Introduction Cystic fibrosis (FC) is a disease caused by mutations that compromise the function of CFTR, a membrane protein with ion channel function permeable to chloride and other anions. FC mutations have been grouped into five different classes depending on the mechanism of action. In the last 15 years, the possibility of a pharmacological approach for the functional recovery of the mutated CFTR protein has become increasingly concrete. However, this approach must be adapted to the different types of mutations. Currently, different types of enhancers (Ivacaftor) and correctors (Lumacaftor, Tezacaftor) of CFTR active on particular types of mutations are already available for use on patients. The introduction of next gen molecules such as Vx445, in association with the aforementioned modulators, has further expanded the population of FC patients eligible for modulatory therapies, as these molecules are active on the most common CF-causing mutation, the F508del, also in single copy. However, a rather large percentage of CF patients with rare genotypes remain (10-25% of the total FC population, given very variable depending on the geographical area), not carriers of the F508del variant, for which at the moment no molecules are available corrective measures on the CFTR function. Objective of the study Our project has proposed to develop an in vitro analysis protocol for the definition of the teratype (in vitro characterization of the response to treatments aimed at modulating CFTR) belonging to Italian FC patients, a particularly important objective given the large number of mutations rare whose class and pharmacological sensitivity are unknown. This analysis was performed on cells collected using the nasal brushing technique. Materials and methods Human nasal cell collection was performed from both nostrils and without local anesthesia, with prior informed consent from the patient. A soft sterile toothbrush was used for the collection, making antero-posterior rotational movements. The toothbrush was then transferred to a 15 mL conical tube containing 6 mL of Ca2 + Mg2 + D-PBS and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in 6 ml of medium consisting of LHC9 and RPMI1640 (1: 1) and then pretreated with a collagen solution. To improve growth and increase cell duplication in vitro, the culture medium was supplemented with a mixture of SMAD and ROCK inhibitors, necessary for the reprogramming and immortalization of human epithelial cells. (Palechor-Ceron et al., 2013; Mouet al., 2016). In order to eradicate the contamination, antibiotics and antifungals have been added to the proliferation medium. The cells were then detached by trypsin and re-plated to continue the culture. The cells were cultured in flasks until 90% of the confluence was reached and then sown on Snapwell permeable supports. After 2 hours the LHC / RPMI 1640 medium was replaced with a differentiation medium containing DMEM / Ham's F12 (1: 1), fetal bovine serum (2%), penicillin (100 U / ml), streptomycin (100 μg / ml) and various hormones and supplements (Galietta et al., 1998). The degree of differentiation of the epithelium was assessed by measuring the difference in potential and transepithelial resistance with an epithelial voltmeter. Subsequently, the soil was added only in the basolateral part; this condition, which lasts for a further 7 days and, favors a more complete differentiation of the epithelium. The effect of individual compounds or combinations of compounds was tested with short circuit current records. In the case of correctors, the epithelia were incubated for 24 hours with the compounds to be tested. The Snapwell porous supports were mounted in the Ussing chamber at 37 ° C. The response to the sequential addition of amiloride, cAMP agonists, VX-770 and inh-17 was measured. Study population 27 patients (F 16, M11) were selected with the diagnosis of Cystic Fibrosis in regular follow-up at our Center with a known CFTR genotype. All 27 selected patients joined the project and provided informed consent. In all cases the sampling procedure was well tolerated (in 3/27 minimal epistaxis with spontaneous resolution) and in 24/27 the cells were isolated in the laboratory without problems. In 2/27 a retest was carried out, in 1 case with success.
|Titolo della tesi:||Verso un approccio personalizzato per il trattamento del difetto di base in Fibrosi Cistica|
|Data di discussione:||20-mag-2020|
|Appare nelle tipologie:||Tesi di dottorato|