Supervisors info:
Ράλλης Μιχαήλ, Επίκουρος Καθηγητής Τομέα Φαρμακευτικής Τεχνολογίας,
Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
(ΣΥΝΕΠΙΒΛΕΠΟΝΤΕΣ:
Λιβανίου Ευαγγελία, Ερευνήτρια Α΄, ΙΠΡΕΤΕΑ, ΕΚΕΦΕ «Δημόκριτος»
Κλέτσας Δημήτριος, Ερευνητής Α΄ - Διευθυντής ΙΒΕ, ΕΚΕΦΕ «Δημόκριτος»
Χαράλαμπος Πράτσινης, Εντεταλμένος ερευνητής ΙΒΕ, ΕΚΕΦΕ «Δημόκριτος»)
Summary:
The subject of this study was the in vitro biological evaluation of peptides, thymosin α1 (Ta1), thymosin β4 (Τβ4) and N-terminal tetrapeptide Ac-SDKP using cell strains which are associated with dermal wound healing.
The Τα1, Tβ4 and Ac-SDKP belong to the family of thymosins, which are endogenous peptides and are present in almost all human cells.
Both Tβ4 and AcSDKP have a positive effect on the wound healing process of various tissues (heart, kidney, liver, skin) according to a large number of in vitro assays (endothelial cells, fibroblasts of cardiac and renal origin etc .) and in vivo assays in animal models. The few studies on the effect of Tα1 on the dermal wound healing phenomenon are mainly related to its angiogenetic effect, in vitro assays as well to its overall effect in vivo assays in animal models.
In an attempt to further investigate the potential effects of the above peptides (in particular Ta1, for which there is little available bibliographic information) on the healing of human skin lesions and in an attempt to an in vitro simulation of the dermal healing process, human cutaneous fibroblasts, which are actively participating in most stages of healing, were selected as study cells. In particular, the effect of the T1, T4 and Ac-SDKP peptides (concentrations: 0.1, 1 and 10 nM) on cell survival, proliferation and collagen synthesis in the above cells was tested.
Initially, the potential cytotoxic effect of Ta1, Tpa4 and Ac-SDKP alone, as well as in combination with the captopril and aprotinin protective agents, on the fibroblasts was evaluated by the MTT assay. No statistically significant effect of the peptides on viability of the fibroblasts was observed.
Subsequently the effect of thymosinic peptides on cell proliferation was studied. The effect of peptides on the proliferation of dermal fibroblasts was studied by quantifying the DNA synthesis by the tritiated thymidine method. In this study, no statistically significant differences were observed, with the sole exception of Te4, which, at the concentration of 10 nM, was shown to lead to a marginal decrease in the incorporation of tritiated thymidine by fibroblasts. Furthermore the total number of cells was counted, with a Coulter analyzer, after 3 and 7 days of thymosinic treatment of the fibroblasts. A statistically significant, stimulatory effect of Ta1, Tβ4 and Ac-SDKP on cell survival was noted. This positive effect of thymosinic peptides represents the overall effect on cell proliferation, including all factors involved in this process.
Finally, the individual effect, of thymosinic peptides as well as their combined effect with the growth factor TGF-β1, on collagen synthesis by cutaneous fibroblasts was tested by means of the tritiated proline method. A statistically significant increase in collagen synthesis was observed following both the individual treatment with Tα1, Tβ4, and Ac-SDKP and their combination with TGF-β1.
In conclusion, the thymosinic peptides Tα1, Tβ4 and Ac-SDKP induce collagen synthesis by human dermal fibroblasts, suggesting a possible stimulatory effect of thymosinic peptides on collagen synthesis during wound healing. Especially with regard to Tα1, it should be noted that this is the first time that the effect of this peptide on collagen synthesis of human dermal fibroblasts is tested, and remarkably with positive results.
