猪粒细胞-巨噬细胞集落刺激因子的原核表达和纯化

粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)是一种具有多种生物学活性的细胞因子。本试验根据GenBank发表的猪的GM-CSF基因序列设计特异性引物,对GM-CSF基因片段进行扩增,扩增后的PCR产物经酶切后克隆入pET-28a载体。重组质粒转化入大肠杆菌杆菌BL21中经IPTG诱导表达,SDS-PAGE结果证明,重组蛋白获得了成功表达,并且主要以包涵体的形式存在。Western blot结果证明重组蛋白具有良好免疫反应性。以上结果为进一步应用重组GM-CSF作为血液干细胞动员剂提供了基础。     

The effect of neutrophils, tumor necrosis factor, and granulocyte macrophage/colony stimulating factor on Babesia bovis and Babesia bigemina in culture.

Bovine neutrophils, human recombinant tumor necrosis factor-alpha (TNF), and bovine recombinant granulocyte macrophage/colony stimulating factor (GM/CSF) were added to microaerophilic cultures of Babesia bovis and Babesia bigemina to determine if those substances could inhibit growth. Incorporation of [3H]hypoxanthine by the Babesia spp. was utilized as an indirect measure of parasite growth. When neutrophils were added to cultures of B. bovis and B. bigemina, the highest percentage inhibition of growth was attained. There was no significant enhancement of neutrophil killing when TNF or GM/CSF or both were added to either Babesia spp. Addition of TNF or GM/CSF or both substances (without neutrophils) resulted in an increase in growth of B. bovis and B. bigemina. For B. bovis, the group that contained neutrophils only and the group that contained neutrophils and TNF resulted in significantly higher growth inhibitions than the treatment group which contained neutrophils and GM/CSF or the group that contained neutrophils, TNF, and GM/CSF. No significant differences in inhibition were observed for the same treatment groups between B. bovis and B. bigemina.     

表皮生长因子的研究进展

表皮生长因子(Epidermal growth factor简称EGF)是Cohen.S于1962年研究神经生长因子时发现的.最初发现它具有促使新生动物睁眼和萌牙的作用,由于EGF的发现及研究成果,Cohen等两位科学家成为1986年度诺贝尔医学和生理学奖获得者,因而这个领域变得更加引入注目,随着对EGF研究的不断深入,在EGF的研究和应用上均取得了很大的进展.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.     

The growth of swine bone marrow cells in the presence of heterologous colony stimulating factor: Characterization of the developing cell population

Conditioned medium prepared from the mouse fibroblast cell line L 929 which is known to produce colony-stimulating factor active on mouse bone marrow cells was also able to stimulate the growth of swine bone marrow cells in a liquid culture system. During the first 4 days of culture mononuclear phagocyte and granulocyte colonies developed. Prolonged cultured cells were classified belonging to the macrophage lineage by morphological and cytochemical criteria. These cells fulfill also functional characteristics for macrophages, like expression of Fc receptors, immune phagocytosis and production of prostaglandins. These bone marrow-derived macrophages could also be activated with LPS and lymphocyte-derived mediators.     

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.     

Epidermal growth factor receptor as a therapeutic target in veterinary oncology

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that stimulates cell proliferation and survival and becomes dysregulated in a range of solid tumours in man. It is recognized as a key oncogenic driver and has become a favoured therapeutic target and a prognostic and predictive marker of cancer in man. In animals, EGFR dysregulation is emerging as a potential factor in the development of a number of naturally occurring tumours including mammary, lung, glial and epithelial cancers. Comparative analyses suggest that these diseases share many features with equivalent diseases in man and EGFR may have value as a prognostic or a biological marker of animal disease. There is still little direct evidence that EGFR is a critical oncogenic driver in naturally occurring animal disease and there are no veterinary trials of EGFR-targeted therapy. These will be critical steps in establishing a role for EGFR in veterinary oncology.     

Epidermal growth factor receptor expression in feline oral squamous cell carcinomas

Feline oral squamous cell carcinomas (SCC) have a poor prognosis despite aggressive treatment with surgery, radiation and anticancer drugs. Overexpression of the epidermal growth factor receptor (EGFR), a membrane‐bound tyrosine kinase receptor, has been found in many human epithelial neoplasms, including oral SCC. EGFR overexpression has been associated with advanced disease and a poor prognosis. The purpose of this study was to determine whether feline oral SCC express EGFR. Thirteen formalin‐fixed paraffin wax‐embedded biopsy samples from feline oral SCC were analysed for EGFR expression using immunohistochemistry. Nine of 13 tumours (69%) were positive for EGFR expression, suggesting that altered EGFR expression plays a role in feline oral SCC and provides a rationale for a potential clinical benefit using EGFR inhibitors in combination with conventional treatments.     

