in accordance with the context, various experiments were conducted to prove the existence and functionality of kinin receptors in both CF and CMF the presence of both receptors in both CF as MFC was observed by Western blot (WB), however, expression of B1R in CMF was greater than in FC, while B2R remained constant. B1R expression increased after treatment of CF with TGF-β1, the main differentiator to CMF. By immunofluorescence techniques and radioligand assays, it was determined that CF B1R was located primarily intracellularly, whereas CMF was markedly located in the plasma membranes. Moreover, by analyzing intracellular calcium movements, B2R was shown to be functional in both cell types, while B1R was functional in CMF, but not in CF. However, when CF were preincubated with the B1R agonist (DAKD) and subsequently stimulated (30 min) with the same agonist, these receptors were also functional. By Immunofluorescence it was shown that after preincubation with the agonist the B1R was relocated to plasma membranes, where they were able to generate intracellular calcium movements, showing they are active receptors. Furthermore, it was demonstrated that B1R and B2R activation in CMF by their respective agonists, induces a reduction in the expression of collagen I (Col I). In contrast in CF, B2R activation induced a decrease in Col I levels, whereas a previous stimulus that redistributes B1R receptor to the plasma membrane is required to induce a reduction in Col I expression after B1R stimulation. Finally, the use of various inhibitors shows that CMF allowed both B1R activation and B2R, leading to a decrease in Col I expression through activation of a signal transduction pathway which is shared by both receptors, and that is mediated by PKC/PLA2/COX-2/PGI2/IPR. In summary, the results of this study argue that B1R and B2R activation in CMF leads to decreased expression of Col I, which is related to antifibrotic effects of kinins in the progress of diseases such as cardiac fibrosis / Conicyt Fondecyt

Memoria para optar al título de Químico Farmacéutico / Fibroblastos y miofibroblastos cardiacos son elementos claves en el desarrollo del remodelamiento cardiaco después de un infarto agudo al miocardio. Una regulación controlada en el crecimiento de la población de fibroblastos y miofibroblastos es importante para una correcta cicatrización y mantención de la función cardiaca. Distintas evidencias muestran que los niveles de angiotensina II (Ang II) y del receptor de angiotensina II tipo 1 (AT1R) aumentan después de un infarto agudo al miocardio. Por ésto, Ang II puede tener un papel central en la regulación del número y función de estas células post-infarto al miocardio. En esta memoria se realizaron ensayos de unión de radioligando, viabilidad y funcionalidad celular, en fibroblastos (FCN) y miofibroblastos cardiacos neonatos (MCN) con y sin expresión ectópica de AT1R o AT2R. Los ensayos de unión de radioligando mostraron que miofibroblastos y fibroblastos cardiacos neonatos expresaban solo AT1R. En ambas condiciones (con y sin expresión ectópica de AT1R o AT2R), los miofibroblastos mostraron mayor unión total de radioligando que los fibroblastos. Ang II (100 nM), estimuló la migración de los fibroblastos controles, y en los que expresaban ectópicamente AT1R disminuyó la viabilidad. Por otra parte, no se observaron efectos en miofibroblastos controles o que expresaban ectópicamente AT1R o AT2R. La muerte de los fibroblastos estimulados con Ang II se previno con losartán e inhibidores de fosfolipasa C (PLC) y proteína kinasa C (PKC), indicando la participación de estas vías de señales intracelulares. Los resultados obtenidos demuestran que Ang II actúa de manera selectiva sobre fibroblastos y miofibroblastos cardiacos neonatos, controlando la función celular en nuestro modelo de estudio / Cardiac fibroblasts and myofibroblasts are key elements of the development of cardiac remodeling after myocardial infarction. A tight regulation of fibroblast and myofibroblast growth is important to correct wound healing and maintain cardiac function. Several lines of evidence have showed an increase in angiotensin II and AT1R levels after myocardial infarction. Thus angiotensin II may play a pivotal role in the regulation of number and function of theses cells. Cell viability and function, and radioligand binding studies, were performed in neonate cardiac fibroblast and myofibroblast with/without ectopically expressed AT1R or AT2R. Radioligand binding studies demonstrated that neonate cardiac fibroblast and myofibroblasts expressed a single class of high affinity angiotensin II AT1R. In both conditions, myofibroblasts revealed a higher maximal binding capacity, compared to fibroblast. Angiotensin II (100 nM) increased the migration in normal fibroblast, and in ectopically expressed AT1R fibroblast reduced the cell viability. No effects were observed in normal or ectopically expressed AT1R or AT2R myofibroblast. Cardiac fibroblast cell death angiotensin II triggered was was prevented by losartan and by phospholipase C (PLC) and protein kinase C (PKC) inhibitors, indicating the participation of that intracellular signaling pathways. The data demonstrate that Angiotensin II acted in a selective manner on cardiac fibroblast and myofibroblast, controlling the cellular number and function

