Human keratinocyte growth factor-1 (hKGF-1), a member of the fibroblast growth factor (FGF) family, plays crucial roles in organ development, cell proliferation, wound healing, and tissue repair, representing one of the most effective and specific growth factors for skin repair. However, obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro. This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1 (hKGF-1) protein, ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line (BmN). In this study, we optimized the hKGF-1 sequence based on the codon preference of baculovirus. By fusing hKGF-1 with polyhedrin (highly expressed in this system) and adding extra promoters and enhancers, we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells. The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells. SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression (accounting for 8.7% of total cellular protein) was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression. The optimal harvest time was determined to be 120 h post transfection. This study achieved the efficient expression of hKGF-1 in Bombyx mori cells, establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1. The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins, but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
Bombyx/metabolism* ; Animals ; Baculoviridae/metabolism* ; Humans ; Fibroblast Growth Factor 7/biosynthesis* ; Recombinant Proteins/genetics* ; Cell Line ; Genetic Vectors/genetics*

BACKGROUND: In our preliminary study, we screened for their potential to inhibit 5α-reductase, and Melandrium firmum (MF) extract showed the most potent activity as confirmed by high-performance liquid chromatography (HPLC). OBJECTIVE: This study aimed to investigate the effects of MF extract on 5α-reductase activity and its mechanisms of action in the prevention or treatment of androgenetic alopecia. METHODS: HPLC was used to measure 5α-reductase activity. The hair growth-promoting effect of MF extract in the shaved dorsal skin of C57BL/6J mice was studied for 30 days. Hair follicles were examined by histological examination. Protein and mRNA levels of growth factors involved in hair growth were determined by western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) and qPCR, respectively. Cell proliferation was measured by (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. RESULTS: MF extract at 0.5 mg/ml showed 43.5% inhibition of 5α-reductase. MF extract promoted hair growth by inducing anagen phase reflected by skin color, hair density, and the number and size of hair follicles. It not only reduces the expression of transforming growth factor-beta 1 (TGF-β1) and Dickkopf-1 (DKK-1), but also markedly upregulated insulin-like growth factor 1 and keratinocyte growth factor in the dorsal dermal tissue. Ursolic acid, ecdcysteron, and ergosterol peroxide were identified as active constituents by activity-guided fractionation to inhibit 5α-reductase. They decreased the gene expression of TGF-β1 and DKK-1 in human hair dermal papilla cells. CONCLUSION: In summary, these finding indicate that MF extract might be a good drug candidate for hair growth promotion.
Alopecia ; Animals ; Blotting, Western ; Cell Proliferation ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Ergosterol ; Fibroblast Growth Factor 7 ; Gene Expression ; Hair Follicle ; Hair ; Humans ; Intercellular Signaling Peptides and Proteins ; Mice ; RNA, Messenger ; Skin ; Skin Pigmentation

OBJECTIVE: In order to investigate the interaction between the cytokines and keratinocyte and determine the role of cytokines in hyperproliferative of chronic otitis media with cholesteatoma, we observe the expression of matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and its receptor (KGFR) in middle ear cholesteatoma. METHOD: We examined the expression of MMP9, VEGF, KGF, KGFR and Ki-67 by immunohistochemistry in 50 specimens from chronic otitis media with cholesteatoma and 15 specimens from the normal skin of external auditory meatus. Ki-67 as an evaluation of cholesteatoma proliferation markers were used to detect the keratinocyte proliferative activity. RESULT: (1) The expression of VEGF and MMP9 in cholesteatoma specimens was higher than normal skin, and the difference was statistically significant (t = 4.914, P < 0.01; t = 3.284, P < 0.01). (2) The expression of KGF and KGFR in middle ear tissues was higher than normal skin, and the difference was statistically significant (t = 4.814, P < 0.01; t = 3.104, P < 0.01); The expression of KGF and KGFR increased, and the expression of Ki-67 also correspondly increased in the cholesteatoma. (3) In the tissue MMP9 and VEGF were positive. Mean optical density increased as well. KGF expression also increased accordingly. CONCLUSION MMP9, VEGF, KGF and KGFR proteins played an important role in hyperproliferation of cholesteatoma tissues. VEGF, MMP9 and KGF had a synergistic effect in hyperproliferation of cholesteatoma tissues.
Cholesteatoma, Middle Ear ; pathology ; Cytokines ; metabolism ; Ear Canal ; metabolism ; Ear, Middle ; metabolism ; Fibroblast Growth Factor 7 ; metabolism ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; Ki-67 Antigen ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Otitis Media ; pathology ; Receptor, Fibroblast Growth Factor, Type 2 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