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…     

Hemogram changes in lactating dairy cows given human recombinant granulocyte colony stimulating factor (r-MethuG-CSF).

As a prelude to mammary gland challenge experiments, this investigation was implemented to assess the hematologic changes in lactating dairy cattle induced by two dosage regimes of human recombinant colony stimulating factor (Hr-GCSF). This study documents the capability of the human recombinant colony stimulating factor to produce hematologic changes in both a time and dose dependent manner when administered to the adult lactating bovine. A screening dose of 1 microgram/kg of Hr-GCSF administered to three study subjects produced a three- to four-fold increase in peripheral blood mature neutrophil counts (P less than 0.043) by day 12 of the trial. The priming dose treatment group of four lactating cows (3 micrograms/kg of Hr-GCSF) exhibited a three- to five-fold increase in peripheral blood mature neutrophil counts (P less than 0.05) and two- to three-fold increases in white blood cell counts by day 5 of the trial. Hematologic examinations of the control group (n = 4; no Hr-GCSF administration) did not detect significant changes in their neutrophil counts over baseline values. The milk somatic cell counts did not statistically shift over baseline values in any of the control or Hr-GCSF treatment groups. When attempting to alter the course of infectious disease processes, potential applications of colony stimulating factors provide interesting speculations about new therapeutic modalities.     

Hematologic improvement in dogs with parvovirus infection treated with recombinant canine granulocyte‐colony stimulating factor

Duffy A., Dow S., Ogilvie G., Rao S., Hackett T. Hematologic improvement in dogs with parvovirus infection treated with recombinant canine granulocyte‐colony stimulating factor. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01153.x. Previously, dogs with canine parvovirus‐induced neutropenia have not responded to treatment with recombinant human granulocyte‐colony stimulating factor (rhG‐CSF). However, recombinant canine G‐CSF (rcG‐CSF) has not been previously evaluated for treatment of parvovirus‐induced neutropenia in dogs. We assessed the effectiveness of rcG‐CSF in dogs with parvovirus‐induced neutropenia with a prospective, open‐label, nonrandomized clinical trial. Endpoints of our study were time to recovery of WBC and neutrophil counts, and duration of hospitalization. 28 dogs with parvovirus and neutropenia were treated with rcG‐CSF and outcomes were compared to those of 34 dogs with parvovirus and neutropenia not treated with rcG‐CSF. We found that mean WBC and neutrophil counts were significantly higher (P < 0.05) in the 28 dogs treated with rcG‐CSF compared to disease‐matched dogs not treated with rcG‐CSF. In addition, the mean duration of hospitalization was reduced (P = 0.01) in rcG‐CSF treated dogs compared to untreated dogs. However, survival times were decreased in dogs treated with rcG‐CSF compared to untreated dogs. These results suggest that treatment with rcG‐CSF was effective in stimulating neutrophil recovery and shortening the duration of hospitalization in dogs with parvovirus infection, but indicate the need for additional studies to evaluate overall safety of the treatment.     

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.     

Epidermal growth factor-like activity in mares' milk.

Epidermal growth factor (EGF)-like activity was measured in mares' colostrum and milk by radioreceptor assay. Milk samples were collected from 22 mares 1 or more times during early lactation. Samples of colostrum were taken after parturition and before the foal first suckled (presuckle), within 6 hours after the foal first suckled (postsuckle), and on days 1, 2, 4, and 8 of lactation. In the 5 mares from which milk samples were obtained at each sampling time, presuckle colostral mean EGF-like activity (17.8 ng/ml) was greatest (P less than 0.05). The mean values for EGF-like activity at all other sampling times were not significantly different from each other (postsuckle colostrum, 9.7 ng/ml; day 1, 9.6 ng/ml; day 2, 8.5 ng/ml; day 4, 8.0 ng/ml; day 8, 7.8 ng/ml).     

Plasma growth hormone concentrations in growth-retarded, cortisone treated chickens.

