OBJECTIVETo build 3D-pharmacophore model of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors using distance comparisons method and design novel EGFR inhibitors.

METHODSThirteen typical EGFR inhibitors were selected, and their biologically active conformations were obtained by using DOCK5.0 program, then 3D-pharmacophore model of EGFR inhibitors was built using distance comparisons method.

RESULTSValidation of the 3D-pharmacophore model was carried out and novel structures with potential inhibitory activity were selected by means of 3D-searching and docking method.

CONCLUSIONThis method can improve hit rate of lead compounds discovery and can be used to design novel EGFR inhibitors.

Drug Design ; Enzyme Inhibitors ; Epidermal Growth Factor ; metabolism ; Models, Chemical ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; chemistry ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; chemistry ; Structure-Activity Relationship

Genistein is a high specific and noncompetitive inhibitor of epidermal growth factor receptor tyramine kinase domain (EGFR-TK). In the paper, a molecular docking between genistein and EGFR-TK was studied to explore the mechanism of their interaction and antitumor mechanism of genistein by AUTODOCK 3.05 program. The results indicated that genistein located in the active cavity of EGFR-TK by high affinity (deltaG = -31.2 kJ/mol), and genistein inhibited EGFR-TK by interfering with forming of Lys721/Glu738 ion pair. The inhibition belonged to noncompetitive interaction, in which hydrophobic force and hydrogen bond played key roles.
Catalytic Domain ; Genistein ; metabolism ; pharmacology ; Models, Molecular ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the mechanism and the accelerating effect of rhEGF and rhbFGF on wound healing.

METHODSTwelve New Zealand rabbits with 72 incised wounds on ventral side of 24 ears were randomly divided into two therapeutic groups (rhEGF of 10 ug/cm(2) and rhbFGF of 100 AU/cm(2)) and a control group (1% silver sulfadiazine cream, SD-Ag). The general conditions of the wound healing was observed grossly. Biopsies were harvested at different time points for the pathomorphological examination, the electron microscopic examination, and for assessment of integrin beta1 mRNA expression by in situ hybridization.

RESULTSThe expressions of integrin beta 1 mRNA in two therapeutic groups were significantly higher than that of control group. The quality of the wound healing was improved in therapeutic group with its healing time shortened when compared with that in control group (P < 0.05). There was an obvious difference in the number of fibroblasts and capillary gemmules between the therapeutic and control groups (P < 0.05).

CONCLUSIONThe wound healing and quality could be improved by both rhEGF and rhbFGF, but rhbFGF seemed better to be employed during the early and middle stages of the wound repair for the growth of granulation tissue, while rhEGF should be applied at the late stage of wound repair to accelerate the re-epithelialization of the wound. Combined application of rhEGF with rhbFGF according to time effect could be more beneficial to the wound repair.

Animals ; Epidermal Growth Factor ; pharmacology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Integrin beta1 ; genetics ; Male ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rabbits ; Recombinant Proteins ; pharmacology ; Wound Healing ; drug effects

In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2 x 2 x 2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 microg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P < 0.05) and the percentages of healthy follicles were reduced (P < 0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P < 0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3% +/- 3.2%), follicles diameter((32.3 +/- 2.3) microm), follicles survival rate (41.6% +/- 3.1%) and the proportion of PCNA stained follicles (26.4% +/- 1.2%, P < 0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5 +/- 5.1) microm), follicles survival rate (59.7% +/- 6.1%) and proportion of PCNA stained follicles (43.5% +/- 4.1%, P < 0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.
Animals ; Ascorbic Acid ; pharmacology ; Culture Techniques ; Epidermal Growth Factor ; pharmacology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Ovarian Follicle ; growth & development ; Ovary ; growth & development ; Proliferating Cell Nuclear Antigen ; analysis ; Sheep

OBJECTIVETo study the possible induction effect of exposure to 50 Hz magnetic field (MF) on clustering of cell membrane surface receptors for epidermal growth factor (EGF) and tumor necrosis factor (TNF), the starting site of signals of biological effects, and its possible intervention effect.

METHODSLung fibroblasts of Chinese hamster (CHL) were exposed to EGF, TNF, 0.4 mT 50 Hz MF, 0.4 mT noise MF, and 0.4 mT 50 Hz MF combined with 0.4 mT noise MF. Respectively, for different durations, following the treatment, EGF and TNF receptors on the cell membrane were marked by corresponding antibodies with immunohistochemical method, then observed under a confocal microscope.

RESULTSClustering of cell membrane receptors could be induced 5 min after treatment with EGF and TNF, as well as with 50 Hz MF at 0.4 mT, which reached the peak in 15 min. While noise MF with the same intensity did not induce clustering of cell membrane receptors. Superposition of noise MF with the same intensity could inhibit clustering of cell membrane receptors induced by 50 Hz MF.

CONCLUSIONClustering of EGF and TNF receptors on the cell membrane could be induced by 50 Hz MF, suggesting that membrane receptors would be one of the sites where MF signals coupled, and noise MF with the same intensity could inhibit these effects.

Animals ; Cell Line ; Cricetinae ; Electromagnetic Fields ; adverse effects ; Epidermal Growth Factor ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; radiation effects ; Noise ; adverse effects ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Tumor Necrosis Factor ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo explore the relationship between the proliferation, differentiation of rat hepatic stem like cell line WB-F344 and cytokines in vitro.

METHODS(3)H thymidine labelling of new synthesized DNA was used to examine the mitogenic responsiveness of WB-F344 cells to cytokines, western blot was used to study the expression of cytokines receptors on hepatic stem cells, and apoptotic cells were detected by Flow cytometry.

RESULTSWB-F344 cells showed a proliferative response to the cytokines of hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Insulin at the dose of 80 ng/ml, and the relative cpm values are 982.95, 906.32, 863.98 and 968.67 respectively, while non response to interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) at the same dose, and an inhibition or apoptosis response to transforming growth factor-beta (TGF-beta) at 80 ng/ml with a 26.89% apoptotic rate. Western blot showed that there were HGF, EGF, FGF, TGF-beta receptors expressed on WB-F344 cells. When WB-F344 cells were cultured in the differential system (DMEM, 10% Fcs, HGF 10 to approximately 50 ng/ml, EGF 20 ng/ml, Insulin 1 microg/ml, Dex 1 micromol/L), the cells could differentiated into hepatocytes. In addition, HGF could scattered WB-F344 cells.

CONCLUSIONThe proliferation and differentiation of liver stem cells are regulated by various cytokines which may play an important role when liver is damaged seriously.

Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; Cytokines ; pharmacology ; Epidermal Growth Factor ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Insulin ; pharmacology ; Liver ; cytology ; Rats ; Rats, Inbred F344 ; Stem Cells ; cytology ; drug effects ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo observe the effects of recombinant human epidermal growth factor (rhEGF) on skin wound healing.

METHODSThe dorsal trauma model of rats was used. A total of 68 dorsal wounds in 34 rats were created and divided into the rhEGF group and the isotonic saline group according to self-control. The process of wound healing was observed and a mean healing time was calculated. Furthermore, the dynamic analysis was performed at different times on wound OHP contents, ratio of collagen I/III and cell DNA cycle.

RESULTSThe wound healing was accelerated obviously in all wounds treated with rhEGF. The mean time of wound healing of the two groups was (17.2 +/- 1.3) days and (20.5 +/- 1.6) days respectively (P < 0.01). In the rhEGF group, the new granulation tissue was more and the re-epithelialization was faster than that of the saline group. External rhEGF increased OHP content, reduced collagen I/II ratio and accelerated cell DNA replication.

CONCLUSIONExternal rhEGF can shorten wound healing time, increase granulation tissue and OHP contents, reduce collagen I/III ratio and accelerate cell DNA replication, thus obviously promoting skin wound repair.

Animals ; Epidermal Growth Factor ; pharmacology ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Skin ; injuries ; Wound Healing ; drug effects

BACKGROUND: Standard protocols are lacking for the preparation of platelet lysates (PL) as an alternative to using fetal bovine serum as a cell culture supplement. This study aimed to establish optimum conditions for preparing PL for use in cell cultures. METHODS: Cell density in three pooled platelet concentrates (PC) were adjusted to 1x10(12)/L and 2x10(12)/L. PL was prepared from PC by 1 to 3 freeze-thaw (FT) cycles. HaCaT cells were cultured in media supplemented with 5% or 10% PL. Cell numbers were estimated using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan). Growth factors were quantified by using the Luminex 200 system (Luminex Corporation, USA). RESULTS: Cell proliferation rates in the presence of PLs were similar when prepared from PCs of both cell densities. The rates were higher in media containing 5% PL than 10% PL when prepared by two FT cycles. Concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor-AB/BB (PDGF-AB/BB), PDGF-AA, and epidermal growth factor (EGF) were significantly higher in PL prepared from PC with a cell density of 2x10(12)/L than 1x10(12)/L PC. However, only VEGF and PDGF-AA concentrations in PLs were correlated with HaCaT cell counts. CONCLUSIONS: The 5% PL from PC with a cell density of 1x10(12)/L prepared by two FT cycles treatment was the most effective condition that supported steady HaCaT cell proliferation. Our finding may be useful for preparing PL-supplemented cell culture media.
Blood Platelets/chemistry/*metabolism ; Cell Line ; Cell Proliferation/drug effects ; Culture Media/pharmacology ; Epidermal Growth Factor/chemistry/pharmacology ; Humans ; Platelet-Derived Growth Factor/chemistry/pharmacology ; Vascular Endothelial Growth Factor A/chemistry/pharmacology

This experiment was designed to study the effect of 131I-recombinant human epidermal growth factor (131I-rhEGF) on the growth of tumor in nude mice loaded with human breast cancer. Bioactivity of 131I-rhEGF and uptake of 131I-rhEGF in breast cancer tissue were verified using biodistribution experiment of 131I-rhEGF in the nude mice loaded with human breast cancer. The effect of 131I-rhEGF on the growth of tumor was assessed via the growth experiment of tumor in the nude mice loaded with human breast cancer. The ultrastructural change of the tumor cell treated with 131I-rhEGF was observed under transmission electron microscope, and the pathological change of the tumor tissue treated with 131I-rhEGF was detected by biopsy. The results showed that the tumor tissue of nude mice bearing human breast cancer obviously takes in 131I-rhEGF; that intravenous administration and intratumoral administration of 131I-rhEGF both obviously inhibit the growth of tumor, the inhibition rates (82.00% and 80.70%) being remarkably higher than that of 131I (7.49%) and that of 131I-HSA (6.91%) (P<0.05); and that intravenous and intratumoral administration of 131I-rhEGF both obviously damage and kill tumor cells. Therefore, 131I-rhEGF can inhibit the growth of human breast cancer cell in nude mice; it is a potential receptor-mediated radioactivity targeting drug for treating breast cancer.
Animals ; Breast Neoplasms ; pathology ; ultrastructure ; Drug Delivery Systems ; Epidermal Growth Factor ; biosynthesis ; genetics ; pharmacology ; Female ; Humans ; Iodine Radioisotopes ; administration & dosage ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo investigate the influence of some topically used antibiotics (amikacin, gentamicin, chloromycetin and sulfamylon), basic fibroblast growth factor (FGF2) , epithelial growth factor (EGF) and recombinant human growth hormone (rhGH) on the growth of fibroblasts in vitro.

METHODSFibroblasts were cultured and passaged. The cultured cells were then divided into control (routine culture of fibroblasts), amikacin (amikacin in respective dose of 0.021, 0.210, 2.100 mg/L), gentamicin (in respective dose of 5, 50, 500 mg/L) , chloromycetin (in respective dose of 0.01, 0.10, 1.00 mg/L), sulfamylon (in respective dose of 5, 10 g/L), FGF2 (2400 U/ml), EGF (2000 U/ml) and rhGH (0.016, 0.160, 1.600 g/L) groups. After the above agents were added to the culture medium respectively, the proliferation of the cultured fibroblasts was determined with MTT method, and the result was expressed as A (absorption) value. The cell cycle was determined with flow cytometry and the morphology of the cells was observed with inverted microscope.

RESULTS(1) MTT method: The A value of fibroblasts cultured with amikacin, gentamicin, chloromycetin and sulfamylon in various doses was obviously lower than that in control group (0.4553 +/- 0.0217, P < 0.05 or 0.01) , and the A value of sulfamylon group was the lowest in two doses (0.1013 +/- 0.0011 for 5 g/L and 0.0950 +/- 0.0041 for 10 g/L, P < 0.01). On the other hand,the A value in FGF2 and rhGH group(0.016 g/L) was much higher than that in the control (P < 0.05). However,theA value in EGF (both doses) and rhGH groups (0.160, 1.600 g/L) was close to that in control (P > 0.05). (2) Cell cycle determination: The proliferation index (PI) of fibroblasts cultured with amikacin in dose of 0.210 mg/L showed no difference compared to that in control (9.63 +/- 0.45)%, (P > 0.05). But the PI of fibroblasts cultured with FGF2, EGF and rhGF in dose of 0.016 g/L was increased significantly (46.76 +/- 2.33)%, (42.30 +/- 1.41)%, and (13.29 +/- 0.47)%, respectively, (P < 0.05 or 0.01). (3) Histological examination of the cells: The number of fibroblasts in elongated or spindle shape was larger, showing a blur contour but high transparency in control as well as in EGF and rhGH groups (both 0.160 and 1.600 g/L doses groups). The number of cells was lower in amikacin, gentamicin, chloromycetin and sulfamylon groups with sharp but irregular contour and lower transparency, and more granule-like materials and vacuoles in the cytoplasm. The cells in the FGF2 and rhGH (in dose of 0.016 g/L) groups exhibited dense with even distribution and slender or spindle shape and with more mitotic figures but blur contour and high transparency.

CONCLUSIONDifferent kinds of the topically used therapeutic agents for burn wounds exert different influence on the biological characteristics of fibroblasts in vitro. The topically used agents for burn wounds should be carefully selected so that wound healing will be promoted and scar formation inhibited.

Anti-Bacterial Agents ; pharmacology ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Human Growth Hormone ; pharmacology ; Humans ; In Vitro Techniques ; Wound Healing ; drug effects