A specialized tissue type, the keratinizing epithelium, protects terrestrial mammals from water loss and noxious physical, chemical and mechanical insults. This barrier between the body and the environment is constantly maintained by reproduction of inner living epidermal keratinocytes which undergo a process of terminal differentiation and then migrate to the surface as interlocking layers of dead stratum corneum cells. These cells provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. Much of this barrier function is provided by the cornified cell envelope (CE), an extremely tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the dead cornified cells. It consists of two parts: a protein envelope and a lipid envelope. The protein envelope is thought to contribute to the biomechanical properties of the CE as a result of cross-linking of specialized CE structural proteins by both disulfide bonds and N(epsilon)-(gamma-glutamyl)lysine isopeptide bonds formed by transglutaminases. Some of the structural proteins involved include involucrin, loricrin, small proline rich proteins, keratin intermediate filaments, elafin, cystatin A, and desmosomal proteins. The lipid envelope is located on the exterior of and covalently attached by ester bonds to the protein envelope and consists of a monomolecular layer of omega-hydroxyceramides. These not only serve of provide a Teflon-like coating to the cell, but also interdigitate with the intercellular lipid lamellae perhaps in a Velcro-like fashion. In fact the CE is a common feature of all stratified squamous epithelia, although its precise composition, structure and barrier function requirements vary widely between epithelia. Recent work has shown that a number of diseases which display defective epidermal barrier function, generically known as ichthyoses, are the result of genetic defects of the synthesis of either CE proteins, the transglutaminase 1 cross-linking enzyme, or defective metabolism of skin lipids.
Animal ; Cell Membrane/metabolism ; Epidermis/metabolism* ; Epidermis/chemistry* ; Human ; Ichthyosis/metabolism ; Ichthyosis/genetics ; Keratinocytes/metabolism* ; Keratinocytes/chemistry ; Membrane Lipids/metabolism* ; Membrane Proteins/metabolism* ; Protein-Glutamine gamma-Glutamyltransferase/metabolism

BACKGROUND: In our previous report, we observed the increaseed epidermal glucose concentrations and decreased hexokinase actiuities of diabetic patients which were ciimpared to those of normal individuals. And we considered that, there were some derrangement of lipid metabolism and glycolysis of diabetic epidermis. OBJECTIVES: This study wns planed to prove the above possible changes of lipid metabolism and glycolysis of diabetic epidermis. METHODS: The epidermal enzymatic activties of glucose-6-phophate dehydrogenase(G6PDH), phosphofructokinase(PFK), 1-glycerophosphate dehydrogenase(GOPDH) and b-hydroxybutyryl CoA dehydrogenase(HBDH) were assayed in the skin samples obtained friom diabetic patients and normal individuals by the fluorometric: method. RESULTS: Teh epidermal PFK activities of diabetic patients were decreased(3.49+1.35(mmole/hr/kg dry weight)) compared to that of normal individuals(5.00+0.56(mmcle/hr/kg dry weight))(p<0.05). The epidermal HBDH activities of diabetic patients were decreised(0.28+0.10(mole/hr/kg dry weight)) compared to that of normal individuals(0.49+0.20(mole/hr/kg dry weight)(p<0.01). The mean epidermal G6PDH activity of diabetic patients was decreasec. compased to that of normal individuals, but there was no statisical significance. The mean epidermal 3OPDH activittes of diabetic patients and normal individual; showed no significant difference. CONCLUSION: We consider that the decreased epidermal HBDH actiities of diabetic patients can decrease keton body formatiori, and the abnormal glycolysis can exist in the diabetic epidermis because the decreased enzymatic activities of diabetic epidermal PFK may decrease the velocity of glycolysis.
Diabetes Mellitus ; Epidermis ; Glucose ; Glycolysis ; Hexokinase ; Humans ; Lipid Metabolism ; Skin

OBJECTIVETo study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo.