No abstract available.
Fibroblast Growth Factor 7* ; Humans* ; Hyperpigmentation* ; Keratinocytes* ; Lentigo* ; Melanosis*

OBJECTIVETo study the influence of heat stimulation on expression of coxsackie-adenovirus receptor (CAR) in keratinocytes (KCs) of mouse skin and the effect of CAR on production of cell growth factors by dendritic epidermal T lymphocytes (DETCs).

METHODS(1) Twenty BALB/c mice were divided into heat stimulation group (HS) and control group (C) according to the random number table, with 10 mice in each group. Mice in group HS were inflicted with scald milder than superficial-thickness by dressing wet hot gauze, which had been soaked in 100°C hot water for 3 min, in the hair removed area on the back for 1 to 3 s, while mice in group C were sham injured by dressing a wet gauze which had been soaked in water of room temperature for 3 min in the hair removed area on the back for 1 to 3 s. Square full-thickness skin specimens measuring 2.0 cm × 2.0 cm in size were obtained from the center of the bare skin. The expression of CAR in skin tissue sections were detected by immunohistochemistry staining. The mRNA and protein expression levels of CAR in skin tissue sections were respectively determined by real-time fluorescent quantitation RT-PCR and Western blotting. (2) KCs were isolated and cultured from full-thickness skin obtained from the trunk of 2 fetal BALB/c mice, and they were divided into 2 groups according to the random number table, with 5 wells in each group. The cells in group HS and group C were respectively cultured in 42°C and 37°C, 5% CO2 incubator for 1 h, and then all the cells were cultured in 37 °, 5% CO2 incubator for 6 h. The apoptosis of the cells and their expression of CAR were detected by flow cytometer. (3) Five BALB/c mice were sacrificed, and full-thickness skin was obtained from the trunk. The DETCs were divided into 7 groups according to the random number table after being isolated and purified from the skin specimens. Cells in group C were cultured without any stimulation, and cells in the 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/L CAR groups were respectively cultured with corresponding concentration of recombinant mice CAR nutrient solutions, with 5 wells in each group. The contents of insulin-like growth factor I (IGF-I) and keratinocyte growth factor (KGF) were determined with ELISA. Data were processed with independent samples t test and one-way analysis of variance.

RESULTS(1) The immunohistochemistry staining showed that there was mild positive staining in the skin tissue sections of mice in group C, while the positive staining was more obvious in group HS. The positive staining was mainly located in KCs, hair follicles, and sweat gland epithelial cells, while no positive staining was observed in fibroblasts. The mRNA expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.157 ± 0.027 and 0.773 ± 0.029. There was statistically significant difference between them (t = 3.052, P < 0.01). The protein expression levels of CAR in skin tissue sections in group C and group HS were respectively 0.23 ± 0.09 and 0.89 ± 0.14. There was statistically significant difference between them (t = 2.556, P < 0.05). (2) The apoptosis rates of KCs in group C and group HS were respectively (5.7 ± 1.3)% and (7.4 ± 1.7)% (t = 0.464, P > 0.05). The expression rates of CAR in KCs in group C and group HS were respectively (48 ± 6)% and (80 ± 8)%. There was statistically significant difference between them (t = 2.585, P < 0.05). (3) The contents of IGF-Iin culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (23.1 ± 1.8), (22.5 ± 2.1), (31.2 ± 2.5), (39.7 ± 2.3), (61.8 ± 3.5), (45.1 ± 2.8), and (29.0 ± 2.0) µg/L. There was statistically significant difference among 7 groups (F = 3.414, P < 0.05). The contents of KGF in culture supernatants in group C and 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/L CAR groups were respectively (131 ± 9), (217 ± 12), (355 ± 21), (563 ± 21), (535 ± 34), (292 ± 20), and (245 ± 10) ng/L. There was statistically significant difference among 7 groups (F = 5.063, P < 0.01).

