[Construction of keratinocyte growth factor phage active peptides for the promotion of epidermal cell proliferation].

OBJECTIVE: To construct and display the keratinocyte growth factor (KGF) phage active peptides so as to detect the promoting effects of epidermal cell. METHODS: KGF sequences were chosen and their primers were designed. The selected genes of P1, P2 and P4 were obtained by reverse transcription (RT)-PCR. P3 was obtained by direct synthesis. And the KGF genes were subcloned into pComb3 vector. The technique of phage display was employed to display the genes on phage surface. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the promoting effects of KGF phage active peptides on the proliferation of epidermal cell. Optical density (A) was determined at 570 nm. Immunofluorescent assay was employed to evaluate the cell affinity of KGF phage active peptides. RESULTS: The four KGF genes were obtained and subcloned into pComb3 vector. The proteins of the KGF genes were expressed on the surface of the pComb3 vector. The MTT data of optical density (A) showed that significant differences existed between the negative control and KGF control (0.293 ± 0.017 vs 0.520 ± 0.043) and KGF phage active peptide groups (0.293 ± 0.017 vs 0.469 ± 0.057, 0.441 ± 0.048, 0.438 ± 0.035, 0.446 ± 0.037) (all P < 0.01). The results of immunofluorescent assay indicated that KGF and KGF phage active peptides had excellent cell affinity. CONCLUSION: KGF phage active peptides are successfully constructed and displayed and they may promote the proliferation of epidermal cell.

[Study of TGF-ß1 phage model peptides on inhibiting keloid fibroblasts proliferation].

OBJECTIVE: To isolate the transforming growth factor-beta 1 (TGF-ß1) phage model peptides from phage 12-mer display peptide library to inhibit the proliferation of keloid fibroblasts. METHODS: The phage display 12-mer peptide library was screened for 4 rounds with monoclonal anti-human TGF-ß1 as the target to yield the specific phage model peptides. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for the quantitative determination of cellular proliferation. Apoptosis was detected by the Annexin V-FITC/PI apoptosis detection kit and the cells were analyzed with flow cytometry. Immunofluorescent assay was employed to show the binding affinity of model peptides for keloid fibroblasts. Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expressions of nuclear factor kappa B (NF-κB) and connective tissue growth factor (CTGF). RESULTS: Ten phage model peptides were obtained and they were similar to TGF-ß1, TGF-ß2, TGF-ß receptor II (TßRII), TGF-ß-induced factor, NF-κB or mitogen-activated protein kinase (MAPK). The results of MTT showed that four phage model peptides (No. 7 - 10) could inhibit the proliferation of keloid fibroblasts (P < 0.05). The results of apoptotic assessment showed that phage model peptides (No. 7 - 10) could slightly trigger the late apoptotic stage of keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the relative expression of NF-κB decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.28, 0.26, 0.46 and 0.30 respectively versus the negative control group. The relative expression of CTGF decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.26, 0.60, 0.34 and 0.17 respectively versus the negative control group. CONCLUSION: Four phage model peptides (No. 7 - 10) isolated from phage display 12-mer peptide library can inhibit the proliferation of keloid fibroblasts via regulating the expressions of NF-κB and CTGF.

[The correlation of the fetal cytokeratin expressing in epidermal cells and the different outcomes of wound repairing].

OBJECTIVE: To investigate the changing regular of specific cytokeratin (CK) markers expressing in human pseudoepitheliomatous hyperplasia (PEH), keloids (Ke) and hypertrophic scar (HS) lesion, and to explore the correlation between such changes and the different outcomes of wound repair. METHODS: Histopathology and immunohistochemistry (IHC) double staining methods were used in samples of human PEH, Ke, HS and NS to determine the distribution characteristics and changing regularity of CKs in epidermal tissues. RESULTS: No CK8&18 and CK17 expressed in epidermis of NS group, while CK8&18(+) cells and CK17(+) cells were detected in epidermis of active-stage Ke, HS and PEH. The quantities of CK8&18(+) cells and CK17(+) cells ranked as follows: PEH > Ke > HS and HS > Ke > PEH (P < 0.05). CK19(+) cells and CK5&6(+) cells expressed similar changing trend, while reverse trend of CK10(+) cells was detected in epidermal cells, with local epidermal hyperplasia, cells morphological changes and sub-epidermal inflammatory reaction. CONCLUSION: Different degree of de-differentiation and terminal differentiation imbalance are found in epidermal cells of active-stage PEH, Ke and HS, which hint the correlation between the abnormal proliferation and differentiation of epidermal cells and the different outcomes of wound repair.

