Les effets de la thrombine sur l'épithélium colique humain, grâce aux organoïdes (modèle ex vivo, 3D)

Abstract : Thrombin, a serine protease known for its role in the coagulation cascade, was described for its effects on the induction of apoptosis in colonic epithelial cell lines, through the activation of Protease Activated Receptors (PARs). However, the effects of thrombin on complex epithelial structures such as the human intestinal epithelium composed of different cell types and cells at different stages of differentiation, has never been investigated. A new cellular model, named organoid, enables to reconstitute a functional epithelium in 3-dimensions (3D), from human resections or biopsies, thanks to the self-renewal and differentiation properties of stem cells isolated from colonic crypts. The first objective of this thesis was to evaluate thrombin's effects on survival, proliferation, apoptosis and differentiation in human colonic epithelium, using the organoid model. Then, we aimed to determining the implication of PAR1 and PAR4 in the thrombin's effects. Thus, thrombin added (at low dose: 10mU/mL and higher dose: 50mU/mL) to organoid cultures from control patients, led to a decrease by half of metabolic activity and cell proliferation. These effects were blocked by the addition of a PAR1 antagonist. Apoptotic process was 8-fold higher in organoid cultures exposed to thrombin (both doses) and this effect was inhibited by the addition of a PAR1 or a PAR4 antagonist. As per epithelial differentiation, thrombin decreased the number of colonospheres (immature structures) favoring the increase of apoptotic structures and colonoids (budding structures considered as more mature). This effect was due to PAR1 and PAR4 activation as again, it was blocked both by PAR1 and PAR4 antagonist. Taken together, these results reveal that exogenous thrombin acts on apoptosis, proliferation and differentiation processes in complex human colonic epithelium. The use of this ex vivo model will allow to compare pathological versus normal organoid cultures, but also to test the effects of pharmacological approaches and new treatment options directly in cultured human tissues. Thus, the 2nd part of this thesis was to setup the best culture conditions and the best imaging conditions to perform a robust and reproducible screening approach, HCS (High-Content Screening), using organoid cultures. Culture conditions in 96-well plates were set up and allowed to acquire images with the HCS system. Operetta HCS coupled to an analysis software (Harmony, PerkinElmer) was used to develop a specific program enabling the recognition of organoids, their counting, their classification according to their differentiation status and enabling to follow organoid growth in cultures. To sum up, the work performed allowed to highlight the effects of thrombin on metabolic status, apoptosis and differentiation of human colon epithelium, using an ex vivo 3D organoid model. The use this model nicely completed epithelial cell line approaches, offering an integrated view of the complex behavior of human epithelium. The HCS approach initiated within this thesis should allow the automated analysis of a number of drugs and treatments. It should help our understanding of epithelial pathologies and the testing of new therapeutic approaches.
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Morgane Sébert. Les effets de la thrombine sur l'épithélium colique humain, grâce aux organoïdes (modèle ex vivo, 3D). Hépatologie et Gastroentérologie. Université Paul Sabatier - Toulouse III, 2018. Français. ⟨NNT : 2018TOU30006⟩. ⟨tel-02022720⟩

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