CONCLUSIONSThe expression of CAR in KCs would rise after HS. The optimum CAR concentration to increase IGF-I and KGF production in DETCs is low.

Animals ; Burns ; metabolism ; Cells, Cultured ; Coxsackie and Adenovirus Receptor-Like Membrane Protein ; metabolism ; Female ; Fibroblast Growth Factor 7 ; metabolism ; Hot Temperature ; Insulin-Like Growth Factor I ; metabolism ; Keratinocytes ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Skin ; cytology ; T-Lymphocytes ; metabolism

The aim of this study was to evaluate the efficacy of palifermin, an N-terminal truncated version of endogenous keratinocyte growth factor, in the control of oral mucositis during antiblastic therapy. Twenty patients undergoing allogeneic stem-cell transplantation for acute lymphoblastic leukaemia were treated with palifermin, and compared to a control group with the same number of subjects and similar inclusion criteria. Statistical analysis were performed to compare the outcomes in the treatment vs. control groups. In the treatment group, we found a statistically significant reduction in the duration of parenteral nutrition (P=0.002), duration of mucositis (P=0.003) and the average grade of mucositis (P=0.03). The statistical analysis showed that the drug was able to decrease the severity of mucositis. These data, although preliminary, suggest that palifermin could be a valid therapeutic adjuvant to improve the quality of life of patients suffering from leukaemia.
Adolescent ; Allografts ; transplantation ; Anti-Inflammatory Agents ; therapeutic use ; Case-Control Studies ; Child ; Cohort Studies ; Cyclophosphamide ; therapeutic use ; Female ; Fibroblast Growth Factor 7 ; therapeutic use ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Male ; Myeloablative Agonists ; therapeutic use ; Parenteral Nutrition ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; therapy ; Radiotherapy Dosage ; Stomatitis ; classification ; drug therapy ; prevention & control ; Time Factors ; Transplantation Conditioning ; methods ; Treatment Outcome ; Whole-Body Irradiation

Sargassum fusiforme has traditionally been widely consumed in Asia as a food, and it has gained much attention due to its high nutritional, pharmaceutical, and industrial value. This study aimed to examine the promotional effects of ethanol extract (ET) and fraction obtained from ethyl acetate (FR) of S. fusiforme on hair growth in C57BL/6 mice and HaCaT cells. Five-week-old mice were used to compare hair regrowth during application of ET and FR for 21 days. Hair regrowth was evaluated by macroscopic observation and verified by hematoxylin-eosin tissue staining. Levels of mRNA expression of factors relevant to the hair growth cycle such as keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were examined by quantitative polymerase chain reaction (qPCR). Our results showed that ET and FR successfully promoted hair regrowth in shaved C57BL/6 mice at a dose >20 mg/kg. Moreover, ET and FR were effective in stimulating expression of KGF and VEGF mRNAs in a dose-dependent manner, whereas TGF-beta1 was not activated. These results indicate that ET and FR of S. fusiforme effectively promoted hair growth and gene expression relevant to hair growth cycles in both in vitro and in vivo models.
Alopecia ; Animals ; Asia ; Ethanol ; Fibroblast Growth Factor 7 ; Gene Expression ; Hair* ; Mice ; Polymerase Chain Reaction ; RNA, Messenger ; Sargassum* ; Transforming Growth Factor beta1 ; Vascular Endothelial Growth Factor A