[Investigation of mesenchymal-epithelial transdifferentiation in the morphogenesis mechanism of embryonic epidermic cells].

OBJECTIVE: To study the relationship between the morphologic mechanism of human embryonic epidermic cells and mesenchymal-epithelial transformation (MET) and its modulation factor. METHODS: Morphological occurrence of epidermis was detected with histologic methods in earlier period [estimated gestational age (EGA) 6-14 weeks] human embryonic skin samples. At the same time, the characteristic expression and their distribution markers of mesenchymal cells [vimentin and alpha-smooth muscle actin (alpha-SMA)], embryonic specific epidermic protein CK8&18, specific protein of epidermic stem cell CK19, transforming growth factor-beta1) (TGF-beta1) and its receptor (TGFbetaRI) in embryonic epidermis were examined with immunohistochemistry and indirect-immunofluorescent doble-labelling method. RESULTS: During EAG 6-8 weeks, ectodermal cells containing Vim+/alpha-SMA(-) were found to transform into epidermal stem cells with CK8&18+/CK19+. In ectodermal cells, protein expression density of TGFbetaRI was moderate (+ +), while positive signal of TGFbeta1 was weak (+/-). After EGA10 weeks, epidermal cells showed typical morphological characteristics. CONCLUSIONS: At EGA 6-8 weeks, human embryonic skin epidermal cells began to form through MET, in which the signal pathway mediated by TGFbetaRI might play important roles, but the role of TGFbeta1 need to be further studied.

Rib Cartilage Assessment Relative to the Healthy Ear in Young Children with Microtia Guiding Operative Timing.

BACKGROUND: The optimal age at which to initiate for auricular reconstruction is controversial. Rib cartilage growth is closely related to age and determines the feasibility and outcomes of auricular reconstruction. We developed a method to guide the timing of auricular reconstruction in children with microtia ranging in age from 5 to 10 years. METHODS: Rib cartilage and the healthy ear were assessed using low-dose multi-slice computed tomography. The lengths of the eighth rib cartilage and the helix of the healthy ear (from the helical crus to the joint of the helix and the earlobe) were measured. Surgery was performed when the two lengths were approximately equal. RESULTS: The preoperative eighth rib measurements significantly correlated with the intraoperative measurements (P < 0.05). From 5 to 10 years of age, eighth rib growth was not linear. In 76 (62.8%) of 121 patients, the eighth rib length was approximately equal to the helix length in the healthy ear; satisfactory outcomes were achieved in these patients. In 18 (14.9%) patients, the eighth rib was slightly shorter than the helix, helix fabrication was accomplished by adjusting the length of the helical crus of stent, and satisfactory outcomes were also achieved. Acceptable outcomes were achieved in 17 (14.0%) patients in whom helix fabrication was accomplished by cartilage splicing. In 9 (7.4%) patients with insufficient rib cartilage length, the operation was delayed. In one (0.8%) patient with insufficient rib cartilage length, which left no cartilage for helix splicing, the result was unsatisfactory. CONCLUSIONS: Eighth rib cartilage growth is variable. Rib cartilage assessment relative to the healthy ear can guide auricular reconstruction and personalize treatment in young patients with microtia.

[Clinical significance of detection of serum hyaluronic acid concentration in severe burn patients].

OBJECTIVE: To study the relationship between the changes in serum hyaluronic acid (HA) concentrations and injury of hepatocytes in severe burn patients. METHODS: Radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) were used to detect HA and tumor necrosis factor-alpha(TNF-alpha) concentrations in serum, and biochemical indexes for hepatic function and renal function in burn patients that were divided into different groups: systemic inflammatory response syndrome(SIRS), multiple organ dysfunction syndrome(MODS). Multiple organ failure(MOF) and non-surviving patient groups. RESULTS: In two weeks after burns, the serum HA concentration attained a high level. With the increase of severity of disease, the values of the serum HA concentration were elevated progressively (P<0.001). At the onset of MOF till death, the serum HA content was rapidly decreased. Lineal correlation analysis indicated that the change in the serum HA concentration was closely correlated with SIRS, burn sepsis, MODS and death (P<0.01). It was positively correlated with TNF-alpha, aspartate aminotransferase (AST) and total bilirubin (P<0.05 or P<0.01), and negatively correlated with plasma albumin and albumin/globulin(both P<0.05). CONCLUSION: The increment in serum HA levels is relate with the impairment of the hepatic endothelial cells due to TNF-alpha etc. The HA content in serum can be used as a sensitive indicator for judging injury of liver, seriousness of the injury, and prognosis of the patient.

