This study was conducted to identify the effectiveness of platelet-rich plasma (PRP) and efficacy of intralesional injection as a method of application to acute cutaneous wounds in dogs. Healthy adult beagles (n = 3) were used in this study. Autologous PRP was separated from anticoagulant treated whole blood in three dogs. Cutaneous wounds were created and then treated by intralesional injection of PRP in the experimental group, while they were treated with saline in the control group on days 0, 2 and 4. The healing process was evaluated by gross examination throughout the experimental period and histologic examination on day 7, 14 and 21. In PRP treated wounds, the mean diameter was smaller and the wound closure rate was higher than in the control. Histological study revealed that PRP treated wounds showed more granulation formation and angiogenesis on day 7, and faster epithelialization, more granulation formation and collagen deposition were observed on day 14 than in control wounds. On day 21, collagen deposition and epithelialization were enhanced in PRP treated groups. Overall, PRP application showed beneficial effects in wound healing, and intralesional injection was useful for application of PRP and could be a good therapeutic option for wound management in dogs.
Animals ; Collagen/metabolism ; Dermis/cytology/injuries/physiology ; Dogs ; Epidermis/cytology/injuries/*physiology ; Female ; Granulation Tissue/cytology ; Injections, Intralesional/veterinary ; Male ; Neovascularization, Physiologic ; *Platelet-Rich Plasma ; Regeneration ; Treatment Outcome ; *Wound Healing ; Wounds and Injuries/therapy/*veterinary

OBJECTIVETo investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs.

METHODShECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture day 1, 3, and 5. (4) Cells were collected and divided into non-transfection group and transfection group, with 12 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in transfection group were transfected with Ad-hEGF gene in the optimum MOI (50). The expression levels of cytokeratin 14 (CK14) and CK19 of cells were measured by immunofluorescence staining at TH 24. (5) Cells were collected, grouped, and treated as in (4), with 6 wells in each group. At post scratch hour (PSH) 0 (immediately after scratch), 12, 24, and 48, the migration distance of cells was observed and measured with inverted phase contrast microscope. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, and LSD test.

RESULTS(1) At TH 24 and 48, morphology of cells in each transfection group and non-transfection group were similar. Compared with that in non-transfection group, the cell debris increased significantly in 200 fold transfection group at TH 72. At TH 24, 48, and 72, the expression of green fluorescent protein was not seen in cells of non-transfection group, whereas it increased in cells of transfection group over transfection time. (2) The transfection rate of Ad-hEGF gene of cells in each transfection group increased gradually over transfection time. At TH 72, the transfection rates of Ad-hEGF gene of cells in 50-200 fold transfection groups were all above 90%, while the transfection rates of Ad-hEGF gene of cells in non-transfection group, 5, and 20 fold transfection groups were (0.51±0.20)%, (62.44±6.23)%, and (75.00±5.43)% respectively, which were obviously lower than the rate in 50 fold transfection group [(93.12±2.55)%, with P values below 0.01]. The mass concentration of EGF in culture supernatant of cells in each transfection group increased gradually over transfection time. At TH 72, the mass concentration of EGF in culture supernatant of cells in 50 fold transfection group was obviously higher than that in each of the other groups (with P values below 0.01). The proliferation activity of cells in each group at TH 24 and 48 was similar (with P values above 0.05). At TH 72, the proliferation activity of cells in 200 fold transfection group was obviously lower than that in other groups (with P values below 0.05). (3) On culture day 1, the biological activity of EGF secreted by cells in two groups was similar (P>0.05). On culture day 3 and 5, the biological activity of EGF secreted by cells in transfection group were obviously higher than that in non-transfection group (with P values below 0.01). (4) At TH 24, the expression levels of CK14 and CK19 of cells in transfection group were higher than those in non-transfection group. (5) The width of scratch in two groups was nearly the same at PSH 0. At PSH 12-48, the migration distance of cells in transfection group was obviously longer than that in non-transfection group (with P values below 0.01).