METHODSA three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohistochemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 microL per wound), SDF-1 group (treated with 100 ng/mL SDF-1, 50 microL per wound), and AMD3100 group [treated with 100 ng/mL AMD3100 (50 microL per wound) for 30 minutes, and then SDF-1 50 microL was added per wound]. The redistribution of ESC around wound was observed.

RESULTSThe expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin beta(1)-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis.

CONCLUSIONSSDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.

Cell Movement ; Chemokine CXCL12 ; metabolism ; Epidermis ; cytology ; Frostbite ; metabolism ; therapy ; Humans ; Stem Cells ; cytology ; Wound Healing

OBJECTIVETo explore the differences of PDGF and EGF expression in the wound healing between fatal and adult.

METHODSWith the established animal model of fetal scarless healing and the adult samples, an immunohistochemical technique was used to evaluate the expression of PDGF and EGF in the normal adult skin, normal fetal skin, and the process of their wound healing.

RESULTS1. The expression of the PDGF was not found in the fetal skin, but a mild amount of the PDGF was shown in the epidermis and the upper dermal layer 12 hours and 1 day after the wounding process. In the normal adult skin, expression of PDGF was shown in the dermal fibroblasts, macrophagocytes and blood capillaries, and a strong expression was presented during its wound healing process. 2. In the fetal skin, the expression of the EGF was seen in the epidermis, hair follicles, sebaceous glands and sweat glands, but there were no markedly changes during the wound healing. In the adult skin, a positive stain of the EGF was shown in the basal layer of the epidermis while the mild stain in hair follicles and sweat glands. The level of the expression became gradually decreasing with the time going in the wounded adult skin.

CONCLUSIONThe different expression of growth factors between fetal and adult skin in wound healing may be one of the important reasons that the fetal wound could produce scarless healing.

Adult ; Epidermal Growth Factor ; metabolism ; Epidermis ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Hair Follicle ; metabolism ; Humans ; Platelet-Derived Growth Factor ; metabolism ; Skin ; injuries ; metabolism ; Sweat Glands ; metabolism ; Wound Healing

OBJECTIVETo establish and optimize the two dimensional gel electrophoresis (2-DE) map of epidermal cells separated from the hypertrophic scar tissues so as to explore the function of the non cultured epidermal cells during the course of the formation of hypertrophic scar.

METHODSTo separate the epidermal cells from the scar tissues, the scar epidermis was digested with Dispase II and trypsin. The total protein of the cells was then extracted and separated with 2-DE and visualized with silver stain. Spots detection and matching were performed with Melaine 3.0 gels analyzing software. The results were then compared with the normal epidermal cells' 2-DE map coming from the Danish Center for Human Genome Research' s 2-DE PAGE Databases.

RESULTSNearly more than 600 protein spots were identified in the final optimized 2-DE map. We found out 24 differentially expressed proteins by comparing the difference in composition, shape or density of all the spots. In the 24 proteins, there are 8 up-regulated ones, 9 down-regulated ones, 4 disappeared ones and 3 newly founded ones.

CONCLUSIONSThe method of digesting the epidermis with Dispase II and trypsin to separate the epidermal cells to establish the 2-DE map is feasible and it made the further study on hypertrophic scar proteomics possible. The 24 differentially expressed proteins revealed that epidermal cells might play a role in the formation of hypertrophic scar.

Cell Line ; Child ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; methods ; Epidermis ; metabolism ; Humans ; Proteome ; metabolism

AIMTo illustrate the non-equilibrium properties of drug permeation through stratum corneum (SC) to provide theory and method for transdermal drug delivery.

METHODSThe system of side-by-side permeation chambers in vitro was isolated, thus conditions for equilibrium state were decided. And a network thermodynamic model was established. Non-equilibrium and leakage experiments were completed with which tinidazole was pattern drug.

RESULTSThe properties of non-equilibrium including: long term to reach equilibrium state; delay-start of the permeation; uncertainty of initial drug permeable flux. The properties of leakage including: degression of drug permeable flux; changeability of permeation time constant.

CONCLUSIONThe permeation cell is believed to be a non-linear and time variable system.

