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Inmunolocalización De Citolisinas En La Anémona De Mar (Stichodactyla Helianthus) Katia Ojito Ramos María Cristina Pico

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Inmunolocalización De Citolisinas En La Anémona De Mar (Stichodactyla Helianthus) Katia Ojito Ramos María Cristina Pico Inmunolocalización de citolisinas en la anémona de mar (Stichodactyla helianthus) Katia Ojito Ramos María Cristina Pico Beltrán Edición: Liset Ravelo Romero Corrección: Fernando Gutiérrez Ortega Diagramación: Roberto Suárez Yera Diseño de cubierta: Claudia María Larrea Marin Katia Ojito Ramos y María Cristina Pico Beltrán, 2010 Editorial Feijóo, 2010 ISBN: 978-959-250-580-3 Editorial Samuel Feijóo, Universidad Central “Marta Abreu” de Las Villas, Carretera a Camajuaní, km 5 ½, Santa Clara, Villa Clara, Cuba. CP 54830 RESUMEN Las sticholisinas I y II son dos proteínas citolíticas provenientes de la anémona de mar (Stichodactyla helianthus) que presentan un gran potencial biomédico. La ubicación de estas citolisinas dentro del tejido del animal sería un elemento de gran utilidad en el estudio de las funciones para las cuales este las emplea y facilitaría el proceso de aislamiento y purificación de las mismas. En este trabajo se obtuvieron anticuerpos policlonales anti-sticholisinas en conejos, los que se purificaron mediante intercambio iónico y cromatografía de afinidad, resultando esta última de bajo rendimiento para la matriz cromatográfica empleada. Se preparó un conjugado anti-IgG de conejo peroxidasa, a partir de anticuerpos anti- IgG de conejos obtenidos en carneros. Este conjugado se empleó en la localización inmunohistoquímica de las sticholisinas en el tejido de la anémona de mar, técnica que fue estandarizada para estos reactivos, permitiendo la localización de las sticholisinas en cnidocitos de tentáculos y boca de Stichodactyla helianthus. ÍNDICE INTRODUCCIÓN / 7 CAPÍTULO 1. CITOLISINAS EN ANÉMONAS DE MAR / 9 Características generales de las anémonas de mar / 9 Características morfológicas / 9 Histologías de las anémonas de mar / 12 Actividades biológicas descritas en anémonas de mar / 15 Stichodactyla helianthus /18 Stichodactyla helianthus como fuentes de compuestos bioactivos / 19 Citolisinas de anémonas de mar / 20 Significado biológico de las citolisinas en las anémonas de mar / 21 Citolisinas de Stichodactyla helianthus / 23 Potencial biomédico de las sticholisinas de Stichodactyla helianthus / 26 CAPÍTULO 2. OBTENCIÓN Y PURIFICACIÓN DE ANTICUERPOS ANTI-STICHOLISINAS I Y II EN CONEJO / 29 Generalidades en la producción de anticuerpos policlonales / 29 Preparación del inóculo (sticholisina I y II- adyuvante) / 31 Preparación de la mezcla sticholisina I y II- adyuvante de Freud / 31 Esquema de inmunización de conejos con el inóculo / 32 Purificación de los anticuerpos anti-sticholisina I y II obtenidos en conejos / 33 Obtención del suero / 34 Precipitación en sulfato de amonio (NH SO ) / 34 4 4 Cromatografía de intercambio iónico / 35 Cromatografía de inmunoafinidad / 36 CAPÍTULO 3. LOCALIZACIÓN INMUNOHISTOQUÍMICA EN LA ANÉMONA DE MAR STICHODACTYLA HELIANTHUS / 39 Técnicas enzimo-inmunohistoquímicas / 39 Antecedentes históricos / 39 Generalidades de la técnica / 41 Selección del procedimiento de procesamiento de los tejidos / 42 Selección de los procedimientos de tinción inmunohistoquímica / 44 La especificidad en las tinciones inmunohistoquímica / 46 Preparación del conjugado anti-IgG de conejo peroxidasa / 48 Obtención de anticuerpos anti-IgG de conejo / 49 Conjugación / 50 Colecta de la anémona Stichodactyla helianthus / 51 Inmunohistoquímica / 51 Procesamiento de las muestras. Fijación y embebimiento en parafina / 51 Tinción inmunoperoxidasa. Método indirecto / 52 Desparafinación y rehidratación / 53 Ensayos preliminares / 53 Eliminación de la reacción inespecífica del conjugado / 55 Efectos de la dilución y de las condiciones de incubación del anticuerpo primario / 58 CAPÍTULO 4. DISCUSIÓN GENERAL / 62 CONCLUSIONES / 67 RECOMENDACIONES / 68 BIBLIOGRAFÍA / 69 Introducción Las citolisinas constituyen una gran familia de proteínas, presentes en bacterias, plantas y animales, que comparten una actividad común, la destrucción de células por rompimiento de la barrera de permeabilidad de sus membranas; estas toxinas, principalmente las provenientes de las anémonas de mar, han sido objeto de muchos estudios. El estudio del mecanismo de acción de estas proteínas tiene gran importancia puesto que puede ser utilizado como herramienta para conocer una gran variedad de procesos de membranas. La relevancia de estas toxinas formadoras de canales, no solo se limita a su estudio y caracterización sino que son consideradas modelos de aquellas que forman los canales propios de membranas naturales. Estas citolisinas, además pudieran ser utilizadas con fines terapéuticos si se consideran sus propiedades cardioestimulatorias, anticoagulantes y sus potencialidades como agentes antitumorales.1, 2 Las sticholisinas I y II (St I y II) son dos proteínas citolíticas provenientes de la anémona de mar Stichodactyla helianthus. Desde hace algunos años son objetos de estudio del grupo de Biomembrana de la Facultad de Biología de la Universidad de La Habana 3-15 y han sido evaluadas en la preparación de una inmunotoxina contra Giardia duodenalis. 