[Analysis of etiological factors of pseudo-epitheliomatous granuloma after cutaneous wound healing: a report of 11 cases of patients with this lesion].

OBJECTIVE: To explore the pathogenetic mechanism, clinical and pathological characteristics, and prevention and treatment of pseudo-epitheliomatous granuloma after skin wound healing. METHODS: The clinical information and the treatment results of pseudo-epitheliomatous granuloma occurring in 11 patients (age 1-67 years) were reviewed and analyzed, their tissue specimens were used for microbial examination and histological observation. RESULTS: Some bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus hemolyticus B, and Streptococcus faecalis could be found in the culture of pseudo-epitheliomatous granuloma. The maJority of these bacteria was tolerant to celbenin but sensitive to vancomycin. The lesions were excised, and the wounds were covered with skin grafting or skin flap, supplemented by local of vancomycin to prevent recurrence of the lesion. Histological examination revealed in pseudo-epitheliomatous granuloma with long epithelial peduncle encapsulating granulation tissue like honeycomb, in which there were many capillaries, macrophages, lymphocytes and mast cells, with only a small amount of extracellular matrix. CONCLUSION: The main pathogenesis of pseudo-epitheliomatous granuloma formation might be improper treatment of wound in earlier period and infection of drug resistant bacteria. Surgical debridement followed by skin coverage and local inJection of vancomycin could be effective and curative.

[Relationship between epithelial-immunologic cells transdifferentiation and pseudoepitheliomatous granuloma lesion].

OBJECTIVE: Inappropriate treatment at early stage of wound could result in the formation of pseudoepitheliomatous granuloma (PEG). The correlation of abnormal transdifferentiation of epithelial cells to immunologic cells and the occurrence of PEG lesion was investigated. METHODS: Morphological change of epithelial tissue was observed with histopathology in 11 specimens of PEG lesions and 6 specimens of normal skins from PEG edge (PEG-N) from 11 patients with damaged skin. The expression characteristics and distribution of pan-cytokeratin (CKp), IV type collagen, laminin (LM), epithelial cadherin (E-Cad), beta-catenin (beta-Cat), focal adhesion kinase (FAK), stem cell factor (SCF) and its receptor-c-Kit, proliferating cell nuclear antigen(PCNA), and cluster of differentiation-14 (CD14), CD68 and mast cell tryptase (MCT) in PEG were detected with the immunohistochemical and the indirect immunofluorescent double-staining. RESULTS: In comparison with PEG-N, epithelial tissue take on squamous metaplasia, and stroma was infiltrated with intensive microvessels and inflammatory cells in the PEG lesion. Poor epithelial basal layer constitution, basal polarization, and migration of basal cells to stroma could be observed. In the ultrastructure, the loose intercellular junction of basal cells and the increased nucleus/cytoplasm ratio and intercellular space could be observed, neonatal monocytoid cells and macrophages and mast cells as a exuviate-like manner brooded from cytoplasm of original epithelial cells and basement membrane. protein expression of CKp and E-Cad by basal cells was significantly decreased, and the IV type collagen and LM protein could not be found in basement membrane of identical locus. By contrast, the immunoreactivity of beta-Cat and FAK was apparently increased. In addition, CD14(+) monocytes, CD68(+) macrophages, MCT(+) mast cells and CD68(+)/MCT(+) cells with various size, and these cells of stronger immuno-staining of SCF, c-Kit and PCNA antigen could be found in epithelial tissue and stroma. CONCLUSION: Epithelial cells in PEG related to wound are characteristized by transdifferentiation of epithelial cells to immunologic cells, wich may be associated with local infectious and inflammatory reaction, ultimately resulting in enhancement the ratio of beta-Cat/E-Cad signal and activation SCF-c-Kit signal pathway. The phenomena of transdifferentiation epithelial cells in the PEG lesion will help to recognize of the neoplatic immune and trauma repair mechanism.

