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Etude De L'effet Du Venin De Scorpion Androctonus Australis Hector Sur La Régulation Des Fonctions Des Polynucléaires Neut

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Etude De L'effet Du Venin De Scorpion Androctonus Australis Hector Sur La Régulation Des Fonctions Des Polynucléaires Neut N° d’ordre : 11/2012-M/S.B REPUBLIQUE ALGERIENNE DEMOCRATIQUE ET POPULAIRE MINISTERE DE L’ENSEIGNEMENT SUPERIEUR ET DE LA RECHERCHE SCIENTIFIQUE UNIVERSITIE DES SCIENCES ET DE LA TECHNOLOGIE HOUARI BOUMEDIENNE FACULTE DES SCIENCES BIOLOGIQUES MEMOIRE Présenté pour l’obtention du diplôme de MAGISTER En : SCIENCES DE LA NATURE Spécialité : Biochimie-Immunologie Par : Dalila KHEMILI THEME Etude de l’effet du venin de scorpion Androctonus australis hector sur la régulation des fonctions des polynucléaires neutrophiles Soutenu publiquement le 31 /10 / 2012, devant le jury composé de : Mme F. LARABA-DJEBARI, Professeur à l’USTHB Présidente Mme D.HAMMOUDI-TRIKI, Professeur à l’USTHB Directrice de mémoire Mme C.TOUIL-BOUKOFFA, Professeur à l’USTHB Examinatrice Mme H.OUMEHDI-OUSSEDIK, Maitre de conférences à l’USTHB Examinatrice Remerciements Je tiens tout d’abord à remercier Dieu pour la force et la patience qu’il m’a donné pour surmonter toutes les épreuves vécues au cours de ce mémoire. J'exprime mes profonds remerciements à ma directrice de thèse, le Professeur Hammoudi-Triki D, je la remercie pour m’avoir proposé ce sujet, pour sa confiance, son soutien et sa constante bonne humeur et surtout ses discussions scientifiques. Je tiens à remercier les membres de jury et particulièrement Mme Lararba-Djebari F, Professeur à la Faculté des Sciences Biologiques, qui nous a fait l’honneur de juger ce travail et de présider le jury. Je la remercie pour m’avoir donné l’opportunité de travailler au sein de son laboratoire. J’adresse mes remerciements à Mme Touil-Boukoffa C, Professeur à la Faculté des Sciences Biologiques, d’avoir aimablement accepté d’examiner ce travail. Je souhaite remercier Mme Oussedik-Oumehdi H, Maître de conférences à la Faculté des Sciences Biologiques. Pour avoir l’amabilité d’accepter d’examiner ce travail. Je tiens à remercier toutes les personnes qui sont intervenues de prêt ou de loin au cours de la réalisation de ce travail, et tout particulièrement le Professeur Kra ϊba R pour son aide. Veuillez trouver ici l’expression de ma très vive reconnaissance et de ma sincère admiration. Je tiens à exprimer ma profonde gratitude à monsieur le Docteur Derrar F, chef de laboratoire Grippe et virus respiratoires à l’Institut Pasteur d'Algérie pour m’avoir accueillie au sein de son laboratoire, mettant à ma disposition l’ensemble des moyens disponibles. Mes sincères remerciements s’adressent également au Docteur Brahimi M et Docteur Issad M. Je voudrais également remercier les membres des deux laboratoires : Vaccins Viraux Vétérinaires et Grippe et virus respiratoires à l’Institut Pasteur d'Algérie. Je n’oublierai évidemment pas de remercier Mr Cherrouf A pour sa disponibilité, ses conseils et ses compétences scientifiques qui m’ont permis de mener à bien mes travaux et d’enrichir mes connaissances. Je remercie les membres du laboratoire de Biochimie des Biomolécules : Mode d’Action, Immunothérapie et Immunodiagnostic pour leur gentillesse. Dédicaces A la mémoire de mes deux grands-pères A mes deux grands-mères A mes très chers parents A mes adorables frères A toute ma famille Sommaire Liste des abréviations Introduction…………………………………………………………..……………………….1 Chapitre I : Données bibliographiques…………………………...…………………………3 I.1. Les scorpions………………………………………………………………………………3 I.2. Les venins de scorpions……………………………………………………………………3 I.2.1. Les toxines longues……………………………………………………………………...5 I.2.1.1. Toxines α………………………………………………………………………………5 I.2.1.2. Toxines β………………………………………………………………………………5 I.2.2. Les toxines courtes………………………………………………………………………5 I.2.3. Les toxines sans ponts disulfure………………………………………………….…...…6 I.3. Les toxines du venin d’ Androctonus australis hector…………………………….……….6 I.4. Action cellulaire des toxines scorpioniques……………………………….………………6 I.4.1. Cellules excitables…………………………………………………………………….…6 I.4.2. Cellules non excitables…………………………………………………………………..7 I.5. Physiopathologie des envenimations scorpioniques……………………………………….8 I.6. Effets des venins de scorpions sur le système immunitaire………………………………..8 I.6.1. Médiateurs de l’inflammation………………………………………………...…………8 I.6.2. Cellules de l’inflammation……………………………………………………………..10 I.6.2.1. Polynucléaires neutrophiles…………………………………………….…………….13 I.6.2.1.1. Origine du neutrophile………………………………………………..…………….13 I.6.2.1.2. Fonctions du neutrophile…………………………………………………………...14 A. Fonctions de base des polynucléaires neutrophiles………………………………………..14 A.1. Chimiotactisme………………………………………………………………………….14 A.2. Phagocytose………………………………………………………………………..........15 A.3. Dégranulation………………………………………………………………………...…16 A.4. Formation d’anions superoxydes…………………………………………………...