Administration, Cutaneous ; Electroporation ; Epidermis ; metabolism ; Humans ; In Vitro Techniques ; Permeability ; Tinidazole ; administration & dosage ; pharmacokinetics

PURPOSE: Ultraviolet (UV) irradiation decreases epidermal hydration, which is maintained by reduction of natural moisturizing factors (NMFs). Among various NMFs, free amino acids (AA) are major constituents generated by filaggrin degradation. This experiment was conducted to determine whether or not dietary supplementation of green tea extract (GTE) in UV-irradiated mice can improve epidermal levels of hydration, filaggrin, free AAs, and peptidylarginine deiminase-3 (PAD3) expression (an enzyme involved in filaggrin degradation). METHODS: Hairless mice were fed a diet of 1% GTE for 10 weeks in parallel with UV irradiation (group UV+1%GTE). As controls, hairless mice were fed a control diet in parallel with (group UV+) or without (group UV-) UV irradiation. RESULTS: In group UV+, epidermal levels of hydration and filaggrin were lower than those in group UV-; these levels increased in group UV+1% GTE to levels similar to group UV-. Epidermal levels of PAD3 and major AAs of NMF, alanine, glycine and serine were similar in groups UV- and UV+, whereas these levels highly increased in group UV+1% GTE. CONCLUSION: Dietary GTE improves epidermal hydration by filaggrin generation and degradation into AAs.
Alanine ; Amino Acids ; Animals ; Diet ; Dietary Supplements ; Epidermis* ; Glycine ; Metabolism* ; Mice ; Mice, Hairless* ; Serine ; Tea*

OBJECTIVETo investigate the abnormalities of human epiderm in keloids and hypertrophic scars.

METHODSBiopsies from ten untreated keloids (duration of disease 3 - 30 years) and ten hypertrophic scars (duration of disease 6 - 10 months) and five normal adult skin specimens. Total RNA was isolated from normal adult skin. A cDNA fragment (base 5941 - 6481 bp) of the full-length human Tenascin-C cDNA was synthesized by polymerase chain reaction and subcloned in pGEM-T-easy. Dioxigen-labeled anti-sense and sense probes were synthesized by using a Sp6/T7 in vitro RNA synthesis kit in the present of Dig-UTP. In situ hybridization was performed on 4% paraformaldehyde-fixed and wax-embedded sections of keloids and hypertrophic scars. NBT-NCIP was used in color detection. Immunohistochemical procedure. The sections were incubated with antibodies (anti-Tenascin-C, anti-CK-16 and anti-Ki-67). Ultrasensitive Streptavidin Peroxidase staining was performed following established procedures.

RESULTSThe study show that the proliferation of epidermal keratinocytes in keloids and hypertrophic scars is very clear. The expressions of Tenascin-C mRNA in keloids epidermal keratinocytes markedly increased in contrast with epidermal keratinocytes of hypertrophic scars and adult skin. The CK-16 and Ki-67 stainings significantly enhanced in the epidermal keratinocytes of keloids and hypertrophic scars.

CONCLUSIONSThe different expressions of Tenascin-C, CK-16 and Ki-67 among normal adult skin, keloids and hypertrophic scars show the abnormalities of epidermal keratinocytes proliferation and differentiation in keloids and hypertrophic scars.

Cicatrix, Hypertrophic ; metabolism ; pathology ; Epidermis ; metabolism ; pathology ; Female ; Humans ; Hyperplasia ; Immunohistochemistry ; In Situ Hybridization ; Keloid ; metabolism ; pathology ; Keratins ; genetics ; metabolism ; Ki-67 Antigen ; genetics ; metabolism ; Male ; Tenascin ; genetics ; metabolism

