OBJECTIVE To search an ideal carrier of transferred keratinocytes for transplantation. METHODS The transferred keratinocytes were seeded on the surfaces of the artificial dermis and the silicone membrane and cultured in vitro for 2 weeks. The growth of the keratinocytes was observed by microscope and scanning electron microscope. RESULTS The keratinocytes implanted on the artificial dermis began to rupture and died after 2 to 3 days. While the keratinocytes adhered well on the surface of silicone membrane with pseudopodia formation after 1 week under scanning electron microscope, and the cells kept normal morphological and proliferative properties 2 weeks later. CONCLUSION The silicone membrane can be applied as an useful carrier for the keratinocytes transplantation.
OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice. METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II. Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension. Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase. After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes. The histological, immunohistochemical examination and electron microscopy were detected. RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia. Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia. CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract. The keratinocyte graft can be used to repair skin defect.
Objective To review the latest research progress on keratinocyte growth factor (KGF), to thoroughlyunderstand its basic characteristics and appl ication methods and to lay a sol id foundation for the research and development of new KGF medicines and improving the qual ity of skin substitutes. Methods Domestical and international l iteratures on KGFin recent years were extensively reviewed and analyzed. Results KGF was secreted by mesenchymal cells and its receptors were distributed in epithel ium to promote the prol iferation, migration and differentiation of epithel ial cell specifically, which closely related to the organ development, wound heal ing, tumorigenesis and immune reconstruction. Conclusion KGF can be used to improve wound heal ing and the performance of skin substitutes. However, the structure of KGF needs to be changed to el iminate its side effects and purify its promoting effect on epithel ial cell growth.
Objective To investigate the effects of heat injured keratinocytes (KC) supernatant on the expressions of collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1) of dermal fibroblasts (Fb). Methods KC and Fb were isolated and cultured. Then the models of heat injured KC and Fb were reproduced in vitro, respectively. The heat injured and normal culture supernatant were collected respectively at 12 hours, and formulated as a 50% concentration of cell-conditioned medium. According to the culture medium, Fb at passage 3-5 was divided into 3 groups. Normal Fb was cultured with the conditioned medium containing 50% heat injured KC culture supernatant (group A), the conditioned medium containing 50% normal KC culture supernatant (group B), and DMEM (group C), respectively. The cells in 3 groups were collected at 24 hours. In addition， the cells in group A were collected at 0, 1, 2, 6, 12, 24, and 48 hours， respectively. Normal Fb was cultured with the conditioned medium containing 50% heat injured Fb culture supernatant. Then, the cells were collected at 0, 1, 2, 6, 12, 24, and 48 hours, respectively. The mRNA levels of the collagen type I, collagen type III, and MMP-1 of Fb were measured by real-time fluorescent quantitative PCR techniques. Results At 24 hours after cultured with supernatant of heat injured KC，mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A were significantly higher than those in groups B and C (P lt; 0.05). The mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A gradually increased with time going, showing significant differences between 0 hour and 2, 6, 12, 24, and 48 hours (P lt; 0.05); significant differences were found between different time points after 2 hours (P lt; 0.05). After Fb was treated with supernatant of heat injured Fb, the mRNA relative expression levels of MMP-1 gradually decreased with time going, showing significant differences between 0 hour and 1, 2, 6, 12, 24, and 24 hours (P lt; 0.05); after 2 hours of culture, significant differences were found among different time points (P lt; 0.05). Conclusion Heat injured KC supernatant may regulate the mRNA expressions of collagen type I, collagen type III, and MMP-1 of Fb.
Objective To observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
【Abstract】 Objective To observe the effects of Angelica dahurica extracts on the biological characteristics of human keratinocytes (KC) in vitro and to explore the possible mechanism in promoting wound healing. Methods HaCaT cells of passage 5 from KC were used during the experiment. Different concentrations (5 × 10-2, 5 × 10-3, 5 × 10-4, and 5 × 10-5 g/L) of Angelica dahurica extracts, which was obtained by 95% ethanol from Angelica dahurica raw material, were prepared by DMEM containing 0.25% fetal bovine serum (FBS). After the extracts at different concentrations were respectively used for KC culture for 5 days, the cell proliferation activities were detected by MTT, and DMEM containing 0.25% FBS served as the negative control. According to the cell proliferation activity, the optimal concentration was determined. KC was further treated with Angelica dahurica extracts of the optimal concentration (experimental group) or with DMEM containing 0.25% FBS (control group) for 48 hours. The cell cycle was tested by flow cytometry. Cyclin D1 and Caspase-3 mRNA levels were also detected by real-time fluorescent quantitative PCR technique. Results Angelica dahurica extracts at concentrations of 5 × 10-4, 5 × 10-3,and 5 × 10-2 g/L could significantly enhance KC proliferation, showing significant differences in absorbance (A) values compared with that of control group (P lt; 0.05) with an optimal concentration of 5 × 10-3 g/L. At this concentration, an increased percentage of S and G2/M phase cells and a decreased percentage of G0/G1 phase cells were detected, showing significant differences when compared with control group (P lt; 0.05). Real-time fluorescent quantitative PCR revealed that the cyclin D1 and Caspase-3 mRNA levels of experimental group was significantly down-regulated, showing significant differences when compared with control group (P lt; 0.05). Conclusion Angelica dahurica extracts can promote the proliferation of KC, accelerate the cell cycle of KC by down-regulating mRNA expressions of cyclin D1, and inhibit apoptosis by down-regulating mRNA expressions of Caspase-3. These effects might enhance the process of wound healing by expediting the process of epithelization.
