神经生长因子的研究进展

1　NGF及其受体的结构NGF是一种由三种不同类型的蛋白质组成的复合物,其分子量为1 4 0 0 0 0左右。其三种蛋白质分别为一个β亚单位和两个α亚单位以及两个γ亚单位以非共价键结合。其组合比例为α2 βγ2 。此外,该分子还包括了2个锌指,增加了复合物的稳定性。α亚单位的分子量为2 6 50 0 ,作用不明。γ亚单位分子量为2 6 0 0 0 ,有脂酶活性,能将无活性的γ亚单位转化为有活性的γ亚单位。先前的研究显示,β-NGF是一种二聚体分子,其单链含有1 1 8个氨基酸残基。近年的研究发现β-NGF的三维空间结构,其单位是由三对反向平行的β股构成…     

The effect of nerve growth factor and antibodies to nerve growth factor on ethylnitrosourea-induced neoplastic proliferation in rat trigeminal nerves

Sprague-Dawley (CD) rats were injected intravenously with ethylnitrosourea at a dose of 20 mg/kg on day 20 of gestation. This exposure resulted in early neoplastic proliferation or development of a neurinoma of the trigeminal nerve in 58% of the offspring at 90 days of age. Implantation of osmotic microinfusion pumps containing 2.5S nerve growth factor prior to ethylnitrosourea administration significantly reduced the incidence of early neoplastic proliferation. Postnatal implantation of microinfusion pumps containing 2.5S nerve growth factor also resulted in a significant but less pronounced reduction of early neoplastic proliferation. Immunoglobulin G directed against nerve growth factor (anti-nerve growth factor) did not influence the incidence of early neoplastic proliferation when administered via microinfusion pumps implanted on day 15 postnatally. These findings suggest that nerve growth factor has a protective effect on the developing nervous system against ethylnitrosourea-induced carcinogenesis.     

Tropism of canine neutrophils to xanthine oxidase

Quantitative evaluation of neutrophil chemotaxis was performed on cells obtained by hypotonic-lysis techniques from heparinized blood samples from clinically normal dogs. The techniques resulted in neutrophil recovery rates between 60 and 80%. Chemotaxis comparisons were based on cellular migration in microchambers equipped with polycarbonate membranes with 5-micron pores. Chemo-attractant comparisons were based on neutrophil migration to medium, normal canine plasma, zymosan-activated plasma, and xanthine oxidase. Cellular migration to zymosan-activated plasma in buffer (1:100 dilution) was significantly (P less than 0.001) enhanced over random baseline medium migration. Neutrophil migrations to normal canine plasma and xanthine oxidase were quantitatively less than to zymosan-activated plasma, but were equivalent to each other and significantly greater than for random migration. Migration to xanthine oxidase was maximal at concentrations near 1 U/ml within 30 minutes.     

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).     

Expression of vascular endothelial growth factor in canine mammary tumors

Vascular endothelial growth factor (VEGF) is a dimeric protein that stimulates angiogenesis in vitro and in vivo by inducing endothelial cell proliferation and migration. In this immunohistochemical study, VEGF-immunolabeled cells were counted in a series of 10 benign and 40 malignant canine mammary tumors. The morphologic pattern of VEGF positivity (intensity of immunolabeling and VEGF granule size and distribution) was also evaluated. A low number of cells weakly positive for VEGF with few and small granules polarized to the luminal pole was detected in benign neoplasms. In contrast, in malignancies a high number of VEGF-positive cells had strong immunolabeling, often with large granules found diffusely in the cytoplasm. This level of immunolabeling was more pronounced in the less differentiated, more malignant phenotypes (grade 3). Macrophages, which can synthesize VEGF, were strongly positive. Stromal and myoepithelial cells were negative. VEGF data were correlated statistically with intratumoral microvessel density (number of newly formed microvessels) and both measures were greater in less differentiated malignant neoplasms, demonstrating that angiogenesis and malignancy increase together. VEGF appears to be a powerful angiogenic factor in canine mammary tumors.     

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours*

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.     

Expression of vascular endothelial growth factor in canine oral malignant melanoma

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P ≤ 0.002). Concentrations of VEGF in the serum and plasma of dogs with melanoma were highly correlated (r = 0.867). Ninety‐five per cent of melanoma tissues expressed VEGF. Two staining patterns were detected: diffuse and granular cytoplasmic staining. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy (P = 0.002). Survival times were significantly longer in dogs receiving definitive therapy versus palliative therapy (median 496 versus 97 days, P = 0.007). Blood concentrations of VEGF were associated with stage (P < 0.05). Dogs with oral melanoma have increased serum, plasma and tissue concentrations of VEGF. Increased expression of VEGF may be a reasonable target for future therapy of canine oral melanoma.     

Vascular endothelial growth factor in diabetic and nondiabetic canine cataract patients

Objective To measure vascular endothelial growth factor (VEGF) levels in aqueous humor, serum, and plasma in diabetic and nondiabetic cataractous dogs. Methods Canine VEGF was assayed in the plasma and serum of 32 dogs (20 diabetics; 12 nondiabetics) and aqueous humor in 57 eyes of those dogs (39 diabetic; 18 nondiabetic) undergoing phacoemulsification, using a commercial canine VEGF assay. Statistical analysis was performed using Fisher’s PLSD, t‐test, and regression analysis to compare values by diabetic status, duration of diabetes, age, weight, gender, left vs. right eye, and blood clarity. Results Plasma, but not serum or aqueous humor VEGF values of diabetics were significantly greater than nondiabetics (P = 0.019). Older nondiabetics (10–15 years) had higher plasma VEGF values than younger (0–5 and 5–10 years) dogs (P = 0.0002 and 0.0001, respectively). There was no significant difference in aqueous humor VEGF between left and right eyes in all patients. Serum and plasma, but not aqueous humor, VEGF values in females were significantly higher than males in both groups. Conclusion Similar to human diabetic patients, VEGF aqueous humor values in all dogs are significantly higher than blood values. Aqueous humor VEGF values in human diabetics are elevated and correlate with the severity of diabetic retinopathy. However, aqueous humor values of VEGF in diabetic dogs are not greater than nondiabetics and may serve to protect the dog against development of diabetic retinopathy.     

