DOI 10.1007/s10517-015-2914-9 Cell Technologies in Biology and Medicine, No. 4, February, 2015
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Dipeptides Increase Functional Activity of Human Skin Fibroblasts V. V. Malinin, A. O. Durnova*, V. O. Polyakova*, and I. M. Kvetnoi* Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 52-55, January, 2015 Original article submitted July 21, 2014 We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes. Key Words: skin; dipeptides; signal molecules; reparation
Reparative medicine is a promising trend in molecular biology and immunology that has great practical importance in repair of the structural and functional integrity of organs and tissues [1,7]. Age, comorbidities and environmental factors can slow down the processes of physiological regeneration. Inhibition of physiological regeneration is associated with metabolic disturbances and various pathologies. Reparative regeneration means restoration of tissues and organs damaged due to traumas, injuries, of degenerative processes [1,11]. In light of this, the search for substances capable of restoring functional activity of skin fibroblasts is an important task of modern regenerative and molecular medicine. The processes of cell and tissue repair represent a chain of intracellular and intercellular communication where signaling molecules carrying neuroimmunoendocrine interactions in various tissues, including the skin, play the key role [1,2]. The leading role of peptidergic regulation in the compensatory and adaptive processes in the body has been demonstrated [5,6,9,10]. Cytomed Medico-Biological Research-and-Production Complex; *D. O. Ott Research Institute of Obstetrics and Gynecology, NorthWestern Division of the Russian Academy of Medical Sciences, St. Petersburg, Russia. Address for correspondence: Vvm-61@mail. ru. V. V. Malinin
Here we studied the effect of short peptides on repair processes in culture of human skin fibroblasts.
MATERIALS AND METHODS Fibroblasts were isolated from facial skin (anterolateral surface) of a woman born in 1974 obtained during plastic surgery. After sampling, the skin was treated under sterile conditions with a solution of dispase II (2.4 U/ml), then the epidermis was mechanically separated from the dermis. For preparing cell suspension, the dermis was minced to 3-4 mm fragments and treated with collagenase I solution (Gibco) in MEM. The cell suspension was cultured in precoated flasks (50 ml; 25 cm2; Sarstedt). The medium for cell culturing contained 87.5% DMEM, 10% fetal calf serum, 1.5% HEPES, 1% PES, and L-glutamine. The cells were passaged on day 4 at a seeding concentration of 3×105 cells per flasks. After passage 3, the cells were seeded to a 24-well plate for immunocytochemical staining. All cultures were divided into 4 groups: without peptides (control group 1) and with addition of isovaleroyl-Glu-Trp (peptide 1; group 2), Glu-Trp (peptide 2; group 3), and Actovegin (group 4). Peptides and Actovegin were added to the culture medium in concentrations of 0.2, 2, and 20 μg/ml during each subculturing procedure. Actovegin was used as the reference preparation. Actovegin, a deproteinized
0007-4888/15/15910164 © 2015 Springer Science+Business Media New York
V. V. Malinin, A. O. Durnova, et al.
165
Fig. 1. Effect of peptides on cell renewal in culture of human skin fibroblasts. a) Expression of Ki-67; b) expression of caspase-9. *p
164
Dipeptides Increase Functional Activity of Human Skin Fibroblasts V. V. Malinin, A. O. Durnova*, V. O. Polyakova*, and I. M. Kvetnoi* Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 52-55, January, 2015 Original article submitted July 21, 2014 We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes. Key Words: skin; dipeptides; signal molecules; reparation
Reparative medicine is a promising trend in molecular biology and immunology that has great practical importance in repair of the structural and functional integrity of organs and tissues [1,7]. Age, comorbidities and environmental factors can slow down the processes of physiological regeneration. Inhibition of physiological regeneration is associated with metabolic disturbances and various pathologies. Reparative regeneration means restoration of tissues and organs damaged due to traumas, injuries, of degenerative processes [1,11]. In light of this, the search for substances capable of restoring functional activity of skin fibroblasts is an important task of modern regenerative and molecular medicine. The processes of cell and tissue repair represent a chain of intracellular and intercellular communication where signaling molecules carrying neuroimmunoendocrine interactions in various tissues, including the skin, play the key role [1,2]. The leading role of peptidergic regulation in the compensatory and adaptive processes in the body has been demonstrated [5,6,9,10]. Cytomed Medico-Biological Research-and-Production Complex; *D. O. Ott Research Institute of Obstetrics and Gynecology, NorthWestern Division of the Russian Academy of Medical Sciences, St. Petersburg, Russia. Address for correspondence: Vvm-61@mail. ru. V. V. Malinin
Here we studied the effect of short peptides on repair processes in culture of human skin fibroblasts.
MATERIALS AND METHODS Fibroblasts were isolated from facial skin (anterolateral surface) of a woman born in 1974 obtained during plastic surgery. After sampling, the skin was treated under sterile conditions with a solution of dispase II (2.4 U/ml), then the epidermis was mechanically separated from the dermis. For preparing cell suspension, the dermis was minced to 3-4 mm fragments and treated with collagenase I solution (Gibco) in MEM. The cell suspension was cultured in precoated flasks (50 ml; 25 cm2; Sarstedt). The medium for cell culturing contained 87.5% DMEM, 10% fetal calf serum, 1.5% HEPES, 1% PES, and L-glutamine. The cells were passaged on day 4 at a seeding concentration of 3×105 cells per flasks. After passage 3, the cells were seeded to a 24-well plate for immunocytochemical staining. All cultures were divided into 4 groups: without peptides (control group 1) and with addition of isovaleroyl-Glu-Trp (peptide 1; group 2), Glu-Trp (peptide 2; group 3), and Actovegin (group 4). Peptides and Actovegin were added to the culture medium in concentrations of 0.2, 2, and 20 μg/ml during each subculturing procedure. Actovegin was used as the reference preparation. Actovegin, a deproteinized
0007-4888/15/15910164 © 2015 Springer Science+Business Media New York
V. V. Malinin, A. O. Durnova, et al.
165
Fig. 1. Effect of peptides on cell renewal in culture of human skin fibroblasts. a) Expression of Ki-67; b) expression of caspase-9. *p