[Study of mechanism on loss of some components from basement membrane in epithelial-interstitial junction in cutaneous pseudoepitheliomatous hyperplasia lesion].

OBJECTIVE: To investigate the relationship between the formation of pseudoepitheliomatous hyperplasia (PEH) and the mechanism of loss of some components from the basement membrane in epithelial interstitial junction (EIJ) in the treatment of cutaneous wounds, when formation of PEH lesion was induced. METHODS: Morphological change in epithelial tissue was observed with histopathologic method and electronic microscopy in 11 specimens of PEH lesions and 6 specimens of normal skin adjacent to PEH (PEH-N) from 11 patients with injured skin. The expression characteristics and distribution of pan-cytokeratin (p-CK), matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9, proliferating cell nuclear antigen (PCNA), epithelial cadherin(E-Cad) and beta-catenin(beta-Cat) in EMJ were detected with immunohistochemical methods. RESULTS: Epithelial cells expressing P-CK presented squamous epithelialization and extended into deep layer of mesenchyma. In epithelium-mesenchyma junction, where IV type collagen and laminin were weakly expressed, the protein contents of p-CK, E-Cad, MMP-2, MMP-3 MMP-9 were decreased, whilst the immunochemical staining of beta-Cat and PCNA was apparently increased. In the junction, epithelial basal cells were observed to migrate and to depart from basal membrane; epithelial islands and isolated epithelial cells expressing p-CK in mesenchyma could be observed. Ultrastructural observation revealed deformation of epithelial basal cells, increment of nucleus/cytoplasm ratio, loosened intercellular junctions, decrement of electronic density of BM and derangement of BM structure could be observed. CONCLUSION: Reduction in the capability of epithelial basal cells adhesion, differentiation and formation of basement membrane and cytokeratin in PEH associated with wound may be the crucial cause which controls epithelial cells migration into mesenchyma. That the contents of ColIV and LN were decreased may not be associated with MMPs, but with enhancement of the ratio of beta-Cat/E-Cad signal might be the important mechanism of dedifferentiation of epithelial basal cells and the loss of ability of structure formation and cellular migration.

[Expression characteristics of transforming growth factor-beta1 in human skin at different development stages of gestation].

OBJECTIVE: To investigate gene expression of transforming growth factor-beta(1) (TGF-beta(1)) and its two upstream signalling factors (smad2 and smad3) in fetal skin at different gestational ages and postnatal skin and its potential biological significance. METHODS: Fetal skin samples of human embryo were obtained from spontaneous abortions at different gestational ages ranging from 13 to 32 weeks, and also skin collected from patients undergoing plastic surgery. After morphological characteristics of skin at different developmental stages were examined histologically, gene expressions of TGF-beta(1), smad2 and smad3 in skin specimens at different developmental stages were examined with reverse transcription-polymerase chain reaction analysis (RT-PCR). RESULTS: Gene expression of TGF-beta(1), smad2 and smad3 could all be detected in fetal skin and skin after birth. In skin from early gestational fetus, gene expressions of TGF-beta(1) and smad2 were weak. Along with advance in gestational age, gene expression of these two genes in skin became progressively stronger. In skin from late gestational fetus and skin after birth, the transcription contents of these two genes were significantly increased compared with early gestation fetus (P<0.05). On the contrary, gene expression of smad3 was apparently higher in younger fetal skin versus elder compared with that of late fetal skin (P<0.05). In skins after birth, the levels of smad3 gene expression were elevated to the level similar to that in early gestational fetal skin. CONCLUSION: The signal pathway mediated by TGF-beta(1) might be involved in regulating development of the skin at embryonic stage and in designating cetaceous structure and function, and also in wound healing after birth. The relative lack in expression of TGF-beta(1) and smad2 genes in skins from younger fetuses might contribute to fetal scar-less healing, in which the role of smad3 needs to be further investigated.

Keratinocyte growth factor phage model peptides can promote human oral mucosal epithelial cell proliferation.