Memoria de título para optar al título profesional de Bioquímico / La reparación y regeneración de tejidos constituyen eventos críticos al momento de enfrentar y corregir injurias que puedan afectar la homeostasis de los mismos. El tabaquismo es una enfermedad muy prevalente en Chile y en el mundo, es factor de riesgo para el desarrollo de enfermedad periodontal y diversos componentes del humo de cigarrillo tienen un alto impacto sobre la integridad y funcionalidad celular. Los fenómenos de reparación pueden estudiarse in vitro utilizando fibroblastos gingivales humanos (FGH) cultivados en matrices tridimensionales (3D) de colágeno, las cuales se pueden someter a diferentes niveles de tensión mecánica simulando distintas etapas de la remodelación del tejido de granulación. En el presente estudio se analizó el efecto del condensado de humo de cigarrillo (CHC) sobre las capacidades de remodelación de FGH en un cultivo 3D. Se examinó el efecto del CHC sobre i) las capacidades contráctiles de las células dentro de geles de colágeno y ii) diversos blancos moleculares implicados en los procesos de reparación de tejidos. Se pudo observar que 100 ug/mL de CHC disminuyó la contracción de geles de colágeno estimulados por suero fetal bovino y por el factor de crecimiento transformante beta 1 (TGF-ß1, “transforming growth factor ß1”). Por otro lado, el CHC disminuyó de manera considerable los niveles de prostaglandina E2 (PGE2) en nuestro modelo 3D de estudio. La carga mecánica presente en la matriz 3D moduló los niveles del factor de crecimiento del tejido conectivo (CTGF, “connective tissue growth factor”) y PGE2, siendo mayores en la condición de alta tensión mecánica (geles ‘tensionados’). Sin embargo, en nuestro modelo 3D, el CHC no modificó los niveles de diversos blancos moleculares que sí se ven alterados por diferentes productos del humo de cigarrillo (nicotina, CHC y extracto del humo de cigarrillo) en placas de cultivo 2D. Estas proteínas fueron actina de músculo liso alfa (α-SMA, “alpha smooth muscle actin”), integrina ß1, metaloproteasa de matriz 2 (MMP-2, “matrix metalloprotease 2”), MMP-3, factor de crecimiento del tejido conectivo (CTGF, “connective tissue growth factor”) e interleuquina 6 (IL-6). Finalmente, el CHC altera los procesos celulares vinculados a los fenómenos de reparación y regeneración de tejidos, modulando tanto los procesos contráctiles como los niveles de proteínas implicadas en estos eventos, afectando de esta forma la salud periodontal / Wound healing and tissue remodeling are critical processes during tissue repair after an injury. Tobacco smoking is a prevalent habit in Chile, is a major risk factor for periodontal disease and several components from cigarette smoke affect cell integrity and functionality. Tissue repair may be studied in vitro using three dimensional (3D) collagen matrices populated by human gingival fibroblasts (HGF). These matrices represent distinct steps during granulation tissue remodeling, when they are exposed to different mechanical tensions. We have analyzed the effect of cigarette smoke condensate (CSC) on HGF remodeling capacities in a 3D model. We evaluated HGF contractile properties in collagen matrices and the production of proteins involved in the regulation of myofibroblasts and granulation tissue contraction. These included α-SMA, ß1 integrin, PGE2, IL-6, CTGF, MMP-3 and MMP-2. Our results show that CSC at 100 ug/mL decreased collagen gel contraction induced by foetal bovine serum and TGF-ß1. Also, PGE2 levels were strongly diminished by CSC in our 3D model. Matrix mechanical load modified levels of CTGF and PGE2, where high mechanical tension increased these proteins levels, compared with low tension matrices. However, different proteins involved in wound healing that are affected by several products of cigarette smoke in a 2D culture model, were unmodified by CSC in our 3D model (α-SMA, ß1 integrin, MMP-2, MMP-3, CTGF and IL-6). Finally, CSC alters cellular responses involved in wound healing, modifying to a minor extent contractile properties and expression of proteins related to tissue repair processes, thereby affecting periodontal health

Doctor en Ciencias Farmacéuticas / Las enfermedades cardiovasculares son, hoy en día, una de las principales causas de muerte. Estas enfermedades generan comúnmente cambios en la estructura del corazón que conlleva a un remodelamiento del tejido generando hipertrofia y fibrosis, lo cual altera la función cardiaca normal y que finalmente se traduce en una insuficiencia cardiaca. La fibrosis cardiaca se genera por un depósito excesivo de proteínas de la matriz extracelular (MEC) producto de la activación de fibroblastos. Estas células, que representan alrededor del 70% del total de las células del corazón humano, frente a una injuria cardiaca, responden a una variedad de citoquinas y factores de crecimiento que promueven su diferenciación a miofibroblastos. Estos a su vez, expresan la proteína α-actina del músculo liso (α-SMA) la que le confiere propiedades contráctiles y durante el proceso de reparación del tejido cardiaco, son los principales responsables de la síntesis de numerosas proteínas de la MEC, las que incluyen colágeno, fibronectina y laminina lo que finalmente se traduce en el cierre de la herida. Asimismo, los miofibroblastos están encargados de liberar citoquinas, factores de crecimiento, como angiotensina II, y en forma adicional, incrementan el número de receptores para estos mismos mediadores. Por otro lado, existen numerosos antecedentes que demuestran que la activación de la vía de transducción del 3’,5’-adenosina monofosfato cíclico (AMPc) podría estar contribuyendo a la reducción de la fibrosis cardiaca, ya que se ha visto que un incremento en los niveles de AMPc en el fibroblasto cardiaco, puede disminuir su función celular así como también inhibir la diferenciación de fibroblasto a miofibroblasto. En este sentido, durante mucho tiempo se ha establecido como regla general que los efectos mediados por el AMPc en las células eucariontes ocurría exclusivamente a través de la activación de la proteína quinasa A (PKA) y que a su vez la PKA mediaba los cambios en la expresión y función de las proteínas. Sin embargo, el descubrimiento de otra proteína llamada EPAC (del acrónimo exchange protein activated directly by cAMP), que regula la actividad de las proteínas G pequeñas Rap 1 y Rap 2, y que también es