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Titre : | Caractérisation de latteinte des voies respiratoires supérieures dun modèle murin de mucoviscidose |
Auteurs : | Iona MARIAN, Auteur ; Angélique Mottais, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
Cystic fibrosis, is the most common lethal autosomal recessive disorder in the Caucasian population, affecting one in 2,500 to 3,000 newborns and 70,000 people worldwide.
Under normal conditions the CFTR protein functions as a Cl- channel pumping Cl- ions out of the cell. CFTR also functions as a regulator of other ions, including Na+, by negatively regulating the epithelial Na+ channel (ENaC). ENaC is an amiloride-sensitive ion channel responsible for the transepithelial transport of Na+ ions. CF is characterized by defective CFTR function, resulting in decreased chloride (Cl-) secretion and hyperabsorption of sodium (Na+). Because of a limited access to relevant patient tissues, genetically modified mice are widely used to study CF pathology and experimental therapeutics. Previous research showed that the phenotype of the Scnn1B-transgenic mouse is notably comparable to the clinical and pathologic descriptions of early cystic fibrosis lung disease like airway obstruction with mucus, neutrophilic inflammation and mucus hypersecretion. The mouse model Scnn1b overexpresses the beta subunit at the bronchial level due to the presence of the CC-16 protein promoter. Nevertheless, overexpression of Scnn1b increased short-circuit across nasal tissue, marking a phenotype of cystic fibrosis disease, of Scnn1b-trangenicmice was observed by my host laboratory. Therefore, the objective of this work is to characterize the pathophysiology of upper respiratory tract of mice over-expressing the subunit βENaC. To achieve this objective, a group of mice was initially genotyped to keep only mice with a heterozygous genotype. After genotyping, a bronchoalveolar lavage was performed on transgenic mice (6 mice) and control mice (6 mice) with a wild phenotype to estimate the inflammatory state of the lung. The same batches were then dissected to isolate the lungs, trachea and nasal epithelium. These tissues were crushed to recover messenger RNAs and total proteins to assess gene expression by quantitative PCR technique and protein expression by Western Blot analysis. Among a total of 33 mice, only nine with the Scnn1b-transgenic gene were observed. The cell count and cell identification performed on the bronchoalveolar lavage fluids of the 11 mice (5 Tg+ and 6 WT) showed a high variability in the results which can be explained by the small number of samples tested. Real-Time PCR quantified the mRNA initially present in the different tissues. As expected, overexpression of the beta subunit in the lungs could be observed. The results obtained from Western Blot showed a more pronounced expression for the β protein in transgenic mice compared to the level of expression of these protein in WT mice for the two tissues studied (trachea and nasal epithelium). As a conclusion, other methods for the study of inflammation such as the ELISA immunological test or the histopathological study of deceased Scnn1b-transgenic mice are planned by the laboratory. To confirm the over-expression of the Scnn1b gene in the nasal epithelium, new Western Blot will be performed. |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | UCLouvain -LTAP |
Département : | Biologie médicale |
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TFE biologie médicale Adobe Acrobat PDF |