Luisetto, Roberto (2008) Effetti dei Microcristalli di Urato Monosodico sull'apoptosi spontanea ed induzione di frammenti di DNA instabile nei sinoviociti. [Tesi di dottorato]
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The deposition of Urate Monosodium crystals (MSU) in the articular and periarticular tissues is cause of the onset of inflammatory states associated to acute gout. Despite several and different pathogenetic hypotheses proposed, the molecular mechanism that controls the acute inflammatory attack induced by the microcrystals is still unknown.
The aim of this study, through a model of acute inflammation in vitro mediated by MSU crystals , is verify the possible cito- and genotoxic effects induced on primary cultures of synoviocites derived from synovial fluid of 6 patients (3 affected by OA and 3 by AP).
The purpose of this research is to determinate if the inflammation can influence the instability of genoma of the involved cells and what are the possible consequences in these cells concern the incidence of the apoptoss, proliferation, DNA integrity, formation of DNA unstable fragments and other aberrant figures as micronuclei.
Experimentally the synoviocites primary cultures have been stimulated for 24 hours with different concentrations of MSU crystals (0,01 mg/ml; 0,025 mg/ml; 0,05 mg/ml; 0,075 mg/ml; 0,1 mg/ml, 0,25 mg/ml and 0,5 mg/ml).
The same conditions have been repeated on CHO cells cultures, from Chinese Hamster, that represents one of the cellular systems more used and standardized for studies about in vitro mutagenesis..
For every experimental point the followings techniques have been applied:
to) Tunel Test in order to analyze and quantify apoptotic cells
b)In situ Incorporation of digoxigenin-dUTP for the staining of DNA breaks in the interphase nuclei and within the fragments of unstable DNA.
c) cytological analysis for the determination of the mitotic index, of the defective mitoses and of other cytological parameters of genomic instability as presence of micronuclei, nuclear vesicles and anaphasic bridges.
The stimulation for 24 hours of the CHO cell lines of Chinese Hamster has underlined as the presence of the MSU crystals induces on this cellular population a clastogenic effect. In fact the incidence of the cells marked by the digoxigenin-dUTP probe, that localizes in correspondence to free 3'-OH inside the DNA, increases in a dose dependent manner . The same results is noticed for other aberrations as the MN , aberrant mitotic figures, NPB and NBUD. Also the Mitotic index shows a meaningful decrease related on the MSU crystals dose used.
The stimulation of the OA and AP derived synoviocytes primary cultures from confirm that , under these conditions, the MSU crystals induced a DNA damage in way analogous to how observed in precedence in CHO cell line used as control system.
Nevertheless for all the types of DNA damage considered , the synoviocites derived from synovial fluid of patients with AP shows, from basal level as in presence of increasing stimulations of MSU crystals, a meaningful incidence of DNA damage greater in comparison to the synoviocites derived from OA. Such greater sensibility for the potentially genotoxic agents could derive from the fact that the primary cultures from AP originate from a synovial liquid, in general, more inflammatory in comparison to OA derived cells. In presence of stimulation with MSU crystals the frequency of apoptotic cells in CHO cells and OA primary cultures shows a meaningful decrement with values inferior to the 1% of incidence on the total population. Increasing the dose of stimulation the frequency of apoptotic cells increases in fucntion of the dose, in linear way for CHO cell line, while for the OA synoviocities , apoptosis increases. Subsequently for doses of MSU greater than 0,1mg/ml apoptosis slightly decreases.
To basal level apoptosia of the AP synoviocites results very lower in comparison to the other cultures; nevertheless this primary culture cells respond in a very analogous way to the CHO cells and AP synoviocites in the presence of increasing doses of MSU crystals.
Observing these data can be hypothesized that, relatively to apoptosis, the presence of MSU crystals, also already to concentrations of 0,01mg/ml acts with a " switch on/switch off "way, inhibiting a molecular apoptotic pathwayi for then subsequently activate an alternative for it in sensitive manner to the dose of stimulation.
The data proposed in this study, even certainly still preliminary ,indicate that this simple experimental model can result very useful to appraise the DNA damage within joint tissues cell populations involved in the articular inflammatory pathologies and to characterize their behavior and evolution of it under these conditions.
In perspective, analogous tests in vitro on cells obtained from the synovial fluid of patients with arthropathies could be predictive, also from the clinical point of view, in to preventively appraise the evolution of a date cellular population under conditions of chronic and acute inflammatory stress and in order to understand the degree of potential citotoxicity that a determined pharmacological treatment could induce to cellular level in a individual patient.
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