OBJECTIVE: The objective of this study was to find keratinocyte growth factor (KGF) mimic peptides by a phage display library screening and to analyze their effects on proliferation of human oral mucosal epithelial cells (HOMECs). STUDY DESIGN: A phage display library was screened by anti-KGF antibody. ELISA was performed to select monoclonal phages with higher binding activity. The promotion of the phage model peptides on HOMEC proliferation were analyzed by MTT and their cell affinities were confirmed by immunofluorescence assay. Their effect on KGFR, human beta-defensin 3, c-Fos, and c-Jun in HOMEC were analyzed by quantitative real-time PCR. RESULTS: Two model peptides with higher affinity with HOMEC were found to have promotive activity on cell proliferation, similar to that of KGF. These 2 model peptides have no KGF-like promotion effect on the expression of c-Fos and c-Jun. CONCLUSIONS: The 2 phage model peptides can promote the proliferation of HOMEC in vitro without tumorigenic effects, which suggests their possible usages in oral mucosal wound healing.

[Advances in the research of the biological activities of degradation products of extracellular matrix].

ECM is a supporting structure for stabilizing the location of cells and preserving the structure of tissues. Recently, it has been discovered that ECM and its degradation products may exert profound influences on tissues and cells, such as activities of inflammatory cells and immune cells. Angiogenesis may be stimulated or inhibited by degradation products of ECM. Matrikines, liberated by partial proteolysis of ECM macromolecules, are found to regulate cell functional activities and play a significant role in wound healing or tumor invasion. Post-burn denatured dermal matrix is being studied in burn healing now. The study of post-burn denatured or necrotic dermal matrix should be emphasized in future.

[Experimental study on the recycling of denatured acellular dermal matrix after burn].

OBJECTIVE: To explore the feasibility of burn denatured acellular dermal matrix (DADM) as dermal substitute in repairing wounds. METHODS: (1) Nine Wistar rats received a deep partial-thickness scald on the back. Full-thickness wounded skin was collected on post scald day (PBD) 1, 2, and 3 (with 3 rats at each time point), and it was treated with 2.5 g/L trypsin/0.5% Triton X-100 to remove cells to prepare DADM, respectively called DADM-1 d, DADM-2 d, and DADM-3 d. Another 3 rats without scald injury were treated with the same method as above to prepare acellular dermal matrix (ADM) to serve as control. Gross and histological observations and microbiological and biomechanical tests, including ultimate tensile strength, maximum tension, stretched length at breaking, stress-strain relationship, were conducted for the resulting ADM and DADM. (2) Another 64 rats were divided into ADM group and DADM-1 d, DADM-2 d, and DADM-3 d groups according to the random number table, with 16 rats in each group. A skin flap in size of 2.0 cm×1.8 cm was raised on the back of each rat. The above-mentioned ADM, DADM-1 d, DADM-2 d, and DADM-3 d were cut into pieces in the size of 1.8 cm×1.5 cm, and they were respectively implanted under the skin flaps of rats in corresponding group. At post surgery week (PSW) 1, 3, 5, or 9, 4 rats in each group were used to observe wound healing condition and change in implants with naked eye, and histological observation of the implants was conducted. Data were processed with one-way analysis of variance and t test. RESULTS: (1) The freshly prepared DADM was milky white, soft in texture with flexibility, but poor in elasticity as compared with ADM. No epithelial structure or cellular component was observed in ADM or DADM under light microscope. Collagen fibers of DADM were seen to be thickened unevenly and arranged in disorder and eosinophilic. All microbiological results of DADM were negative. There was no statistically significant difference among DADM-1 d, DADM-2 d, and DADM-3 d in levels of ultimate tensile strength, maximum tension, stretched length at breaking, and stress-strain relationship (with F values from 0.088 to 3.591, P values all above 0.05). Values of the above-mentioned four indexes were the highest in DADM-3 d, they were respectively (13.0 ± 2.4) MPa, (61 ± 4) N, (173 ± 7)%, (45.7 ± 2.0)%. Values of the four indexes of ADM were respectively (19.0 ± 2.6) MPa, (95 ± 4) N, (201 ± 5)%, (62.5 ± 2.2)%, which were higher than those of DADM-1 d, DADM-2 d, and DADM-3 d (with t values from 6.424 to 17.125, P values all below 0.01). (2) No exudate or swelling in the wounds of rats, and no contraction or curling of implants were observed in every group at PSW 1, but inflammatory cells infiltration and Fbs inward migration were observed in the wound. At PSW 3, the growth of hair was normal in the wound in DADM-1 d, DADM-2 d, and ADM groups, but few and scattered hair grew in DADM-3 d group. The inflammatory cells decreased, while Fbs increased, and new capillaries were found to grow inwardly in each group. The decrease in inflammatory cells was slightly delayed in DADM-3 d group. At PSW 5, hair growth became normal, and implants shrank and thinned with fiber membrane wrapped densely and bundles of ingrowing large caliber blood vessels in all groups. The dermal matrix in each group merged with the surrounding normal tissue. At PSW 9, ADM and DADM became white, thin, and soft tissue sheet which was closely connected with the inner side of the flap. There was no infiltration of inflammatory cells in implants in either group. The collagen fibers arranged regularly and densely, and they were integrated with normal collagen tissue. CONCLUSIONS: The burned DADM does not have obvious immunogenicity, but with good biocompatibility. It is prospective to become as a dermal substitute in repairing wounds.