activada por el AMPc, comenzó a cambiar el campo de investigación relacionado con el estudio de la vía de transducción de este segundo mensajero. A pesar de que los fibroblastos cardiacos expresan EPAC-1, poco se conoce acerca de los mecanismos que regulan la expresión de las isoformas de EPAC en los fibroblastos y miofibroblastos, y de que factores (péptidos, citoquinas, hormonas, entre otros) son los que están involucrados en la regulación de la misma. Estudios han demostrado que los niveles de expresión de EPAC-1 se encuentran aumentados en ratones con hipertrofia cardiaca y que la vía AMPc-EPAC1-Rap1 se encuentra activa bajo estas circunstancias. Estos antecedentes estarían dando cuenta de que EPAC-1 podría estar participando de forma activa en procesos asociados al remodelamiento cardiaco. Paralelamente, se ha observado que el factor de crecimiento transformante β1 (TGF-β1), citoquina estrechamente relacionada con el proceso de fibrosis cardiaca, promueve la disminución de la expresión de EPAC-1 en el fibroblasto cardiaco de rata adulta. Sin embargo, se desconoce el mecanismo por el cual TGF-β1 regula la expresión de EPAC-1 tanto en el fibroblasto como en los miofibroblastos cardiacos. Desde el punto de vista de la funcionalidad de EPAC-1, existen algunos estudios que demuestran la participación de esta proteína en algunos procesos celulares asociados al remodelamiento cardiaco, tales como la migración de fibroblastos y secreción de colágeno, pero se desconoce totalmente su participación en los mecanismos asociados a adhesión y contracción de geles de colágeno, procesos celulares importantes en el remodelamiento del miocardio, en donde participan activamente tanto fibroblastos como miofibroblastos. Bajo este contexto, en la presente tesis, se estudió en una primera instancia cómo el TGF-β1 era capaz de regular los niveles de EPAC-1 tanto en fibroblastos como en miofibroblastos de rata neonata. Para ello se realizaron estudios mediante westernblot con el fin de determinar los niveles basales de EPAC-1 en nuestro modelo de estudio, para luego estudiar la influencia del TGF- β1 sobre estos niveles a través de la intervención de sus vías de señalización canónica (Smad2/3) y no canónica (MAPK y Akt). Los resultados obtenidos dieron cuenta de que los miofibroblastos poseían mayores niveles de EPAC-1 comparado a los fibroblastos y que en la regulación de EPAC- 1 en fibroblastos cardiacos participaron proteínas tales como Smad-2 y JNK, mientras que en el miofibroblasto ambas vías de señalización, canónica y no canónica, participaron de la regulación de los niveles de EPAC-1. Posteriormente y con el fin de dilucidar el rol que juega este factor intercambiador de nucleótidos de guanina sobre las funciones de fibroblastos y miofibroblastos, se evaluó la capacidad de EPAC-1 de fosforilar a su efector río abajo Rap-1, utilizando para ello un análogo del AMPc con capacidad de activar selectivamente a EPAC-1. Los resultados demostraron que en ambos fenotipos celulares EPAC-1 fue capaz de activar a su efector río abajo Rap-1, y más aún, que en los miofibroblastos está activación era mucho mayor en comparación a sus precursores los fibroblastos. Finalmente y con el objetivo de dilucidar la participación de EPAC-1 en procesos asociados al remodelamiento cardiaco tales como adhesión, migración, contracción de geles de colágeno y expresión de colágeno; se llevaron a cabo una serie de experimentos destinados a evaluar los procesos antes mencionados en nuestro modelo de estudio. Para ello se utilizaron herramientas mediante las cuales fue posible dilucidar los efectos mediados principalmente por EPAC-1 y a la vez los efectos mediados por PKA, ya que al ser ambas proteínas efectoras del AMPc, era importante poder establecer la contribución real de cada una de ellas a los procesos antes señalados. Los resultados obtenidos sugieren que EPAC-1 participaría aumentando la adhesión de fibroblastos y miofibroblastos a fibronectina, promoviendo la migración de fibroblastos, aumentando la contracción de geles de colágeno tanto en fibroblastos como en miofibroblastos y disminuyendo los niveles de colágeno en ambos fenotipos celulares. Estos resultados se complementan con los obtenidos en relación a la PKA, en donde se logró dilucidar que esta proteína estaría participando en los procesos de contracción de geles de colágeno y expresión de colágeno. PKA al parecer aumentaría la contracción de geles de colágeno en miofibroblastos y además, sería capaz de disminuir la expresión de colágeno tanto en fibroblastos como en miofibroblastos. En resumen, los resultados de la presente tesis sugieren por un lado que, el TGF- β1 regularía diferencialmente los niveles de EPAC-1 en fibroblastos y miofibroblastos cardiacos y por otro lado, que EPAC-1 estaría regulando importantes funciones asociadas al remodelamiento cardiaco tales como adhesión, migración, contracción de geles de colágeno y expresión de colágeno en ambos fenotipos celulares. / Throughout the last decades cardiovascular diseases has become one of the most important causes of death. These diseases commonly generate changes in the structure of the heart which leads to tissue remodeling and fibrosis, which alters the normal cardiac function and eventually resulting in cardiac failure. Cardiac fibrosis is generated by excessive deposition of extracellular matrix proteins (ECM) produced by the activation of fibroblasts. These cells represent about 70% of total human heart cells and following a cardiac injury, they respond to a wide range of cytokines and growth factors promoting their differentiation into myofibroblasts. These myofibroblasts express the protein α-smooth muscle actin (α-SMA) synonymous with increased contractile force. Enhanced contractility that attends this protein’s expression is believed to be important in allowing these cells to contract during the wound