16 Estas toxinas tienen gran potencial biomédico, son las proteínas que interactúan con proteínas canales de membrana, como las que actúan sobre canales de sodio, éstas se proponen para el tratamiento del dolor; otras, actúan sobre canales de potasio los cuales están implicados en la regulación de funciones celulares, tales como excitabilidad del músculo liso, y neuronas y proliferación celular. Obviamente el bloqueo de estos canales conduce a condiciones patofisiológicas, las sticholisinas de Stychodactyla helianthus se encuentran en la fase II con su propiedad de bloquear canales de potasio (Kv 1.1 y Kv 1.3), han demostrado ser eficaz en modelos animales de esclerosis múltiple y otras enfermedades autoinmunes, como son, diabetes tipo I, psoriasis y artritis reumatoide.17 Las St I y II interfieren con la unión de dendrotoxinas marcadas en la membrana de los sinaptosomas, y bloquea la corriente a través de los canales con varias subunidades KV1 y también la conductancia intermedia de los canales de potasio dependientes de calcio .18 entre otras investigaciones. 19-24 En todas estas investigaciones, las proteínas han sido aisladas a partir del extracto crudo del cuerpo integro del animal. 7 La ubicación de estas toxinas dentro del tejido del animal sería un elemento de gran utilidad en el estudio de las funciones para las cuales este las emplea y en la aplicación de estas toxinas, a la par que facilitaría el protocolo de aislamiento y purificación. Una de las técnicas más utilizadas con el objetivo de detectar histológicamente antígenos, son las técnicas inmunohistoquímicas. Dada la disponibilidad y especificidad de anticuerpos como reactivos y la gran sensibilidad del método, constituyen procedimientos sencillos, rápidos y económicos. Los objetivos del trabajo fueron obtener y purificar anticuerpos anti-sticholisinas en conejos, Localizar las sticholisinas en el cuerpo de la anemona de mar Stichodactyla helianthusis y estandarizar la técnica inmunohistoquímica para la localización de toxinas en anémonas de mar. CAPÍTULO 1. Citolisinas en anémonas de mar Anémonas de mar 8 Los cnidarios pertenecen a los invertebrados metazoarios inferiores y se caracterizan por ser el único filo que posee células urticantes denominadas cnidocistos. Los cnidocistos tienen importancia taxonómica, ecológica y zoogeográfica, ya que a través del estudio de estas células es posible conocer el status de una especie y si ésta se encuentra representada por clines. 25 Dentro de este grupo zoológico se encuentran las anémonas de mar. Características morfológicas La clase Anthozoa, a la cual pertenecen las anémonas de mar (Cuadro 1), son los miembros más avanzados de los cnidarios, y su estructura corporal es mucho más compleja que los pólipos simples de las hidras. 26 Dominio: Eukaryota - Whittaker & Margulis,1978 Reino: Animalia - Linnaeus, 1758 - animals Subreino: Radiata - (Linnaeus, 1758) Cavalier-Smith, 1983 Infrarreino: Coelenterata - Leuckart, 1847 Phylum: Cnidaria - Hatschek, 1888 - Cnidarians Subphylum: Anthozoa - (Ehrenberg, 1831) Cavalier-Smith, 1998 - Corals, Flower Animals Clase: Anthozoa - Ehrenberg, 1831 - Corals, Flower Animals Subclase: Zoantharia Orden: Actiniaria - Sea Anemones Familia: Stichodactylidae Género: Stichodactyla - Brandt, 1835 Nombre específico: helianthus - (Ellis, 1768) 9 Nombre científico: Stichodactyla helianthus (Ellis, 1768) Cuadro 1. Ubicación taxonómica de la anémona de mar Sticodactyla heialnthus. El cuerpo de una anémona de mar tiene una forma más o menos cilíndrica y carece de esqueleto. Esta formado por una gruesa columna, en cuyo extremo aboral se destaca un disco pedal aplanado que le sirve para la fijación al sustrato (Figura 1). En el extremo oral de la columna se estrecha ligeramente para formar el disco oral o perisoma en el cual se aprecian varios tentáculos huecos. En el centro del disco oral se observa una boca en forma de hendidura que presenta en ambos extremos un surco ciliado llamado sifonoglifo. Este facilita la circulación del agua hacia la cavidad gastrovascular. La función de la corriente de agua es mantener el esqueleto interno líquido o hidrostático contra el cual actúa el sistema muscular. Sigue a la boca una faringe aplanada que se prolonga unas 2/3 partes a lo largo de la columna y que contiene las mismas capas de células que la pared del cuerpo. 27 La cavidad gastrovascular de las anémonas de mar es más compleja que en el resto de los cnidarios, se halla dividida en cámaras por mesenterios longitudinales dispuestos radialmente: 6 cámaras situadas entre
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