Transforming growth factor-ß1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-ß1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts. METHODS: A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-ß1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-ß1. Enzyme-linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB (NF-κB) mRNA, connective tissue growth factor (CTGF) mRNA and TGF-ß receptor II (TßRII) mRNA in keloid fibroblasts. RESULTS: Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-ß1 and one phage model peptide (No. 4) could promote keloid fibroblasts proliferation, however, three phage model peptides (No. 1 - 3) could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TßRII mRNA slightly increased. CONCLUSIONS: Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-ß1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TßRII.

Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect.

BACKGROUND: Keratinocyte growth factor (KGF) significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. METHODS: A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. RESULTS: Thirty-three out of fifty-eight (56.9%) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor (EGF). MTT assay data showed that four (No. 1 - 4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No. 1 and No. 2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1 - 4). CONCLUSION: Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.

Focal injection of vancomycin combined with surgical debridement-dermatoplasty in the treatment of pseudo-epitheliomatous granuloma.

BACKGROUND: Pseudo-epitheliomatous granuloma (PEG) can occur in some small skin wounds with secondary infections resulting from improper treatments. It is difficult to heal and can easily relapse. OBJECTIVES: This study explores the clinical and pathological characteristics of PEG and effective treatments. PATIENTS AND METHODS: Tissue specimens of PEG obtained from 11 patients (age range: 2-67 years) were sent for microbial examination and histological observation. The local lesions were treated by focal injection of vancomycin combined with surgical debridement-dermatoplasty. RESULTS: The diagnosis of PEG was based on histological examination, which revealed long epithelial peduncle encapsulated granulation tissue-like honeycomb in which more vessels, macrophages, lymphocytes and mast cells and less extracellular matrix were distributed. Bacteria such as Staphylococcus aureus, Bacillus pyocyaneus, ethylene-type Streptococcus, stool Streptococcus and F-citric acid Bacillus were found in the microbial culture of the specimens. They were tolerant to celbenin but sensitive to vancomycin. PEG could be cured by focal application of vancomycin combined with free skin or skin flap after thorough debridement. The relapse of PEG could be prevented by the therapy. CONCLUSION: Focal injection of vancomycin combined with surgical debridement-dermatoplasty is an effective therapy for PEG.

[The changing pattern of stem cell markers of sweat gland in deep partial-thickness burn wound].

OBJECTIVE: To investigate the rules of proliferation of epithelial cells of sweat glands in deep partial-thickness burn wound and its transdifferentiation towards epidermal cells during healing process to explore its mechanisms. METHODS: Twenty-eight patients with limbs and trunk burn hospitalized in the Fourth People's Hospital of Taizhou City of Jiangsu Province and the Second Hospital of Shandong University from January 2004 to December 2007 were enrolled in the study. Tissue samples of deep partial-thickness burn wound (DPBW, n = 37), superficial partial-thickness burn wound (SPBW, n = 21), and normal skin (NS, n = 10) were harvested. Expressions of cytokeratin 10 (CK10), bcl-2, P63, CK14 and CK19 of epithelial cells in glandular secretory portion (GSP) in DPBW, SPBW and NS were detected with immunohistochemical double staining method. RESULTS: In NS, CK19, CK14 and CK10 expressed in medium intensity in GSP epithelial cells, P63 and CK14 weakly expressed in basal myoepithelial cells, while no expression of bcl-2 or P63 was observed in all CK10 positive terminally differentiated cells. In SPBW, no change of the construction of GSP and above-mentioned proteins during healing process was observed. In DPBW, as examined on 7(th) post burn day (PBD), expression of P63 and bcl-2 in GSP epithelial cells was enhanced. In DPBW on 8 - 10 PBD, bcl-2, P63, CK19 and CK14 strongly positive solid island-like epithelial structure was formed by proliferation, migration and squamous epithelization of basal cells. Such structure, along with granulation tissue, migrated towards the superficial layer of wounds. The hyperplasia of squamous epithelium resulted in complete reepithelialization. In DPBW, bcl-2, CK14, CK19 and P63 still strongly expressed in hyper-proliferative epidermal basal and suprabasal layers on 13 - 30 day after healing. CONCLUSIONS: During the natural healing process of DPBW, monolayer epithelium (CK19 and CK10 positive) of GSP slowly develops into stratified squamous epithelium (bcl-2, P63, CK19, and CK14 positive), suggesting that the epithelial-epidermal transdifferentiation of GSP undergoes slow retrodifferentiation process of stem cells and transient amplifying cells, resulting in the imbalance between lagged growth of epithelium and the hyperplasia of granulation tissue, constituting one of the important mechanisms of disturbance in DPBW repair.