healing in the damaged heart. Myofibroblasts are the primary mediators responsible for synthesis of many ECM proteins, including collagen, fibronectin and laminin, among others. During transition of fibroblast into myofibroblasts they acquire the capability to be avid producers of cytokines and growth factors, and in addition, increase the receptor number for the same mediators. A growing body of literature over the last years has made evident that activation of the transduction pathway of 3', 5'-cyclic adenosine monophosphate (cAMP) may be contributing to the reduction of cardiac fibrosis. It has been demonstrated that an increase in cAMP levels in cardiac fibroblasts can reduce their cellular function as well as inhibit fibroblast differentiation into myofibroblasts. In this regard, it has become generally accepted, that the effects mediated by cAMP in eukaryotic cells occurred only by activation of protein kinase A (PKA) which once activated was able to mediate expression and protein function. However, the discovery of another protein, called EPAC (exchange protein activated directly by cAMP), which regulates the activity of small G proteins Rap 1 and 2, has begun to innovate the research field of the cAMP transduction pathway. It is known that cardiac fibroblast express EPAC-1, however little is known about the mechanism regulating the expression of this protein in fibroblast and myofibroblasts. In this regard, studies in this area have demonstrated that levels of EPAC-1 expression were increased in mice with cardiac hypertrophy and cAMP pathway EPAC1-Rap1 was very active. These results support the idea that EPAC-1 could be actively involved in processes associated with cardiac remodeling. In parallel, it has been observed that the transforming growth factor β1 (TGF-β1), cytokine closely related to the process of cardiac fibrosis, promotes the decrease of EPAC-1 expression in cardiac fibroblasts. However, the mechanism by which TGF-β1 regulates fibroblasts and myofibroblasts EPAC-1 expression still remains unclear. There are few studies demonstrating the involvement of EPAC-1 in cellular processes associated with cardiac remodeling. There are some investigations related to fibroblast migration and collagen secretion, nevertheless EPAC-1 participation in processes associated to cardiac remodeling such as cellular adhesion and those related to capability to contract collagen gels matrix are completely unknown. In this context, we studied EPAC-1 levels and the effect caused by TGF-β1 on EPAC-1 expression in both cardiac fibroblasts and myofibroblasts through the intervention of TGF-β1 canonical (Smad) and no canonical (MAPK and Akt) signaling pathways. The data obtained showed that cardiac myofibroblasts have greater amounts of EPAC-1 than cardiac fibroblasts. In the presence of chemical inhibitors the results showed that fibroblast EPAC-1 expression is regulated by JNK-MAPK and Smad2 protein, and the myofibroblast EPAC-1 expression is regulated by both signaling pathways (Smad, MAPKs and Akt). Subsequently, in order to determine the EPAC-1 capability to activate its downstream effector Rap1, we stimulated this protein using a cAMP analog able to specifically activate EPAC-1. The results showed that EPAC-1 was able to phosphorylate its downstream effector Rap-1 in both fibroblast and myofibroblasts, however myofibroblasts demonstrated to have higher levels of Rap1-GTP compared to cardiac fibroblasts. Finally and in order to elucidate the EPAC-1 involvement in cardiac remodeling, we performed a series of experiments designed to evaluate cellular adhesion, migration, collagen gel contraction and collagen expression. To evaluate these functions we used selective molecules ables to recognize specifically both EPAC-1 and PKA. The results suggest that EPA-1 increases fibroblast and myofibroblast fibronectin adhesion, promotes fibroblasts migration, enhances collagen gel contraction in both fibroblast and myofibroblast and decrease collagen levels measured by western blot. On the other hand, the results obtained using a PKA agonist suggest that this protein apparently increases myofibroblasts collagen gel contraction and reduces collagen expression measured by western blot in both fibroblast and myofibroblasts. In summary, the results obtained in this thesis suggest TGF-β1 participation in regulating differentially EPAC-1 levels in cardiac fibroblasts and myofibroblasts and EPAC-1 participation in cellular processes related to adhesion, migration, collagen gel contraction and collagen expression; all of them important cellular processes involved in cardiac remodeling in both fibroblast and myofibroblasts. / Fondecyt

Orientador: Alexandre Augusto Zaia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T23:03:14Z (GMT). No. of bitstreams: 1 Santos_CarolinaCarvalhodeOliveira_D.pdf: 6938585 bytes, checksum: 0b73617c9f542daafb6815d1e62fb4cb (MD5) Previous issue date: 2015 / Resumo: A polpa dentária contém uma heterogeneidade de células, dentre elas destacam-se os fibroblastos e odontoblastos. Quando este tecido é agredido por micro-organismos ou seus bioprodutos, fibroblastos e odontoblastos secretam diversas citocinas inflamatórias para ativação do sistema imune, dentre estas TNF-'alfa', IL-6 e MCP-1. Este estudo avaliou a expressão gênica e a produção de citocinas inflamatórias após o contato in vitro das espécies Sreptococcus mutans, Porphyromonas gingivalis, Enterococcus faecalis e seus bioprodutos com odontoblastos e fibroblastos. A expressão gênica foi avaliada por RT-qPCR e as proteínas foram quantificadas por meio de citometria de fluxo, tipo CBA, nos períodos de 4, 8 e 24 horas. As bactérias estudadas promoveram sensibilização das células para expressão e produção das citocinas IL-6, TNF-'alfa', e MCP-1 principalmente nas primeiras 8 horas de contato. Tanto o contato direto com as bactérias como