7 research outputs found
Π Π΅ΠΊΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½Π½ΡΠΉ ΡΠΏΠΈΠ΄Π΅ΡΠΌΠΈΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° in vitro β ΠΌΠΎΠ΄Π΅Π»Ρ Π΄Π»Ρ ΡΡΠ½Π΄Π°ΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΡΡ ΠΈ ΠΏΡΠΈΠΊΠ»Π°Π΄Π½ΡΡ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ ΠΊΠΎΠΆΠΈ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°
Background. The reconstructed human epidermis (RE) is an in vitro tissue-engineering construct similar to the native epidermis.
Objective. To develop a full-layer RE. Describe its structure: determine the presence of all layers of the epidermal component, including basal, spinous and granular layers and stratum corneum of the epidermis; detect the basement membrane, the border between the epidermal and mesenchymal component.
Materials and methods. Isolation of keratinocytes and fibroblasts from human donor skin. Cultivation of keratinocytes and fibroblasts in vitro under 2D conditions, cell subculturing and 3D modeling of RE, obtaining cryosections, histological staining, immunohistochemical (IHC) study with antibodies to cytokeratins 14 and 10, Ki67 protein, loricrin, laminin 5 and plectin.
Results. A technique was developed for the formation of RE. Histological examination showed that the stratification of keratinocyte layers occurs during the formation of RE. Layers are formed including basal, spinous and granular layers and stratum corneum. The IHC study has shown the proliferative activity of keratinocytes of the basal layer and has detected the presence of marker proteins of keratinocytes at different stages of differentiation. RE basal keratinocytes, like native ones, form hemidesmosomes and synthesize basement membrane proteins.
Conclusions. A full-layer human RE was obtained in vitro. RE meets all the characteristics of the native epidermis and it is suitable for basic and practical research in the field of skin biology, dermatology, and cosmetology.Π Π΅ΠΊΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½Π½ΡΠΉ ΡΠΏΠΈΠ΄Π΅ΡΠΌΠΈΡ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° (Π Π) ΡΠΊΠ°Π½Π΅ΠΈΠ½ΠΆΠ΅Π½Π΅ΡΠ½Π°Ρ ΠΊΠΎΠ½ΡΡΡΡΠΊΡΠΈΡ in vitro, ΠΏΠΎΠ΄ΠΎΠ±Π½Π°Ρ Π½Π°ΡΠΈΠ²Π½ΠΎΠΌΡ ΡΠΏΠΈΠ΄Π΅ΡΠΌΠΈΡΡ.
Π¦Π΅Π»Ρ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ. Π Π°Π·ΡΠ°Π±ΠΎΡΠ°ΡΡ ΠΏΠΎΠ»Π½ΠΎΡΠ»ΠΎΠΉΠ½ΡΠΉ Π Π. ΠΡ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΠ·ΠΎΠ²Π°ΡΡ Π΅Π³ΠΎ ΡΡΡΡΠΊΡΡΡΡ: ΠΎΠΏΡΠ΅Π΄Π΅Π»ΠΈΡΡ Π½Π°Π»ΠΈΡΠΈΠ΅ Π²ΡΠ΅Ρ
ΡΠ»ΠΎΠ΅Π² ΡΠΏΠΈΠ΄Π΅ΡΠΌΠ°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠ°, Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ Π±Π°Π·Π°Π»ΡΠ½ΠΎΠ³ΠΎ, ΡΠΈΠΏΠΎΠ²Π°ΡΠΎΠ³ΠΎ, Π³ΡΠ°Π½ΡΠ»ΡΡΠ½ΠΎΠ³ΠΎ ΠΈ ΡΠΎΠ³ΠΎΠ²ΠΎΠ³ΠΎ ΡΠ»ΠΎΠ΅Π² ΡΠΏΠΈΠ΄Π΅ΡΠΌΠΈΡΠ°; Π΄Π΅ΡΠ΅ΠΊΡΠΈΡΠΎΠ²Π°ΡΡ Π±Π°Π·Π°Π»ΡΠ½ΡΡ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ, Π³ΡΠ°Π½ΠΈΡΡ ΠΌΠ΅ΠΆΠ΄Ρ ΡΠΏΠΈΠ΄Π΅ΡΠΌΠ°Π»ΡΠ½ΡΠΌ ΠΈ ΠΌΠ΅Π·Π΅Π½Ρ
ΠΈΠΌΠ°Π»ΡΠ½ΡΠΌ ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠΎΠΌ.
ΠΠ°ΡΠ΅ΡΠΈΠ°Π»Ρ ΠΈ ΠΌΠ΅ΡΠΎΠ΄Ρ. ΠΡΠ΄Π΅Π»Π΅Π½ΠΈΠ΅ ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ² ΠΈ ΡΠΈΠ±ΡΠΎΠ±Π»Π°ΡΡΠΎΠ² ΠΈΠ· Π΄ΠΎΠ½ΠΎΡΡΠΊΠΎΠΉ ΠΊΠΎΠΆΠΈ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°. ΠΡΠ»ΡΡΠΈΠ²ΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ² ΠΈ ΡΠΈΠ±ΡΠΎΠ±Π»Π°ΡΡΠΎΠ² Π² 2D-ΡΡΠ»ΠΎΠ²ΠΈΡΡ
in vitro, ΠΏΠ΅ΡΠ΅ΡΠ΅Π² ΠΊΠ»Π΅ΡΠΎΠΊ ΠΈ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠ΅ 3D-ΠΌΠΎΠ΄Π΅Π»ΠΈ Π Π, ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΠ΅ ΠΊΡΠΈΠΎΡΡΠ΅Π·ΠΎΠ², Π³ΠΈΡΡΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠ°Ρ ΠΎΠΊΡΠ°ΡΠΊΠ°, ΠΈΠΌΠΌΡΠ½ΠΎΠ³ΠΈΡΡΠΎΡ
ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΎΠ΅ (ΠΠΠ₯) ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ Ρ Π°Π½ΡΠΈΡΠ΅Π»Π°ΠΌΠΈ ΠΊ ΡΠΈΡΠΎΠΊΠ΅ΡΠ°ΡΠΈΠ½Π°ΠΌ 14 ΠΈ 10, Π±Π΅Π»ΠΊΡ Ki67, Π»ΠΎΡΠΈΠΊΡΠΈΠ½Ρ, Π»Π°ΠΌΠΈΠ½ΠΈΠ½Ρ 5 ΠΈ ΠΏΠ»Π΅ΠΊΡΠΈΠ½Ρ.