CONCLUSIONSThe suitable range of MOI of hECs transfected with Ad-hEGF gene is 50-150, and 50 is the optimum. hECs transfected with Ad-hEGF gene with MOI 50 can effectively express the EGF gene and keep its good abilities of proliferation, differentiation, and migration, as well.

Adenoviridae ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; EGF Family of Proteins ; genetics ; metabolism ; Epidermis ; cytology ; Genetic Vectors ; Humans ; Keratins ; metabolism ; Male ; Transfection

OBJECTIVETo observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism.

METHODS(1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 μL dimethyl sulfoxide, 1 μL 17β-estradiol (100 nmol/L), 1 μL LY294002 (10 μmol/L), and 1 μL PD98059 (30 μmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 μL 17β-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the protein levels of phosphorylated Akt (p-Akt), phosphorylated ERK (p-ERK), and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one-way analysis of variance, analysis of variance of factorial design, and LSD test.

RESULTS(1) The epidermis thickness of mice in ovariectomized group was (33.5±3.0) μm, which was obviously thinner than that in estrus group [(51.4±3.1) μm, t=20.7, P<0.01]. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovariectomized group was 37±12 per 200 fold visual field, obviously fewer than that in estrus group (96±15 per 200 fold visual field, t=15.3, P<0.01). (2) During PCH 0 to 48, there were no significant differences in the proliferation activity of cells between group PE and group NC (with P values above 0.05). At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously increased (P<0.01). The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups (with P values below 0.01). (3) Compared with that in group NC [(51.6±1.1)%], the PI of cells in group PE was obviously increased [(58.5±0.8)%, P<0.05]. The PI values of cells in groups AI and EI were (34.9±0.8)% and (48.2±0.4)% respectively, both obviously lower than those in the previous two groups (with P values below 0.01). (4) Compared with that of group NC (0.566±0.034), the protein level of p-Akt in cells of group PE was significantly increased (1.048±0.077, P<0.01). Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI (respectively 0.682±0.095 and 0.672±0.019, with P values below 0.01). Compared with that of group NC (0.469±0.013), the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI (respectively 1.064±0.089, 1.010±0.038, 0.778±0.065, with P values below 0.01). The protein level of p-ERK in cells of group EI was obviously lower than that in group PE (P<0.01). Compared with that of group NC (0.386±0.053), the protein level of PCNA was obviously increased in cells of group PE (0.743±0.043, P<0.01). The protein levels of PCNA in cells of groups AI and EI were 0.264±0.019 and 0.223±0.065 respectively, both obviously lower than those in the previous two groups (with P values below 0.01).

CONCLUSIONSLack of estrogen damages the growth ability of epidermis of mice. Estrogen (17β-estradiol) can promote the proliferation of HaCaT cells by increasing the expression of PCNA via activating ERK/Akt signaling pathway.

Animals ; Cell Cycle ; Cell Line ; Cell Proliferation ; drug effects ; Epidermis ; cytology ; drug effects ; growth & development ; Estradiol ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; Signal Transduction

BACKGROUNDTrichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dermatophytosis caused by T. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin ®, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection.

METHODSThe reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation.

RESULTSThe histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of T. rubrum infection in an intuitionistic way.

CONCLUSIONSWe established the T. rubrum infection model on RHE in vitro successfully. It is a promising model for further investigation of the mechanisms involved in T. rubrum infection.

Animals ; Disease Models, Animal ; Epidermis ; microbiology ; Humans ; Keratinocytes ; cytology ; Tissue Culture Techniques ; Trichophyton ; pathogenicity

Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.
Adult ; Cell Proliferation ; Chemokine CXCL12 ; genetics ; Epidermal Cells ; Epidermis ; pathology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; metabolism ; Gene Expression Regulation ; Humans ; Keratinocytes ; cytology ; pathology ; Signal Transduction ; Skin Diseases ; genetics ; pathology

OBJECTIVETo investigate the effect of epidermal stem cells from human hypertrophic scar (HS-ESCs) on the skin wound healing in nude mice.