o contato com seus bioprodutos resultaram em estímulo para as células, contudo, o contato direto com as bactérias S. mutans e E. faecalis se mostrou mais eficiente para a estimulação de expressão e produção de citocinas. Por outro lado, o contato indireto com P. gingivalis mostrou ser mais efetivo para estimular a produção das citocinas. Desta forma, pode-se concluir que odontoblastos e fibroblastos são capazes de expressar e produzir citocinas pró-inflamatórias a partir de diferentes contatos quando estimulados por tipos bacterianos distintos. / Abstract: Dental pulp contains a heterogeneity of cells in its composition, among them stand out from fibroblasts and odontoblasts. When this tissue is attacked by bacteria or their by-products, fibroblasts and odontoblasts can secrete several inflammatory cytokines in order to attract and activate immune system to contain infectious agents. TNF-'alfa', IL-6 and MCP-1 are proteins secreted by fibroblasts and odontoblasts mainly involved in the origin and activation of the inflammatory process. This study aimed to evaluate gene expression and secretion of inflammatory cytokines after in vitro contact of bacteria Sreptococcus mutans, Porphyromonas gingivalis, Enterococcus. faecalis and its by-products with odontoblasts and fibroblasts. The proteins were quantified by flow cytometry type CBA and gene expression by Real Time - PCR in periods of 4, 8 and 24 hours. The results showed that the studied bacteria promoted sensitizing cells for expression and production of the cytokines IL-6, TNF-'alfa', MCP-1, particularly during the first 8 hours of contact. Both direct contact with bacterias or their by-products resulting in stimulation to the cells, however, direct contact with the bacteria S. mutans and E. faecalis was more efficient for stimulating expression and cytokine production. On the other hand, the indirect contact with P. gingivalis shown to be more effective to stimulate the production of cytokines. Thus, we may conclude that fibroblasts and odontoblasts are able to express and produce proinflammatory cytokines from different contacts when stimulated by different bacterial types / Doutorado / Endodontia / Doutora em Clínica Odontológica

Memoria para optar al título de Químico Farmacéutico / Una consecuencia común de las enfermedades cardiovasculares es la remodelación cardíaca, que se caracteriza por cambios en el tamaño, forma y función del corazón. El infarto agudo al miocardio es la causa más común que desencadena un proceso de remodelado. El daño causado por el proceso isquémico conduce a la activación de receptores de reconocimiento de patrones moleculares asociados a daño (PRR), y de mediadores celulares y humorales de la respuesta inflamatoria en el tejido. En relación a esto, se ha reportado que el receptor tipo toll-4 (TLR-4), un tipo de PRR, tiene un papel clave en la función cardiaca luego de un infarto, mientras que el aumento en la expresión de las moléculas de adhesión intercelular 1 (ICAM-1) y vascular 1 (VCAM-1), en el tejido cardiaco se ha asociado a una mayor adhesión e infiltración leucocitaria y a un mal pronóstico de la función cardiaca. En fibroblastos cardiacos (FC) se ha demostrado que TLR-4 reconoce patrones moleculares asociados a daño y que su activación conduce a la secreción de citoquinas proinflamatorias, sin embargo los efectos sobre la expresión de moléculas de adhesión no han sido determinados. En esta memoria se estudió el rol del TLR-4 en la expresión ICAM-1 y VCAM-1 en FC de rata adulta e indagó las vías de señalización involucradas en dicha expresión. El tratamiento con lipopolisacárido (LPS) 1 μg/mL aumentó los niveles de ICAM-1 y VCAM-1 de manera tiempo dependiente, observándose en cada caso un aumento significativo a las 24 hrs. Por otra parte, LPS aumentó de forma temprana la fosforilación de NF-κB, ERK1/2, y JNK, mientras que p-38 y Akt sólo mostraron una tendencia a aumentar a partir de los 5 min. La utilización de los inhibidores TAK-242, PD98059 y SP600125, revirtieron el aumento en los niveles de ambas proteínas, mientras que los inhibidores BAY117085, LY294002 y SB202190 no lo los revirtieron. En resumen, nuestros resultados demostraron que la activación del TLR-4 en FC, gatilla un aumento en los niveles de ICAM-1 y VCAM-1 a través de las vías de señalización ERK1/2 y JNK / The cardiac remodeling is a common consequence of the ECV, and is characterized by changes in the size, shape and function. Moreover the acute myocardial infarction is the most common cause of remodeling. The damage caused by the ischemic process leading to activation of pattern recognition receptors (PRR), and humoral and cellular mediators of the inflammatory response in the tissue. In regards to this Toll like receptor-4 (TLR-4) has been described has a key mediator of cardiac function after myocardial infarction, while the increase in intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in cardiac tissue has been linked to increased adhesion and leukocyte infiltration and poor prognosis of cardiac function. In cardiac fibroblast TLR4 is capable to recognize danger associated molecular patterns and release pro-inflammatory cytokines, however their role in ICAM-1/VCAM-1 expression remain unknown. In this study we have determined the ability of TLR-4 to increased ICAM-1/VCAM-1 protein level in adult rat cardiac fibroblast and investigated the potential mechanisms underlying this induction. Treatment with LPS 1 μg/ml resulted in a time-dependent increased of ICAM-1 / VCAM-1 protein level. Furthermore, NF-κB, ERK1/2 and JNK were rapidly phosphorylated by LPS, while p-38 and Akt showed a tendency to increase from 5 min. We also found that inhibition of ERK1/2 and JNK pathways prevented LPS–induced ICAM-1 and VCAM-1 protein level, whereas NF-κB, p-38 and Akt inhibition did not. In summary, these findings demonstrate that the increased ICAM-1/VCAM-1 protein level by TLR-4 activation is regulated via the ERK1/2 and JNK pathways, in cultured cardiac fibroblasts / Fondecyt FONDAP ACCDiS