OBJECTIVES: The purpose of this study was to investigate the wound healing effect of primary cultured oral mucosal keratinocytes (OMKs) and to assess their roles in skin wounds. MATERIALS AND METHODS: OMK labeled with BromodeoxyUridine were scattered onto 1.5x1.5 cm skin defects of adult female nude mice (OMK group, n=15). For the control, culture media were placed on the wound (control group, n=15). Mice in both groups were sacrificed at three days (n=5), one week (n=5), and two weeks (n=5), and histomorphometric and immunoblot analyses with keratinocyte growth factor (KGF), interleukin (IL)-6, and IL-1alpha antibody were performed for the biopsied wound specimen. To verify the effect of the cytokine, rhIL-1alpha was applied instead of OMK transplantation, and the OMK and control groups were compared with regard to re-epithelialization. RESULTS: Histomorphometric analyses demonstrated faster re-epithelialization in the graft group than in the control group at the third day, first week, and second week. Newly forming epithelium showed maintenance of the histological character of the skin epithelium. The graft group showed superior expression of KGF, IL-6, and IL-1alpha protein, compared with the control group. Similar faster re-epithelialization was observed after treatment with rhIL-1alpha instead of OMK transplantation. CONCLUSION: We successfully confirmed that the graft of primary cultured OMKs promoted regeneration of skin defects. The mechanism of accelerated wound healing by primary cultured OMKs was attributed to inducement of cytokine expression as required for re-epithelialization.
Adult ; Animals ; Bromodeoxyuridine ; Culture Media ; Epithelium ; Female ; Fibroblast Growth Factor 7 ; Humans ; Interleukin-6 ; Interleukins ; Keratinocytes ; Mice ; Mice, Nude ; Primary Cell Culture ; Re-Epithelialization ; Regeneration ; Skin ; Tissue Engineering ; Transplants ; Wound Healing

OBJECTIVETo study the function of keratinocyte growth factor (KGF) on apoptosis of oral mucosal epithelial cells and to provide a basis for further investigation of the role of KGF in the occurrence and development of oral mucosal diseases.

METHODSDifferent concentrations of KGF (control group, 0 ng x mL(-1); experiment 1 group, 5 ng x mL(-1); experimental 2 group, 25 ng x mL(-1); experiment 3 group, 50 ng x mL(-1)) were added in oral mucosa epithelial cells cultured in vitro. After training for 12, 24, and 48 h, cell morphology was observed under an inverted microscope. Apoptosis was detected by using a flow cytometry instrument, and mRNA expression of apoptosis-related genes Bcl-2 and Bax was detected by using Real-Time fluorescent quantitative detection.

RESULTSCell adherence of the experimental group was more obvious than that of the control group, and the cell nucleolus of the experiment 3 group was obviously cultured at 48 h. After culturing for 48 h, the apoptosis rate and Bcl-2 and Bax mRNA expression among the four groups were statistically significant. The increase of KGF concentration, apoptosis rate, and Bax mRNA expression gradually reduced, whereas Bcl-2 mRNA expression increased (P < 0.05).

CONCLUSIONKGF may inhibit epithelial cell apoptosis through upregulation of Bcl-2 mRNA and downregulation of Bax mRNA.

Apoptosis ; Epithelial Cells ; Fibroblast Growth Factor 7 ; Humans ; Mouth Mucosa ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger

Carcinoma, Squamous Cell ; metabolism ; pathology ; Chemokine CCL2 ; metabolism ; Fibroblast Growth Factor 7 ; metabolism ; Fibroblasts ; metabolism ; pathology ; Gelatinases ; metabolism ; Humans ; Membrane Proteins ; metabolism ; Mouth Neoplasms ; metabolism ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Serine Endopeptidases ; metabolism