…..16 B. Rôle des polynucléaires neutrophiles dans la régulation de l’immunité adaptative..…….17 C. Rôle des polynucléaires neutrophiles dans la résolution de l’inflammation………...…....19 I.6.2.2. Phagocytes mononucléés……………………………………………………...……..21 I.6.2.2.1. Origine du monocyte………………………………………………………...…….21 I.6.2.2.2. Sous populations de monocyte……………………………………………..……...21 I.6.2.2.3. Propriétés fonctionnelles du monocyte…………………………………...……….22 I.6.3. Réponse inflammatoire et production de dérivés réactifs de l’oxygène et du monoxyde d’azote………………………………………………………………………………………..23 I.7. Cytotoxicité induite par les venins de scorpions…………………………………………24 I.7.1. Mécanismes impliqués dans la cytotoxicité induite par les venins de scorpions………25 I.7.1.1. Génération des formes réactives de l’oxygène (ROS)…………………………...…..26 I.7.1.2. les dommages provoqués par les ROS…………………………………………...…..27 • La peroxydation des lipides…………………………………………………………...……27 • Modifications des protéines…………………………………………………………...……27 • Modification de l'ADN……………………………………………………………..……....29 I.5. Traitement des envenimations scorpioniques…………………………………...…….….29 Chapitre II : Matériel et Méthodes……………………………………………...…….…...31 II.1. Matériel……………………………………………………………………..……..…….31 II.1.1. Matériel biologique……………………………………………………..…….….……31 II.1.1.1. Le venin d’ Androctonus australis hector…………………………...…………..…...31 II.1.1.2. Les fragments F(ab)'2……………………………………………………….…...….31 II.1.1.3. Les cellules…………………………………………………………………….…….31 II.1.1.4. Le sang humain………………………………………………………………….…..31 II.1.2. Réactifs et produits chimiques…………………………………………………….…..31 II.2. Méthodes………………………………………………………………………….....….32 II.2.1. Préparation, mise en culture et induction des cellules……………………………..….32 II.2.1.1. Préparation des cellules mononuclées du sang périphérique……………….….…....32 II.2.1.2. Préparation des polymorphonucléaires neutrophiles……………………………......32 II.2.1.3. Contrôle de la viabilité cellulaire……………………………………………..……..32 II.2.1.4. Induction des cellules……………………………………………………..…….......33 II.2.2. Etude de l’effet du venin d’ Androctonus australis hector sur la viabilité des PBMC et les PNs humains……………………………………………………………………..…….….33 II.2.3. Etude de l’état d’activation des PBMC et des PNs en présence de concentrations non cytotoxiques de venin d’ Aah …………………………………………………….…...……....33 II.2.3.1. Effet du venin d’ Aah sur la prolifération des PBMC humains…………...………...34 II.2.3.2. Effet du venin d’ Aah sur la dégranulation des polynucléaires neutrophiles et des monocytes……………………………………………………………………………...…......34 II.2.3.3. Effet du venin d’ Aah sur le volume du système lysosomal des polynucléaires neutrophiles et des monocytes……………………………………………………...…….......35 II.2.3.4. Effet du venin d’ Aah sur la production du peroxyde d’hydrogène………………...35 II.2.3.5. Effet des fragments sur le métabolisme oxydatif des PNs et des PBMC après stimulation avec le venin d’Aah …………………………………………………………..…36 II.2.3.6. Effet du venin d’ Aah sur la production du monoxyde d’azote………………...…..36 II.2.4. Etude de l’effet cytotoxique du venin d’ Androctonus australis hector sur les PBMC et les PNs humains…………………………………………………………………………..….36 II.2.4.1. Effet du venin d’ Androctonus australis hector sur l’intégrité mitochondriale…..….37 II.2.4.2. Effet du venin d’ Androctonus australis hector sur l’intégrité membranaire……..…37 II.2.5. Effet du venin d’ Androctonus australis hector sur la balance pro/anti-oxydante….....37 II.2.5.1. Dosage du monoxyde d’azote…………………………………………………...….37 II.2.5.2. Evaluation de la peroxydation lipidique par dosage du malondialdéhyde……...….38 II.2.5.3. Evaluation de l’activité de la catalase…………………………………………...….38 II.2.6. Etude de l’effet du venin d’Androctonus australis hector sur la fragmentation de l’ADN………………………………………………………………………………………..38 II.2.7. Effet du temps d’incubation en présence du venin sur les PNs………………………39 II.2.8. Exploration statistique des résultats…………………………………………...……...40 Chapitre III : Résultats et Discussion………………………………………………..…….42 III.1. Etude de l’effet du venin d’ Androctonus australis hector sur la viabilité des PNs et des PBMC humains………………………………………………………………………...…….42 III.2. Etude de l’état d’activation des PBMC et des PNs en présence de concentrations non cytotoxiques de venin d’ Aah ……………………………………………………………...…44 III.2.1. Effet du venin d’ Aah sur la prolifération des PBMC humains………………….…...44 III.2.2. Effet du venin d’ Aah sur la dégranulation des polynucléaires neutrophiles…………46 III.2.3. Effet du venin d’ Aah sur le volume du système lysosomal des polynucléaires neutrophiles et des PBMC……………………………………………………………….…...48 III.2.4. Effet du venin d’ Aah sur la production du peroxyde d’hydrogène……………..…..50 III.3.5. Effet du venin d’ Aah sur la production du monoxyde d’azote……………………...52 III.3. Etude de l’effet cytotoxique du venin d’ Androctonus australis hector sur les PBMC et les PMNs humains……………………………………………………………………………54
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