[Relationship between the proliferation and immunoinduction of epithelial cells and the destruction of hair follicles and sebaceous glands in keloids].

OBJECTIVE: To investigate the influence of biological behavior of epithelial cells on the hair follicles and sebaceous glands (HFSG) structure in keloids (K). METHODS: The expression of intercellular adhesion molecule (ICAM)-1, D-related human leucocyte antigen (HLA-DR), and cytokeratin (CK) 14 on epithelial cells and the amount of activity T-lymphocytes were detected in specimens of keloid edge and normal skin with immunohistochemical and histological methods. RESULTS: In comparison with normal skin specimens, epithelial cells were proliferated in K-HFSG presented structural aberration and disintegrate or abnormally to form solid-epithelial island-like structure, and the density of HSFG with hyperplasia and the ageing scar in keloids was apparently decreased. They strongly expressed ICAM-1, HLA-DR, and CK14 in the epithelial cells, there were many immunologic cells which expressed CD4, CD45RO, and interferon (IFN)-gamma around the K-HFSG. The expressed level of epithelial cells was positively correlated with the density of immunologic cells nearby K-HFSG. CONCLUSION: It could be concluded that the reactivity with hyperplasia and immunoinduction of epithelial cells might be associated with the destruction of the some HFSG structure in the keloids.

[Clinical study on the immunoregulation effects of cytokines on the acellular xenogenic dermal matrix].

OBJECTIVE: To investigate the relationship between some cytokines and early outcome of grafts in burn patients receiving xeno-/allo-ADM grafting. METHODS: Nineteen xeno-ADMs were grafted onto the wounds of 12 patients after escharectomy in extremities, and 18 allo-ADMs were grafted onto the escharectomy wounds of 15 patients in extremities. All the grafts were covered with thin split thickness skin autografts. Six patients grafted with split thickness skin autografts (auto-TTS) were employed as control. After the grafts survived for 4 to 8 weeks, immunohistochemistry and ELISA methods were employed to determine the contents of IL-1 beta, IL-4, IL-6, TNF-alpha and IFN-gamma in the exudation fluid from wounds after the rejection of xeno-ADM, local skin and peripheral blood. RESULTS: It was exhibited by immunochemistry staining that the positive cellular density and staining intensity of the IL-1 beta, IL-4, IL-6, TNF-alpha and IFN-gamma in the grafts were ranked as following: Xeno-ADM > allo-ADM > auto-TTS (P < 0.05 - 0.01). The levels of IL-4, IL-6, TNF-alpha and IFN-gamma in the exudation fluid during the rejection of xeno-ADM were obviously higher than those in the blood of the patients as determined by ELISA, while the serum levels of IL-4 and IFN-gamma in xeno-ADM group were lower and higher than those in xeno-ADM when without rejection respectively. The serum levels of IL-4, TNF-alpha and IFN-gamma in xeno-ADM group were significantly higher than those in allo-ADM and auto-TTS groups (P < 0.05 - 0.01). CONCLUSION: Local detection of high levels of IL-1 beta, IL-4, IL-6, TNF-alpha and IFN-gamma might be related to the immune augmentation mediated by cytotoxic lymphocytes and cytokines. The dynamic changes of these cytokines might be helpful to the explanation of the bad outcome of xeno-ADMs.