1. Weekly variations in body weight, relative body-weight gain and plasma growth hormone (GH) concentrations were determined in control and cortisone-treated (5 mg cortisone acetate/bird d) chickens between 3 and 10 weeks of age. 2. Treatment with cortisone between 3 and 5 weeks of age markedly suppressed growth but did not inhibit GH secretion. 3. After the cortisone injections were stopped these birds grew more rapidly than the controls. However, this period of "catch-up growth" was not accompanied by alterations in plasma GH concentrations.     

Expression of nerve growth factor and its receptors, TrkA and p75, in porcine ovaries

The cellular localization of nerve growth factor (NGF) and its receptors (TrkA, p75) was investigated during the estrous cycle in gilts. Also, the levels of expression of these factors in walls of tertiary follicles and corpora lutea (CLs) were determined using Western blot. The ovaries from days 3, 7, 16 and 20 of the cycle revealed the presence of NGF and its receptors in oocytes of secondary and tertiary follicles, follicular cells of primary and secondary follicles, thecal and granulosa cells of tertiary follicles and steroidogenic cells of CLs. In wall cells of primary follicles, NGF, TrkA and p75 staining was strongest on day 16, while in secondary follicles, only p75 was more intensely stained on day 16 and 20. In walls of small (to 3 mm in diameter) and medium (4-6 mm in diameter) follicles, NGF staining was lower on day 16, and the p75 reaction was strongest on day 20. On day 20, NGF staining in large follicles (7-10 mm in diameter) was higher than in smaller follicles. The levels of NGF and p75 in small and medium follicles were highest on day 20. The contents of NGF and TrkA in large follicles on day 20 were higher than in smaller follicles. NGF and TrkA contents in CLs were highest on day 7. Our study demonstrates that NGF, TrkA and p75 are expressed in the ovary during the estrous cycle in gilts. These results suggest that NGF and its receptors may be important for ovarian function in cycling gilts.     

Age-related effects on motor nerve conduction velocity in chickens.

Compound motor-nerve action potentials evoked by supramaximal stimulation of the proximal and distal aspects of the tibial nerve were evaluated in chickens 1 to 15 weeks old. Motor-nerve conduction velocity increased from a mean of 22.6 m/s at week 1 to a mean of 52.7 m/s at week 15. The increase in conduction velocity was greatest for the first few weeks, and reached a plateau at 10 weeks. Subcutaneous limb temperature, limb length, and proximal latency measurements also increased with age; however, distal latency measurements were not significantly influenced by age. A quadratic equation was calculated to predict mean motor-nerve conduction velocity for maturing chickens.     

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.     

Effect of daily injections of growth hormone-releasing factor and thyrotropin-releasing hormone on growth and endocrine parameters in chickens

The control of growth is a complex mechanism regulated by several metabolic hormones including growth hormone (GH) and thyroid hormones. In avian species, as well as in mammals, GH secretion is regulated by hypothalamic hypophysiotropic hormones. Since thyrotropin-releasing hormone (TRH) and growth hormone-releasing factor (GRF) are potent GH secretagogues in poultry, we were interested in determining the influence of daily intravenous administration of either peptide or both simultaneously on circulating GH and IGF-I concentrations and whether an improvement in growth rate or efficiency would be obtained.

Male broiler chicks were injected once daily for a period of 21 days with either GRF (10 μg/kg), TRH (1 μg/kg) or both GRF and TRH (10 and 1 μg/kg respectively) between four and seven weeks of age. On the last day of the experiment, following intravenous injection of TRH, GRF or a combination of GRF and TRH, plasma GH levels were significantly (P<.05) increased to a similar extent in control chicks and in those which had received daily peptide injections for the previous 21 days. Circulating GH levels between 10 and 90 min post-injection were significantly (P<.05) greater and more than additive than GH levels in chicks injected with both GRF and TRH when compared to those injected with either peptide alone. Mean plasma T₃ concentrations during that same time period were significantly elevated (P<.05) above saline-injected control chick levels in birds treated with TRH or GRF and TRH respectively, regardless of whether the chicks had received peptide injections for the previous 21 days. There was no evidence of pituitary refractoriness to chronic administration of either TRH or GRF injection in terms of growth or thyroid hormone secretion.

Despite the large elevation in GH concentration each day, growth rate, feed efficiency and circulating IGF-I concentrations were not enhanced. Thus the quantity or secretory pattern of GH secretion induced by TRH or GRF administration was not sufficient to increase plasma IGF-I concentration or growth.     

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).