Neste trabalho tentamos traçar quais seriam os efeitos funcionais e de expressão gênica de mutações patogênicas no gene TCOF1 em células não embrionárias. A partir do estabelecimento de culturas celulares oriundas de periósteo facial de quatro pacientes portadores da síndrome de Treacher Collins (STC), obtivemos populações celulares com mais de 95% de marcação positiva para antígenos que caracterizam células de origem mesenquimal. Demonstramos que as células-tronco mesenquimais e fibroblastos provenientes de pacientes com STC apresentaram redução da expressão de TCOF1 de aproximadamente 31% quando comparados aos controles. Tal redução se mostrou compatível com a diminuição da expressão dos transcritos portadores de mutação patogênica observada no seqüenciamento do cDNA dos pacientes. Portanto, estes resultados sugerem que há degradação do transcrito mutado, que muito possivelmente deve ser regulada pelo mecanismo de non-sense mediated mRNA decay (NMD) e corroboram o modelo de haploinsuficiência da Treacle. Como o pico de expressão do gene Tcof1 é detectado durante o estágio embrionário de formação das células da crista neural e embriões de camundongos Tcof1+/- apresentam redução da proliferação das células neuroepiteliais, testamos se haveria alteração da capacidade proliferativa de células mesenquimais adultas com mutações no TCOF1. A análise das taxas de crescimento das culturas celulares provenientes de pacientes com STC se mostrou semelhante aos controles normais, indicando que menores níveis de transcritos do gene não interferem na capacidade proliferativa celular. Além disso, avaliamos o potencial de diferenciação óssea in vitro, mostrando que menores níveis de TCOF1 também não parecem influenciar a capacidade de diferenciação celular. Apesar de ainda não termos identificado um fenótipo celular, a deficiência de TCOF1 em células não embrionárias resulta na alteração da expressão gênica, já que a análise da expressão genômica das culturas celulares identificou uma série de genes diferencialmente expressos. Ademais, genes de parada do ciclo celular e apoptose com expressão aumentada em células embrionárias de camundongo Tcof1+/- não apresentaram expressão aumentada nas células humanas com mutações patogênicas no TCOF1, sugerindo que a via da p53 não está ativa nestas células não embrionárias. Dentre os genes diferencialmente expressos encontrados, o aumento dos transcritos de DAXX nas células-tronco mesenquimais com mutações patogênicas no TCOF1 poderia modular as funções da p53 nessas células, constituindo um bom alvo de investigações futuras para a elucidação da função do TCOF1 em células não embrionárias. Os resultados obtidos sugerem que deficiência de TCOF1 em células mesenquimais leva à ativação de outras vias de sinalização, cujo efeito funcional é ainda desconhecido. Quais seriam as funções e em quais vias celulares o TCOF1 agiria, são questões a serem elucidadas a respeito do papel do gene na fase adulta. / In this work we tried to outline the effects of functional and gene expression of pathogenic mutations in the gene TCOF1 in non-embryonic cells. From the establishment of facial periosteum derived cell culture of four patients with Treacher Collins syndrome (TCS), we obtained cell populations that are more than 95% positive for mesenchymal origin characteristic antigens. We demonstrated that mesenchymal stem cells and fibroblasts from patients with TCS showed reduction of approximately 31% in the TCOF1 expression when compared to controls. This reduction was consistent with the decrease in the expression of transcripts carrying the pathogenic mutations observed when sequencing the cDNA of the patients. Therefore, these results suggest that degradation of the mutated transcript occurs and it may possibly be governed by the mechanism of non-sense mediated mRNA decay (NMD), thus corroborating the Treacle haploinsufficiency model. As the peak of TCOF1 gene expression is detected during the embryonic stage of the formation of neural crest cells and Tcof1+/- mice embryos had shown reduction of neuroepithelium cells proliferation rate, we tested if there is a change in proliferative capacity of adult mesenchymal cells with mutations in TCOF1. The analysis of the growth rate of TCS patients cells was similar to normal controls, indicating that lower levels of transcripts of the gene does not interfere with cellular proliferative capacity. Furthermore, we evaluated the bone differentiation potential of these cells in vitro, showing that lower levels of TCOF1 also seem does not influence cell differentiation ability. Although a cellular phenotype has not yet been identified, deficiency of TCOF1 in non-embryonic cells results in gene expression change, since genomic analysis of the expression of cell cultures identified a number of differentially expressed genes. Furthermore, arresting cell cycle and apoptosis related genes with increased expression in embryonic cells of Tcof1+/- mice showed no increased expression in human cells with pathogenic mutations in TCOF1 suggesting that the p53 pathway is not active in these non-embryonic cells. Among the differentially expressed genes found, the increase of DAXX transcripts in mesenchymal stem cells with pathogenic mutations in TCOF1 could modulate the function of p53 in these cells, providing a good target for future investigations to elucidate the function of TCOF1 in non-embryonic cells. The results suggest that deficiency of TCOF1 in mesenchymal cells leads to activation of other signaling pathways, whose functional effects are still unknown. The functions and cellular pathways in which the TCOF1 act, are issues yet to be elucidated concerning the role of the gene during adulthood.