METHODS40 mice were randomly divided into two groups as experimental group (n = 20) and control group (n = 20). Wounds, 1 cm in diameters, were made on every mouse back. The wounds were treated with HS-ESCs and erythromycin ointment in experimental group, or only with erythromycin ointment in control group. The wound healing was observed during the following 14 days. The expression of collagen-I, collagen-III, epidermal growth factor (EGF), fibroblast growth factor (FGF2) , transforming growth factor (TGFbeta1, and TGFbeta2) were studied.

RESULTSThe wound healing time in the experimental group was (20.8 +/- 0.84) d, which was (25.6 +/- 0.89) d in the control group. HE staining revealed that the extent of vascularization in the experimental group was 11.60 +/- 0.55, while it was 8.04 +/- 0.33 in the control group. Immunochemistry analysis showed the expression of collagen-I, collagen-III, EGF, FGF2, TGFbeta1, and TGFbeta2 in the experimental group were significantly higher, compared with those in control group (P < 0.05).

CONCLUSIONHS-ESCs may promote wound healing through enhancement of the vascularization of the wound tissue and the expression of growth factors.

Animals ; Cicatrix, Hypertrophic ; pathology ; Epidermis ; cytology ; Female ; Humans ; Male ; Mice ; Mice, Nude ; Skin ; injuries ; Stem Cell Transplantation ; Stem Cells ; Wound Healing

OBJECTIVETo observe the changes in expression of microRNA-203 and P63 in human epidermal stem cells and KCs, and to investigate their effects and significance in the epidermal proliferation and differentiation.

METHODS(1) Five normal foreskin tissue specimens were collected from 5 patients by circumcision in Department of Urinary Surgery of the First Affiliated Hospital of Nanchang University from March to June in 2013. Then single cell suspension was obtained by separating epidermis with trypsin digestion method. The cells were divided into quick adherent cells and non-quick adherent cells by type IV collagen differential adherent method. The biological characteristics of cells were observed by inverted phase contrast microscope immediately after isolation and on post culture day (PCD) 3. The expression of CD29, keratin 19, keratin 1, and keratin 10 was identified by immunocytochemical staining. The expression of microRNA-203 and mRNA of P63 was determined by real-time fluorescent quantitative RT-PCR. The protein expression of P63 was determined by Western blotting. Data were processed with t test and Pearson correlation analysis.

RESULTS(1) Immediately after isolation, quick adherent cells were small, round, and dispersed uniformly. On PCD 3, the cells adhered firmly, and they grew in clones. Immediately after isolation, non-quick adherent cells appeared in different shapes and sizes, and dispersed unevenly. On PCD 3, the cells adhered precariously and did not show clonal growth. Quick adherent cells showed positive expression of CD29 and keratin 19, while non-quick adherent cells showed positive expression of keratin 1 and keratin 10. Quick adherent cells were identified as epidermal stem cells, and non-quick adherent cells were identified as KCs. (2)The expression level of microRNA-203 in epidermal stem cells (0.74 ± 0.20) was lower than that in KCs (3.66 ± 0.34, t =16.582, P <0.001). The mRNA expression level of P63 in epidermal stem cells (4. 16 ± 0.28) was higher than that in KCs (2.90 ± 0.39, t =5. 850, P =0.001). The protein expression level of P63 in epidermal stem cells (1.42 ± 0.05) was higher than that in KCs (0.73 ± 0.03, t =26.460, P <0. 001). (3) The expression level of microRNA-203 was in significantly negative correlation with the expression levels of mRNA and protein of P63 (with r values respectively - 0. 94 and -0.98 , P values below 0.05).

CONCLUSIONSThe expression levels of microRNA-203 and P63 in human epidermal stem cells and KCs were significantly different, which might be related to the different characteristics of proliferation and differentiation of the cells.