Increased expression of vascular endothelial growth factor in neo-vascularized canine brain tissue

This retrospective study was done to characterize the levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1 (HIF-1α) in dog brains with neo-vascularization in the cerebral cortex of frontal, temporal, and parietal lobe by using immunohistochemistry (IHC) and Western blot. In neo-vascularized (NV) brains, we analyzed the number and area of blood vessels and the expression of VEGF and HIF-1α. The IHC results showed that the number and area of blood vessels, as assessed by immunolabeling for von Willebrand factor, was higher in the NV brain than in the control brain. The Western blot results showed that the level of VEGF was increased, predominantly in NV brain of the cerebral cortex relative to the clinically normal cerebral cortex, whereas the expression of HIF-1α in NV brains was not different from the control brains. Our study showed that dilatation of vessels and development of new vessels in the cerebral cortex were observed in cases of canine CNS disease and found increased expression of VEGF in canine brains with neo-vascularization.     

In vitro and in vivo bioactivity of recombinant canine hepatocyte growth factor

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

Epidermal growth factor receptor expression in canine transitional cell carcinoma

Transitional cell carcinoma (TCC), a urinary bladder tumor with high mortality, is encountered commonly in dogs. Whereas overexpression of epidermal growth factor receptor (EGFR) is associated with development of human urinary bladder cancer, information on EGFR expression in canine TCC is lacking. In this study, EGFR protein and mRNA expression in canine normal bladder (n=5), polypoid cystitis (n=5) and TCC (n=25) were examined by immunohistochemistry and real-time polymerase chain reaction. EGFR protein expression was significantly higher in TCC than that in normal healthy bladder (P<0.001) and polypoid cystitis (P<0.005). High EGFR protein expression was significantly (P<0.01) associated with TCC with a sensitivity of 72% and specificity of 100%. Comparative analysis of protein and mRNA expression levels in TCC showed significant positive correlation (r=0.88, P<0.05) between mRNA and protein expression. These findings suggest that intense expression of EGFR protein could be used as a marker to help canine TCC diagnosis.     

In vitro function of canine neutrophils during experimental inflammatory disease

The effect of acute inflammation on neutrophil function in the dog was studied by measuring in vitro phagocytosis and killing of Staphylococcus aureus. Phagocytosis was not impaired after 30 or 60 minutes and bactericidal activity was not impaired after 60 minutes incubation. However, average bactericidal activity after 30 minutes incubation was diminished significantly (P less than 0.01). Wide variations in bactericidal activity after 30 minutes incubation during the course of the inflammation did not correlate with neutrophil count, number of toxic neutrophils, or clinical course of the inflammation. These results indicate that a defect in bactericidal activity can occur in dogs with severe inflammatory disease, and that repeated assays, rather than single determinations, may be needed to detect this dysfunction.     

Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines

Canine mammary gland tumours (CMTs) are the most common malignancies in female dogs. The receptor tyrosine kinase EGFR (erbb1), a receptor for epidermal growth factor (EGF) and related factors, mediates multiple oncogenic functions in human epithelial neoplasms. While previous studies have demonstrated EGFR expression in canine tumours, its function has not been studied in canine cancer. The purpose of this study was to determine the in vitro effects of EGF and vandetanib (ZD6474), a small molecule inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, on proliferation, invasion, survival and chemosensitivity in CMT cells. In low serum, EGF enhanced proliferation and chemotaxis, attenuated apoptosis, and stimulated vascular endothelial growth factor (VEGF) production. Vandetanib dose-dependently inhibited EGFR phosphorylation as well as PI3K/Akt activation, and inhibited all EGF-induced phenotypic effects. In conclusion, EGF stimulates multiple features promoting the malignant phenotype in CMT. Thus, CMT may be an important translational model for the investigation of novel EGFR-directed therapies.     

Reverse effects of tetraarsenic oxide on the angiogenesis induced by nerve growth factor in the rat cornea

To compare the antiangiogenic effects of tetraarsenic oxide (As4O6) with those of diarsenic oxide (As2O3) in the rat cornea, rat cornea micropocket assay was conducted to induce angiogenesis by implantation of the pellet contained 1.0 ng of nerve growth factor (NGF). Ten of thirty eyes of Sprague-Dawley rats were randomly assigned to one of three groups, namely, control group (no medication), As2O3 group (50 mg/kg As2O3, PO, s.i.d.), and As4O6 group (50 mg/kg As4O6, PO, s.i.d.). After implantation, the number of new vessels, vessel length and clock hour of neovascularization were examined under the microscope from day 3 to day 7. The area of neovascularization was calculated using a mathematical formula. Although new vessels in control and As2O3 groups were first noticed at day 3, whereas those of As4O6 group were first observed on day 5. The number, length, clock hour of neovascularization and areas of the vessels in As4O6 group showed more significant inhibition than those of control and As2O3 groups from day 5 (P<0.05). However, there were no differences in all parameters between control group and As2O3 group during the entire study period. These results showed that As4O6 had antiangiogenic effects on the new vessels induced by NGF in the rat cornea.     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.