N,N-Dimethylglycine (DMG) is a glycine derivative, and its sodium salt (DMG-Na) has been demonstrated to possess various biological activities, including immunomodulation, free radical scavenging, and antioxidation, collectively contributing to the stability of tissue and cellular functions. However, its direct effects and underlying mechanisms in wound healing remain unclear. In this study, a full-thickness excisional wound model was established on the dorsal skin of mice, and wounds were treated locally with DMG-Na. Wound healing progression was assessed by calculating wound closure rates. Histopathological analysis was conducted using hematoxylin-eosin (HE) staining, and keratinocyte proliferation, migration, and differentiation were evaluated using CCK-8 assays, scratch wound assays, and quantitative reverse transcription PCR (qRT-PCR). Inflammation-related cytokine expression in keratinocytes was analyzed via ELISA and qRT-PCR. Results revealed that DMG-Na treatment significantly accelerated wound healing in mice and improved overall wound closure quality. The wound healing rates on days 3, 6, and 9 were 49.18%, 68.87%, and 90.55%, respectively, with statistically significant differences compared to the control group (P<0.05). DMG-Na treatment downregulated the mRNA levels of keratinocyte differentiation markers while enhancing cell proliferation and migration (P<0.05). Furthermore, DMG-Na decreased the secretion of LPS-induced keratinocyte inflammatory cytokines, including IL-1β, IL-6, IL-8, TNF-α, and CXCL10 (P<0.05). These findings indicate that DMG-Na regulates inflammatory responses and promotes keratinocyte proliferation and migration, thereby facilitating the healing of skin wounds.
OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P lt; 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P gt; 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P lt; 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.
Objective To find a feasible method that can fast isolate seed cells, keratinocyte stem cell and fibroblasts, for composite tissue engineered skin. Methods The foreskin could be attained from posthectomy, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2 mm×2 mm in size, then the pieces were submerged into a centrifuge tube containing collagenase Ⅰ in a oscillator. After 3-hour digestion at 37℃, the dermis was dissolved completely with all the fibroblasts in the digestion solution and the epidermis could be separated easily.With more than 10minute digestion in trypsin at 37℃, the epidermal cells could be harvested. Then flowcytometry and FITCimmunofluorescence for cytokeratin 19 of epidermal cells and FITC-immunofluorescence for vimentin of fibroblast were conducted to identify keratinocyte stem cells in the epidermal cells and fibroblasts in the digestion solution. Moreover, epidermal cells and fibroblasts were cultured in vitro for 7 days to investigate their biological behavior. Results Using collagenase Ⅰ combined with trypsin, epidermal cells andfibroblasts could be isolated at one time within 3 hours. Up to 17% cells demonstrated cytokeratin 19 positive in the epidermal cells, with fibroblast vimentin positive. The amount of fibroblast could be enlarged to more than 100 times within 6 days, but the putative keratinocyte stem cells were difficult for subculture. Conclusion Seed cells for composite tissue engineered skin could be harvested fast at one time, that made it possible to reconstruct composite tissue-engineered skin in vitro.
Objective To find new ways for wound healing and tissue expansion by reviewing of progress in recent years in functional molecules which are used for signaling channels of mechanical stress perception and mechanotransduction of keratinocyte. Methods The domestic and international articles were reviewed to summarize the functional molecules and signaling channels of mechanical stress perception and mechanotransduction of keratinocytes. Results The mechanism of mechanical stress perception includes mechano-sensitive channels, growth factor receptor-mediated mechanical stress perception, and mechanical stress perception by protein deformation. The mechanism of mechanotransduction includes cell adhesion-mediated signaling, mitogen-activated protein kinase signaling, the cytoskeleton and extracellular matrix, and so on. Conclusion Keratinocytes can response to the mechanical stress and transfer the effective information to undergo shaping, migration, proliferation, differentiation, and other biological behavior in order to adjust itself to adapt to the new environment.