O conhecimento do potencial citotóxico, das reações histológicas e propriedades clínicas é imprescindível para a escolha do material na terapia pulpar de dentes decíduos. O estudo teve como objetivo avaliar o efeito de fármacos desta terapia quanto à citotoxicidade e distribuição in vitro de proteínas da matriz extracelular e do citoesqueleto de fibroblastos da polpa humana. Os grupos foram: pasta Guedes- Pinto, pasta Óxido de Zinco e Eugenol (OZE), Vitapex®, Calen® e Calen PMCC®. Os extratos brutos dos fármacos foram testados na concentração 0,2g/ml de meio DMEM/F12 (ASTM, 1992), nas diluições 10, 100 e 1000x. A citotoxicidade foi analisada pela viabilidade (24hs) e sobrevivência celular (24, 48 e 72hs) que se baseou na atividade mitocondrial de fibroblastos da polpa humana (FP5) pelo método de redução do MTT. O grupo controle foi utilizado como 100% de células viáveis. Os resultados foram submetidos à análise de variância, e teste de Tukey como contraste. O efeito dos fármacos na distribuição in vitro de proteínas da matriz extracelular (fibronectina, tenascina, colágeno I) e de citoesqueleto (vimentina) nas FP5 também foi analisado por imunofluorescência. Os resultados demonstraram que na viabilidade celular, as pastas Guedes-Pinto, pasta OZE, e Calen® foram mais citotóxicas que o grupo controle somente na maior concentração (p<0,05), sendo que esse efeito perdurou para a pasta OZE nas menores concentrações em relação ao grupo controle (p<0,05). As diferenças de citotoxicidade entre os fármacos só foram observadas na maior concentração, onde a pasta Guedes-Pinto teve maior efeito tóxico que a pasta Calen®, Calen PMCC® e Vitapex® (p<0,05), mas similar à pasta OZE. Na análise da sobrevivência celular em 72hs, todos os grupos apresentaram mesma capacidade proliferativa que o grupo controle em 24 e 48hs (p>0,05). As diferenças entre os fármacos foram observadas ao final do tempo experimental quando a Pasta Guedes-Pinto não manteve a mesma capacidade de proliferação celular que o grupo controle na maior e menor concentração (p<0,05). Na imunofluorescência, não houve diferença entre os grupos para a distribuição de proteínas da matriz extracelular e citoesqueleto nas FP5. A vimentina, proteína do citoesqueleto de células mesenquimais, encontrou-se distribuída na forma de filamentos ao longo do citoplasma celular. A fibronectina obteve marcação positiva, formando uma rede reticular no citoplasma. A tenascina e colágeno I apareceram como pequenos pontos (vesículas) distribuídos homogeneamente no citoplasma e na região perinuclear. Concluiu-se que, todos os fármacos estudados foram biocompatíveis em relação à citotoxicidade e à distribuição in vitro de proteínas da matriz extracelular e citoesqueleto de fibroblastos da polpa humana. / When electing a material to be used in deciduous pulp therapy, it is essential to acquire knowledge regarding the materials potential toxicity, histological reactions and clinical properties. This study aims at analyzing the effect of different drugs used in pulp therapy in relation to their cytotoxicity and in vitro protein distribution of extracellular matrix and cytoskeleton from human dental pulp fibroblasts. The groups were: Guedes-Pinto Paste, Zinc Oxide and Eugenol paste (ZOE), Vitapex®, Calen® e Calen PMCC®. The materials were tested using the following concentration: 0.2g/mL of culture medium (DMEM/F12) (ASTM, 1992), diluted in 10x, 100x and 1000x. The cytotoxicity was evaluated by cellular viability (24hs) and survival (24, 48 and 72hs), which was based on mitochondrial activity (MTT reduction test) of human dental pulp fibroblasts (FP5). The control group was considered to have 100% of viable cells. Data were submitted to variance analysis, using Tukey test as contrast. The drugs effect on in vitro expression of extracellular matrix proteins (fibronectin, tenascin, type I collagen) and cytoskeleton (vimentin) from FP5 were also evaluated using immnunofluorescence. The results demonstrated that, concerning cellular viability, Guedes-Pinto paste, ZOE paste, and Calen® were more cytotoxic than the control group only in their highest concentration (p<0.05), an effect observed for ZOE paste in all dilutions (p<0.05). Differences regarding cytotoxicity between groups were only observed in the highest concentration where Guedes-Pinto Paste was more toxic than Calen®, Calen PMCC® e Vitapex® (p<0.05), but was similar to ZOE paste (p>0.05). Cellular survival analysis after 72 h showed that all groups presented a similar proliferative capacity compared to the control group at 24 and 48h (p>0.05). Differences were only observed in the end of experimental period (72hs), when Guedes-Pinto paste did not maintain the same proliferative capacity than the control group in its lowest and highest concentrations (p<0.05). In immnunofluorescence tests, there was no difference between all groups for extracellular matrix and cytoskeleton proteins distribution from FP5. Vimentin, a protein from the cytoskeleton of mesenchymal cells, was distributed as filaments throughout the cytoplasm. Fibronectin was positively marked, forming a reticular net in the cytoplasm. Tenascin and Collagen I appeared punctually (as vesicles) and were homogeneously distributed in the cytoplasm and peri-nuclear region. It was concluded that all studied drugs investigated were biocompatible regarding cytotoxicity and in vitro distribution of extracellular matrix proteins and cytoskeleton proteins from human dental pulp fibroblasts.

