Valutazione degli effetti del Dexametasone e del Cisplatino nei modelli sperimentali in vitro (OC-k3)e in vivo(Ratti Wistar)

In this thesis I want to evaluate the Dexamethasone and Cisplatin effects in vitro (OC-k3) and in vivo (Rats Wistar) model. On more I want to get the protocol for future gene therapy, using the new non-viral transposable element Sleeping Beauty in vitro model (Human Fetal Auditory Stem Cells (hFASC). The Cisplatin is an antineoplastic agent used to contrast different types of malignant tumors (testicolaris, ovaric, bladder, neck and head). This drug is very toxic and it generates many side effects like ototoxicity. This agent destroys the inner ear tissues, but they are not able to regenerate for this manner it is very important to study the otoprotective drugs mechanisms. There are many agents otoprotective and I want to remember the Dexamethasone. Dexamethasone is a glucocorticoid and usually it is used to contrast the inflammatory processes. The aim of this thesis is evaluating the drugs effects and protective effect of Dexamethasone against the Cisplatin toxicity. I used the cell line OC-k3 for in vitro model, because these cells derive from immortal mouse inner ear. The OC-k3 don’t show the neurological markers, but epidermal markers. For evaluating the drugs toxicity I used the flow citometry and I noted that the Cisplatin toxicity is time dependent. Dexamethasone is not toxic and is able to protect the cells from Cisplatin action. Another method for toxicity and protection evaluation is studying the cytoskeleton morphology. I noted that after 24h Cisplatin and Dexamethasone are not toxic for OC-k3, but after 48h Cisplatin destroys the cytoskeleton, while Dexamethasone is not toxic and protects the cells from Cisplatin action. Finally I studied the apoptosis marker after Cisplatin and Dexamethasone treatment. Cisplatin generates apoptosi intrinsecal via, but Dexamethasone is not toxic and it protects the cells, because they don’t show the apoptosis marker. I can conclude that Cisplatin is toxic for OC-k3 and Dexamethasone is not toxic and it is able to protect the cells from Cisplatin action. In vivo model I used the Wistar rats and I injected the drugs introperitoneal via alone and in co-treatmennt. To evaluate the drugs effects I used the microscope (SEM) and Immunoistochemistry. I noted that Cisplatin destroys the inner ear tissues and Dexamethasone is not toxic and protects the tissues from Cisplatin action. In vitro and in vivo model, Cisplatinand Dexamethasone generate the same effects; Cisplatin is toxic as for the cells as for the tissues. Dexamethasone is not toxic and it is protective as for cells as for tissues. For gene therapy I used hFASC and I trasfected them with new non viral transposable element: Sleeping Beauty. Two plasmids derive from this transposable element (pT2/veus: gene for GFP, SB100: gene for transposase). I used different concentrations of these plasmids for evaluating their toxicity, I noted that high concentrations of plasmids are toxic for the cells and the green cells number decreases during the incubation time. I decided to decrease the plasmids concentrations and I noted that the green cells number increases. I can conclude that high concentrations of plasmids are toxic for the cells, but I can transfect the cells without problem if I use low concentrations. After I evaluate if the cells are able to express the typical marker of undifferentiated cells (SOX 2, PAX 2, OCT4). With immunoistochemistry I noted the cells show these typical markers. Finally I decided to differentiate the transfected cells to bipolar neurons and cells like hair cells. I got that no transfected cells are able to differentiate to neuronal cells and cells like hair cells only. It is not significant result because I had one only single cell transfected in that cells pool.