[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.

Dura cells in the etiopathogenesis of Crouzon syndrome: the effects of FGFR2 mutations in the dura cells on the proliferation of osteoblasts through the hippo/YAP mediated transcriptional regulation pathway.

BACKGROUND: FGFR2 (fibroblast growth factor receptor 2) mutations are implicated in the etiopathogenesis of syndromic craniosynostosis, and C278F- or C342Y-FGFR2 mutations can lead to Crouzon syndrome. The dura mater exerts crucial effects in the regulation of cranial suture development. However, the underlying mechanisms of these biological processes are rarely studied. This research explored and analyzed the biological function of FGFR2 overexpressed by dura cells on cranial osteoblasts. METHODS: Dura cells and cranial osteoblasts from C57BL/6 mice aged 6 days were obtained and cultured respectively. Lentivirus-FGFR2 constructs were engineered with C278F- and C342Y-FGFR2 mutations. The dura cells were infected with the constructs and co-cultured with osteoblasts in a trans-well system. Four experimental groups were established, namely the Oste group, the Oste+Dura-vector group, the Oste+Dura-C278F group, and the Oste+Dura-C342Y group. FACS, CCK8, and EdU assays were used to evaluate the osteoblast proliferation levels. Western blot and RT-qPCR were used to measure the expressions of the factors related to proliferation, differentiation, and apoptosis. Furthermore, the expression levels of the key factors in the Hippo/YAP-PI3K-AKT proliferation pathway were measured and analyzed. Finally, rescue experiments were performed with an RNA interfering assay. RESULTS: The proliferation and differentiation levels of the osteoblasts in the Oste+Dura-C278F and Oste+Dura-C342Y groups were significantly up-regulated, but the apoptosis levels in the four groups were not significantly different. The YAP, TEADs1-4, p-PI3K, and p-AKT1 expressions in the mutant FGFR2 groups were higher than the corresponding expressions in the control groups, and the results of the rescue experiments showed a reverse expression tendency, which further confirmed the effects of the FGFR2 mutations in the dura cells on the proliferation of the osteoblasts and the underlying possible mechanisms. CONCLUSION: Our studies suggest that the Crouzon mutations (C278F- and C342Y-) of FGFR2 in dura cells can enhance osteoblast proliferation and differentiation and might influence the pathogenesis of craniosynostosis by affecting the Hippo/YAP-PI3K-AKT proliferation signaling pathway.

[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.

[Effects of recombinant adenovirus-mediated double suicide genes on implanted human keloid: experiment with athymic mice].

OBJECTIVE: To detect the effects of the recombinant adenovirus-mediated double suicide genes constructed by Escherichia coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) and herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-CDglyTK on implanted human keloids and mechanisms thereof. METHODS: Twenty nude mice were implanted with human keloid obtained during operation so as to establish mouse keloid models and then were randomly divided into 4 equal groups: Group A, injected with normal saline (NS) into the keloid once per 3 days for 18 days totally, Group B injected with NS into the keloid and injected intraperitoneally with 5-Fc and GCV; Group C injected with CDglyTK into the keloid, and Group D injected with CDglyTK into the keloid and 5-Fc and GCV injected intraperitoneally. The volume of the implanted keloid tissue was measured 2, 7, 14, 21, 28, 35, and 42 days after operation. On day 42 the keloid tissues were removed to undergo morphological examination, TUNEL method was used to examine the apoptosis of the fibroblasts, and the expression of Bcl-2 and BAX, products of apoptosis-related genes, were detected by immunohistochemistry. RESULTS: Compared to those before treatment the volume of the implanted keloid of Group D began to decrease since 14 days after treatment time-dependently (all P < 0.05), and the volumes of the other 3 groups continued to increase and peaked on days 21, 14, or 7 respectively (all P < 0.05). Microscopy showed infiltration of a larger quantity of histiocyte in the keloid tissue, and more obvious collagen disorganization and apoptosis of fibroblasts in Group D than in the other 3 groups. The protein expression of Bcl-2 was more remarkable and the protein expression of BAX was less remarkable in Group D than in the other 3 groups. CONCLUSIONS: The recombinant adenovirus-mediated double suicide gene therapy is effective on the implanted keloid tissue. The main mechanism may be induction of apoptosis in the keloid fibroblasts.

Surgical outcome and patient satisfaction after Z-epicanthoplasty and blepharoplasty.

AIM: To evaluate surgical outcomes of modified Z-epicanthoplasty with blepharoplasty that we previously reported from the patient's perspective using patient-reported outcome measures (PROMs) and patient satisfaction scores. METHODS: A total of patients (n=180) who underwent the surgery between January 2013 and June 2016 were randomly selected. Standardized patient satisfaction forms (total score, 40) and validated PROMs questionnaires (total score, 12) were sent to patients for completion. PROMs assesses the severity of scarring, pain and asymmetry, as well as functional and appearance issues. RESULTS: All patients were female, ranging from 18 to 35 years old (mean=24). The response rate was 73.3% (n=132). The majority of patients reported good or excellent outcomes based on PROM analysis. Patients reported minimum or non-visible scarring at both the double eyelid surgical scar (85.6%) and the inner canthus (80.3%). Issues concerning function and appearance were minimal as 80.3% reported satisfaction with both domains. Notably, the majority of patients reported either a high or very high satisfaction rate to yield a mean score of 104 out of 120 (P<0.05). CONCLUSION: Integration of our modified Z-epicanthoplasty with blepharoplasty produces good outcomes based on PROM results, which shows a positive linear relationship with patient satisfaction scores.

[The clinical classification method research of keloid].

OBJECTIVE: To explore the clinical classification method of keloids and providing a thread for the treatment of keloids. METHODS: To summarize the 600 cases of keloid patients we accepted and diagnosed from November 2004 to October 2012, and filling in keloid patients information sheet, recording the keloids form by photographs, analyzing the treatment, putting forward the classification method of keloids in clinic. RESULTS: According to the position and quantity that keloids grow, the keloid patients are divided into four major categories:one in single site, one in each site, more than one in single site and more than one in each site; According to the area and thickness of keloids, the keloid single lesion is divided into four subclasses: type of small area and thin, type of small area and thick, type of large areas and thin,type of large areas and thick; According to the number of lesions, keloid multiple lesions is divided into two subgenera: isolated multiple and dispersion multiple, different kinds of keloids suit different methods of treatment. CONCLUSION: The clinical classification method of keloids can be used to provide thought for the treatment of keloids, and have a good application value.

[Skin needle roller importing triamcinolone acetonide into scar to treat hypertrophic scars].

OBJECTIVE: To evaluate the effect of importing triamcinolone acetonide into hypertrophic scars with skin roller needles. METHODS: Thirty-two cases with burn hypertrophic scar were treated. The skin roller needles were moved back and forth on the hypertrophic scars with triamcinolone acetonide dropping on the scar surface at the same time. So the triamcinolone acetonide could be imported into the scar through needles and needle holes. The effect was evaluated as cured, effective, and no effect. The Vancouver scaring criteria and visual analogue scale was used to assess the scar color, thickness, texture and feeling before and after treatment, as well as at the untreated scar area (control). RESULTS: Thirty-two cases were treated 1-3 times, including 28 cases with cured result and 4 cases with effective result. The total effective rate was 100%. The scar color, thickness, texture and feeling was significantly different between the scar before and after treatment, or between the treated and untreated scar (P < 0.05). CONCLUSIONS: Importing triamcinolone acetonide into hypertrophic scars with skin roller needles is effective. It is a new method for the treatment of large hypertrophic scar with medicine.

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.