It is well estabilshed that the xerosis is closely related to diabeic pruritus. Although the causes of xerosis are thought to bie the abnormalities of sweating and autonomic nervous system, the exact mechanism of the xerosis in diabetic skin is still unknown. This study was designed to investigate the possible derangement of glucose metabolism in the skin of diabetes mellitus patients with xerosis. The epidemal glucose content and hexokonase activities were masured in the skin samples obtained from normal individuals and diabetes mellitus patients with xerosis The epidermal glucose content was measured by the enzymatic cycling method. The enzymatic activities of hexokinase were assayed by fluorometric method. The epidermal glucose content of diabetic patients increaed approximately twice [27.46+9.52 (mmole/kg /dry weight) that of normal individuals [13.90+4.79(mmole/kg /dry weight)] (p<0. 0001). The epidermal hexokinase activities of diabetes patients were significantly decreased [0.56+0.15(mole/hr/kg/dry weight)] compared to that of normal indivduals [0.96+0.24(mole/hr/kg dry weight)] (p<0.0001). There were no significant differences in the epidermal glucose content and enzyme activities of hexokinase between the diabetic patients with: erosis and diabetic patients without xerosis. These data indicated that decreased activities of hexokinase could reduce the glucose phopkiorylation and uptake into keratinocytes, and which could lead to accumulat. glucose in the interstitial space of diabetic epidermis. And the decreased hexokinase activities may exert on lipid metabolism and glycolysis of diabetic epiidermis, because hexokinase is a key enzyme of hexose monophosphate pathway and glycolysis.
Autonomic Nervous System ; Diabetes Mellitus ; Epidermis ; Glucose ; Glycolysis ; Hexokinase* ; Humans ; Keratinocytes ; Lipid Metabolism ; Metabolism ; Pruritus ; Skin ; Sweat ; Sweating

OBJECTIVETo study the effect of calcium on the activity and protein expression of integrin beta1 promoter in human immortal keratinocyte colony HaCaT cell and cell migration.

METHODS(1) HaCaT cells were cultured in vitro (12-slot plate) and divided into 5 groups according to the random number table, with 18 slots in each group: reporter plasmid pGL3 promoter (positive control group, PC), pGL3 empty vector (negative control group, NC), pGL3-1756 bp (total length promoter group, TL), pGL3-1442 bp (distal promoter group, D), and pGL3-261 bp (proximal promoter group, P) was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0.00, 0.03, 0.09, 0.30, 0.80, or 1.20 mmol/L calcium (3 slots in each group with each concentration). Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. (2) HaCaT cells steadily transfected with small interfering RNA-integrin beta1 vector (steadily transfected in brief) constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin beta1 protein was determined with Western blot. (3) Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media (divided according to the random number table, with 3 slots of cells for each concentration) for 12 hours after scratch test. Cell migration rate was observed and determined. (4) Data were processed with one-way analysis of variance and independent samples t test.

RESULTS(1) The luciferase activity of cells in TL group increased from 0.16+/-0.09 to 0.39+/-0.09 and 0.35+/-0.05 (with t value respectively 3.143, 3.140, P values all below 0.05) as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0.30 mmol/L, and it decreased as calcium concentration increased to 0.80 and 1.20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity (0.56+/-0.32, 0.64+/-0.06) at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group (with t value respectively 0.887, 6.122, P values all below 0.05). There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. (2) The expression amount of integrin beta1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium (0.58+/-0.09, 1.40+/-0.29, 1.41+/-0.09, 0.99+/-0.10, 1.16+/-0.15) were all increased as compared with that cultured with 0.00 mmol/L calcium (0.53+/-0.10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6.199, P values all below 0.05). (3) Migration speed of normal HaCaT cells cultured with 0.09, 0.30 mmol/L calcium increased obviously as compared with that cultured with 0.00 mmol/L calcium, and it slowed down when cultured with 0.80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium.

CONCLUSIONSDistal promoter region of integrin beta1 plays a vital role in regulating integrin beta1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin beta1 promoter and cell migration.

Calcium ; pharmacology ; Cell Line ; Cell Movement ; drug effects ; Epidermis ; cytology ; metabolism ; Humans ; Integrin beta1 ; metabolism ; Promoter Regions, Genetic ; Transfection