Cell Differentiation ; Cells, Cultured ; Epidermis ; cytology ; growth & development ; Epithelial Cells ; cytology ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Humans ; Integrin beta1 ; Keratin-10 ; genetics ; metabolism ; Keratin-19 ; genetics ; metabolism ; Keratinocytes ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Stem Cells ; cytology ; metabolism

Based on the research of plant taxonomy and botanical investigation, microscopic characteristics of the root, stem, leaf transverse section and powder of Atropa belladonna were studied for identification of the herb. The research detailed and made clear to the description identification and microscopic characteristics of officinal parts of the herbs. The work provided reference for the identification of A. belladonna herbs and pieces of work in the future, as well as a theoretical basis for the further research, development, medicinal use and the upgrading of quality standards.
Atropa belladonna ; anatomy & histology ; cytology ; Microscopy ; methods ; Plant Epidermis ; anatomy & histology ; cytology ; Plant Leaves ; anatomy & histology ; cytology ; Plant Roots ; anatomy & histology ; cytology ; Plant Stems ; anatomy & histology ; cytology ; Plant Stomata ; anatomy & histology ; cytology ; Plants, Medicinal ; anatomy & histology ; cytology

OBJECTIVETo explore the mechanisms of hair follicle regeneration by injection of follicular cells isolated from murine skin.

METHODSEpidermis was peeled off from the dermis of 3-5 d C57BL/6J mouse by 0.2% Dispase digestion at 37 degrees C for 2 hours. Dermis was cut into small pieces and digested in 0.2% collagenase at 37 degrees C for 30 minute with low speed stirring to isolate hair follicles from dermis. Hair follicles were collected through filtration, low-speed centrifugation and density gradient centrifugation. Collagenase and trypsin were added to digest hair follicles into dissociated cells which were marked by Dio and injected into the nude mouse skin.

RESULTS2 d after intradermal injection of hair follicle cells, a cyst was formed containing lots of round and elliptical cells and homogeneous eosin stained cell-free tissues. The cyst wall was composed of many spindle shaped fibroblast cells and showed sparsely localized green fluorescence. The contents of the cyst showed bright green fluorescence. 4 d after injection, the skin became slightly thicken with grey appearance, a lots of hair follicles formed with black bulb. 1 weeks after injection, the injection site became black and evaluated with a lots of black hair follicles and hyperproliferation of capillary blood. Newly formed hair follicles showed bright green fluorescence. 3 weeks after injection, a cyst containing lots of black hairs formed in the injection site. Newly formed hair follicles showed positive for Dio. Sebaceous gland can be seen accompanied with hair follicles. 6 weeks after injection, the cyst contained lots of sheded club hair shafts and hair follicles on the stage of anagen. Cultured follicular cells and injection below 1 x 10(5) failed to regenerate hairs. While the regenerated hair follicle was few when the hair follicle cells were injected subcutaneously.

CONCLUSIONSFollicular cells can aggregate spontaneously and develop synergistically into hair follicles with normal growth cycle after implantation. The regeneration depends on the interactions between follicular cells, as well as on the recipient sites and cell numbers.

Alopecia ; surgery ; Animals ; Cell Transplantation ; methods ; Dermis ; cytology ; Epidermis ; cytology ; Hair Follicle ; cytology ; Injections, Intradermal ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Regeneration ; Skin ; cytology ; Tissue Engineering ; methods

OBJECTIVETo investigate the feasibility of differentiation of human induced pluripotent stem cells (iPSCs) into epidermal-like stem cells.

METHODS(1) Human strain of iPSCs were plated on-to trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase IV digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase (AKP). iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. (2) Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin beta1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining.

RESULTSHuman iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positive. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin beta1 and CK19 positive. Most of the cells died, and no integrin beta1 and CK19 positive cells were found in control group.

CONCLUSIONSHuman iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane induction, and it lays an experimental basis for providing new source of seed cells of skin tissue engineering.

Animals ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Epidermis ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Mice