Π Π΅Π·ΡΠ»ΡΡΠ°ΡΡ. Π Π°Π·ΡΠ°Π±ΠΎΡΠ°Π½Π° ΠΌΠ΅ΡΠΎΠ΄ΠΈΠΊΠ° ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΡ Π Π. ΠΠΈΡΡΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΎΠ΅ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ ΠΏΠΎΠΊΠ°Π·Π°Π»ΠΎ, ΡΡΠΎ ΠΏΡΠΈ ΡΠΎΡΠΌΠΈΡΠΎΠ²Π°Π½ΠΈΠΈ Π Π ΠΏΡΠΎΠΈΡΡ
ΠΎΠ΄ΠΈΡ ΡΡΡΠ°ΡΠΈΡΠΈΠΊΠ°ΡΠΈΡ ΡΠ»ΠΎΠ΅Π² ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ². Π€ΠΎΡΠΌΠΈΡΡΡΡΡΡ ΡΠ»ΠΎΠΈ Π±Π°Π·Π°Π»ΡΠ½ΡΡ
, ΡΠΈΠΏΠΎΠ²Π°ΡΡΡ
, Π³ΡΠ°Π½ΡΠ»ΡΡΠ½ΡΡ
ΠΈ ΠΎΡΠΎΠ³ΠΎΠ²Π΅Π²Π°ΡΡΠΈΡ
ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ². Π‘ ΠΏΠΎΠΌΠΎΡΡΡ ΠΠΠ₯-ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΠΏΠΎΠΊΠ°Π·Π°Π»ΠΈ ΠΏΡΠΎΠ»ΠΈΡΠ΅ΡΠ°ΡΠΈΠ²Π½ΡΡ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ² Π±Π°Π·Π°Π»ΡΠ½ΠΎΠ³ΠΎ ΡΠ»ΠΎΡ ΠΈ Π΄Π΅ΡΠ΅ΠΊΡΠΈΡΠΎΠ²Π°Π»ΠΈ Π½Π°Π»ΠΈΡΠΈΠ΅ Π±Π΅Π»ΠΊΠΎΠ²-ΠΌΠ°ΡΠΊΠ΅ΡΠΎΠ², Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠ½ΡΡ
Π΄Π»Ρ ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΠΎΠ² Π½Π° ΡΠ°Π·Π½ΡΡ
ΡΡΠ°Π΄ΠΈΡΡ
Π΄ΠΈΡΡΠ΅ΡΠ΅Π½ΡΠΈΡΠΎΠ²ΠΊΠΈ. ΠΠ°Π·Π°Π»ΡΠ½ΡΠ΅ ΠΊΠ΅ΡΠ°ΡΠΈΠ½ΠΎΡΠΈΡΡ Π Π ΠΏΠΎΠ΄ΠΎΠ±Π½ΠΎ Π½Π°ΡΠΈΠ²Π½ΡΠΌ ΡΠΎΡΠΌΠΈΡΡΡΡ Π³Π΅ΠΌΠΈΠ΄Π΅ΡΠΌΠΎΡΠΎΠΌΡ ΠΈ ΡΠΈΠ½ΡΠ΅Π·ΠΈΡΡΡΡ Π±Π΅Π»ΠΊΠΈ Π±Π°Π·Π°Π»ΡΠ½ΠΎΠΉ ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Ρ.
ΠΠ°ΠΊΠ»ΡΡΠ΅Π½ΠΈΠ΅. ΠΠΎΠ»ΡΡΠ΅Π½ ΠΎΡΠ²Π΅ΡΠ°ΡΡΠΈΠΉ Π²ΡΠ΅ΠΌ Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠΈΡΡΠΈΠΊΠ°ΠΌ Π½Π°ΡΠΈΠ²Π½ΠΎΠ³ΠΎ ΡΠΏΠΈΠ΄Π΅ΡΠΌΠΈΡΠ° ΠΏΠΎΠ»Π½ΠΎΡΠ»ΠΎΠΉΠ½ΡΠΉ Π Π ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° in vitro, ΠΏΡΠΈΠ³ΠΎΠ΄Π½ΡΠΉ Π΄Π»Ρ ΡΡΠ½Π΄Π°ΠΌΠ΅Π½ΡΠ°Π»ΡΠ½ΡΡ
ΠΈ ΠΏΡΠ°ΠΊΡΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ Π² ΠΎΠ±Π»Π°ΡΡΠΈ Π±ΠΈΠΎΠ»ΠΎΠ³ΠΈΠΈ ΠΊΠΎΠΆΠΈ, Π΄Π΅ΡΠΌΠ°ΡΠΎΠ»ΠΎΠ³ΠΈΠΈ ΠΈ ΠΊΠΎΡΠΌΠ΅ΡΠΎΠ»ΠΎΠ³ΠΈΠΈ
Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET
SummaryWe have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor forΒ a photochromic FΓΆrster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor