Objective: To investigate the regulatory effects of bio-intensity electric field on directional migration and microtubule acetylation in human epidermal cell line HaCaT, aiming to provide molecular theoretical basis for the clinical treatment of wound repair. Methods: The experimental research methods were used. HaCaT cells were collected and divided into simulated electric field group (n=54) placed in the electric field device without electricity for 3 h and electric field treatment group (n=52) treated with 200 mV/mm electric field for 3 h (the same treatment methods below). The cell movement direction was observed in the living cell workstation and the movement velocity, trajectory velocity, and direction of cosθ of cell movement within 3 h of treatment were calculated. HaCaT cells were divided into simulated electric field group and electric field treatment 1 h group, electric field treatment 2 h group, and electric field treatment 3 h group which were treated with 200 mV/mm electric field for corresponding time. HaCaT cells were divided into simulated electric field group and 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group treated with electric field of corresponding intensities for 3 h. The protein expression of acetylated α-tubulin was detected by Western blotting (n=3). HaCaT cells were divided into simulated electric field group and electric field treatment group, and the protein expression of acetylated α-tubulin was detected and located by immunofluorescence method (n=3). Data were statistically analyzed with Kruskal-Wallis H test,Mann-Whitney U test, Bonferroni correction, one-way analysis of variance, least significant difference test, and independent sample t test. Results: Within 3 h of treatment, compared with that in simulated electric field group, the cells in electric field treatment group had obvious tendency to move directionally, the movement velocity and trajectory velocity were increased significantly (with Z values of -8.53 and -2.05, respectively, P<0.05 or P<0.01), and the directionality was significantly enhanced (Z=-8.65, P<0.01). Compared with (0.80±0.14) in simulated electric field group, the protein expressions of acetylated α-tubulin in electric field treatment 1 h group (1.50±0.08) and electric field treatment 2 h group (1.89±0.06) were not changed obviously (P>0.05), while the protein expression of acetylated α-tubulin of cells in electric field treatment 3 h group (3.37±0.36) was increased significantly (Z=-3.06, P<0.05). After treatment for 3 h, the protein expressions of acetylated α-tubulin of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group were 1.63±0.05, 2.24±0.08, and 2.00±0.13, respectively, which were significantly more than 0.95±0.27 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of acetylated α-tubulin in 200 mV/mm electric field group and 300 mV/mm electric field group were increased significantly (P<0.01); the protein expression of acetylated α-tubulin of cells in 300 mV/mm electric field group was significantly lower than that in 200 mV/mm electric field group (P<0.05). After treatment for 3 h, compared with that in simulated electric field group, the acetylated α-tubulin of cells had enhanced directional distribution and higher protein expression (t=5.78, P<0.01). Conclusions: Bio-intensity electric field can induce the directional migration of HaCaT cells and obviously up-regulate the level of α-ubulin acetylation after treatment at 200 mV/mm bio-intensity electric field for 3 h.
Humans ; Acetylation ; Tubulin/metabolism* ; Microtubules/metabolism* ; Electricity ; Epidermal Cells/metabolism*

OBJECTIVETo elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma cell line.

METHODSThe levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment.

RESULTSIn pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense.

CONCLUSIONEB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.

Blotting, Western ; Epidermal Growth Factor ; metabolism ; Herpesvirus 4, Human ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; pathology ; virology ; Phosphorylation ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; metabolism

Epidermal growth factor receptor (EGFR) cell signaling plays a central role in beta-cell mass/function regulation, and provides a new strategy for the treatment of diabetes, but its mechanisms of action remain poorly understood. In developmental biology, pancreatic islet development is impaired in lacking EGFR of mice. The attenuation of EGFR signaling in the islets leads to markedly reduced beta-cell proliferation. EGFR ligands BTC can increase proliferation and neogenesis. In this article EGFR and their ligands in the pancreas, EGFR cell signaling, and EGFR effects in pancreatic beta-cell mass/function regulation were reviewed.
Betacellulin ; Humans ; Insulin-Secreting Cells ; metabolism ; Intercellular Signaling Peptides and Proteins ; metabolism ; Ligands ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; physiology

OBJECTIVE: To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process. METHODS: The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot. RESULTS: When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05). CONCLUSION Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
Humans ; beta Catenin/metabolism* ; Bone Marrow Cells ; Cell Differentiation ; Cells, Cultured ; Epidermal Growth Factor/metabolism* ; Mesenchymal Stem Cells ; Wnt Signaling Pathway ; Neurons ; Fibroblast Growth Factor 2/metabolism*

OBJECTIVETo explore the roles of epidermal growth factor receptor (EGFR) signaling pathway on cultured human nasal epithelial cells RPMI-2650.

METHODSRPMI-2650 cells were cultured in vitro, the growth curve was measured and the ultrastructure was observed using scanning electron microscope. When the cells were significantly confluent, they were divided into 4 groups, group A: maintained in Eagle's minimum essential media (EMEM) medium without adding any stimulators; group B: added with epidermal growth factor (EGF) 25 ng/ml; group C: added with AG1478 (EGFR selective inhibitor) 10 micromol/L followed by EGF 25 ng/ml 30 minutes later; group D: added with PD98069 (p44/42MAPK selective inhibitor) 30 micromol/L followed by EGF 25 ng/ml 30 minutes later. After incubated for 24 hours, the expression of EGFR and MUC5AC proteins in the cells of these 4 groups was studied using cytoimmunity and Western blotting.

RESULTSRPMI-2650 cells were significantly confluent after incubated for 5 to 7 days. The shape of cells was round or oval, and a large number of microvilli covered to their surface but without cilia under scanning electron microscope. The EGFR protein was expressed in the cells of group A and D, abundantly in group B, while weakly in group C. The values of comparative absorbance had significant difference between group A, B, D and group C, respectively (P < 0.01). For the MUC5AC protein, its expression was strong in the cells of group A, abundant in group B, and weak in group C and D. Significant difference of the values of comparative absorbance was analyzed between group B and group C, D, respectively (P < 0.01), while no difference between group C and group D (P > 0.05).

CONCLUSIONSThe production of MUC5AC in human nasal epithelial cells RPMI-2650 is regulated via the expression and activation of epidermal growth factor receptor signaling pathway.

Cell Proliferation ; Epithelial Cells ; metabolism ; Humans ; Mucin 5AC ; genetics ; metabolism ; Mucins ; metabolism ; Nasal Cavity ; cytology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Tumor Cells, Cultured

Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P-selectin was then transfected to CHO(dhfr-) cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided a necessary basis for the following study of selection the antibodies targeting P-selectin.
Animals ; Antibodies ; metabolism ; CHO Cells ; Cell Membrane ; metabolism ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Epidermal Growth Factor ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; Lectins ; metabolism ; P-Selectin ; biosynthesis ; genetics ; immunology ; RNA ; metabolism ; Transfection

OBJECTIVETo investigate the regulation of epithelium growth factor receptor (EGFR), pan-Ras, and extracellular regulated protein kinase (ERK) with both a ras homologue member I (ARHI) suppression and epithelium growth factor (EGF) stimulation.

METHODSAfter identification and implication, the constructed plasmid pIRES2-EGFP-ARHI was transfected into Panc-1. The untransfected cell was also explored as controls. The growth curve was drawn to indicate the proliferation effect of ARHI. EGFR-ELISA was performed to investigate the expression of EGFR. Western blot analysis was used to investigate the expression of protein MAPK/ERK1/2, pan-Ras in Panc-1.

RESULTSThe proliferation rate of Panc-1 was inhibited by ARHI compared with both empty plasmid and untransfected cell. The amount of EGFR was parallel in both transfected and untransfected cell but affected by EGF stimulation. The amount of pan-Ras was decreased after ARHI transfection. The optimum concentration of EGF effect on P-ERK was 50 ng/ml.

CONCLUSIONBoth ARHI and EGF play roles in the EGF-EGFR-Ras-Raf-MAPK/ERK1/2 pathway.

Cell Proliferation ; Epidermal Growth Factor ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; Transfection ; Tumor Cells, Cultured ; ras Proteins ; metabolism ; rho GTP-Binding Proteins ; genetics

AIMTo explore the changes of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) expressions in cryptorchid children and their clinical significance.

METHODSThe level of serum EGF was measured by radioimmunoassay (RIA) and the expression of EGFR by immunohistochemistry.

RESULTS(1) The level of serum EGF was significantly lower in cryptorchid children than in normal subjects at age group of 5-9 years (P<0.01) and 10-14 years (P<0.01), (2) The level of EGF was significantly lower in boys with impalpable testis than in those with extracanalicular and intracanalicular testes (P<0.01), (3) The serum EGF level increased significantly 6 months after orchiopexy (P<0.05), (4) The EGFR expression in testicular Leydig's cells was lower in 2 approximately 4 year-old boys than in those over 5 years (P<0.05) and (5) the EGFR expression was less positive in the impalpable group and the intracanalicular group than that of the extracanalicular group (P<0.01).

CONCLUSIONThe EGF and EGFR expressions may correlate with the age and the position of testes; orchiopexy improves the EGF and EGFR expressions in cryptorchid boys.

Adolescent ; Aging ; Biomarkers ; analysis ; Child ; Child, Preschool ; Cryptorchidism ; blood ; metabolism ; pathology ; Epidermal Growth Factor ; blood ; metabolism ; Humans ; Immunohistochemistry ; Leydig Cells ; metabolism ; pathology ; Male ; Receptor, Epidermal Growth Factor ; blood ; metabolism

OBJECTIVETo investigate the effects of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the biological activity of glioblastoma cell line GL15.

METHODSGlioblastoma GL15 cells were cultured and transfected with LRIG3-EGFP plasmid. The location of LRIG3 in GL15 cells was observed with confocal microscopy. The proliferation and invasiveness of GL15 cells were detected with methyl thiazolyl tetrazolium (MTT) and Transwell methods respectively; the expression of epidermal growth factor receptor (EGFR) and LRIG3 mRNA and protein were detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot respectively.

RESULTAfter transfection with the plasmid LRIG-EGFP, LRIG3 fusion protein was found in cytoplasm of GL15 cells and cell proliferative and invasiveness were reduced. The expression of EGFR and LRIG3 varied with the duration of EGF treatment (100 ng/ml): the expression of EGFR decreased while the expression of LRIG3 increased as time prolonged.

CONCLUSIONLRIG3 can inhibit the proliferation and invasiveness of glioblastoma cells and may be used as a target gene in gene therapy of glioblastoma.

Brain Neoplasms ; pathology ; Cell Proliferation ; Epidermal Growth Factor ; genetics ; Glioblastoma ; pathology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Neoplasm Invasiveness ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

Wnt signaling pathway is a complex protein interaction network, and its function can most commonly or often be seen in embryonic development and cancer treatments, and meanwhile it is also involved in normal physiological processes in adult animals. Recently, with the rapid development of skin tissue engineering, there have been more and more researches on signal pathway in skin wound healing. At present, it is known that Wnt signaling pathway plays a vital role in the epidermal stem cells, epidermal growth factors, hair follicle development and other important factors related to the epidermal repair. The systemic research on Wnt signaling pathway has important clinical significance in the demonstration and functional process of the skin tissue. In this paper, we review the research development of the Wnt signaling pathway in the epidermal repair process.
Epidermal Growth Factor ; metabolism ; Epidermis ; cytology ; injuries ; Humans ; Stem Cells ; cytology ; Wnt Signaling Pathway ; physiology ; Wound Healing ; physiology