O objetivo deste estudo foi avaliar a viabilidade in vitro e in vivo de embriões bovinos reconstruídos com oócitos enucleados em Metáfase II e núcleos de células somáticas (fibroblastos) fetais e adultas. Para tanto, oócitos de ovários colhidos em matadouro foram maturados in vitro por 17 horas e enucleados pela remoção do primeiro corpúsculo polar (CP) e da região do oolema contendo a placa metafásica. Como núcleo doador, foram utilizados fibroblastos de orelha de vaca da raça Nelore e de feto colhido em abatedouro. Para a reconstrução dos embriões, cada célula doadora de núcleo, após indução à G0, foi inserida sob a zona pelúcida de cada oócito enucleado e o complexo citoplasma receptor - núcleo doador (CCN) fundido e ativado por eletrofusão (2 pulsos de 4 KV/cm durante 20µs). Após ativação elétrica, cada CCN foi incubado em solução de ciclohexemide (10µg/ml) e citocalasina D (2,5µg/ml) por 1 hora e, em seguida, em solução de ciclohexemide (10µg/ml) por mais 4 horas. Os embriões reconstruídos e ativados, assim como os fecundados in vitro (controle), foram co-cultivados em monocamada de células da granulosa e TCM 199 acrescido de 10% de SFB por 7-9 dias. Após o co-cultivo por 7-9 dias, parte dos embriões (controle e reconstruídos) foi fixada e corada para determinação do número de células e parte transferida para receptoras. Um total de 668 embriões foram reconstruídos com célula fetal e 569 com fibroblasto adulto. Após eletrofusão, 212 embriões reconstruídos com célula fetal e 181 com célula adulta fundiram e 32 (15,1%) e 30 (16,6%) atingiram o estádio de blastocisto, respectivamente. O número médio de células dos blastocistos foi 129,3, 101,3 e 114,3, respectivamente, para célula fetal, adulta e embriões FIV (controle), não havendo diferença estatística significante entre os grupos (P<0,05). Após a transferência de 18 blastocistos de célula fetal e 21 de célula adulta, as taxas de prenhez aos 90 dias foram 16,7% (3) e 19% (4), respectivamente, não havendo diferença estatística significante entre os grupos (P<0,05). A primeira prenhez com célula fetal deu origem a um bezerro saudável, aos 290 dias, pesando 34kg. Uma das receptoras morreu aos 229 dias de gestação em conseqüência de hidroalantóide e outra abortou aos 252 dias. As prenhezes de embriões reconstruídos com célula adulta ainda estão em andamento. Estes resultados indicam que fibroblastos fetal e adulto podem ser usados como doadores de núcleo com semelhantes taxas de desenvolvimento in vitro e in vivo. / The aim of this study was to evaluate the in vitro and in vivo viability of bovine nuclear transferred embryos from metaphase II oocytes and fetal and adult fibroblasts. Oocytes from ovaries collected at slaughterhouse were matured in vitro for 17 hours and enucleated after aspiration of first polar body (PB) and small volume of cytoplasm containing metaphase plate. Fibroblasts from Nelore cow and foetus collected at slaughterhouse were used as nuclei donor. In Nuclear Transfer, each nuclei donor cell, after serum starvation, was inserted under the zona pellucida of the each enucleated oocyte and the enucleated oocyte- nuclei donor cell complexes were electrofused and activated (2 pulses of 4KV/cm for 20µs). After electrical activation, the couplets were incubated in TCM199 plus 10% FCS supplemented with cycloheximide (10µg/ml) and cytochalasin D (2.5µgml) for 1 hour and cycloheximide alone for further 4 hours. The activated reconstructed embryos, as well as IVF embryos (control group), were co-cultured with granulosa cells in TCM 199 + 10% FCS for 7–9 days. After co-cultured, part of embryos (control and reconstructed) was fixed and the number of cells counted and part was transferred into recipients. A total of 668 couplets were reconstructed from fetal and 569 from adult fibroblasts. After electrofusion, 212 (fetal cells) and 181 (adult cells) embryos got fused and 32 (15.1%) and 30 (16.6%) reached blastocyst stage, respectively. The blastocyst cell number means were 129.3, 101.3 and 114.3, respectively, for fetal, adult and IVF (control) embryos. There was no significant difference (P<0.05) in the number of cells of blastocysts among the groups. After transferring 18 (fetal cells) and 21 (adult cells) blastocysts, pregnancy rates at day 90 were 16.7% (3) and 19% (4), respectively. There was no significant difference (P<0.05) between pregnancy rates. The first pregnancy from fetal cells delivered a healthy male calf at day 290, weighting 34kg. One of the remaining recipients died with hydrallantois at day 229 and the other aborted at day 252. The pregnancies of adult cells reconstructed embryos are still in course. These results indicated that fetal and adult fibroblasts could be used as nuclei donor, with similar rates of in vitro and in vivo developments.