动物乳腺生物反应器生产人乳铁蛋白的研究进展

利用动物乳腺生物反应器生产人乳铁蛋白产量高,工艺简单,在医药方面具有广泛的应用前景。本文就人乳铁蛋白的生物学功能,人乳铁蛋白基因的表达调控与载体的构建,生产人乳铁蛋白的方法、现状,存在的问题及其展望加以综述。     

动物乳腺生物反应器生产人乳铁蛋白的研究进展

利用动物乳腺生物反应器生产人乳铁蛋白产量高，工艺简单，在医药方面具有广泛的应用前景。本文就人乳铁蛋白的生物学功能，人乳铁蛋白基因的表达调控与载体的构建，生产人乳铁蛋白的方法、现状，存在的问题及其展望加以综述。     

乳铁蛋白对细胞增殖与分化的影响

乳铁蛋白（LF）作为一种天然的具有免疫功能的糖蛋白，其多种生物学功能早在20世纪60年代就引起了人们普遍的关注。在食品方面，乳铁蛋白作为牛乳中天然的功能性强化剂已经成为众多乳品研发人员关注的焦点。乳铁蛋白在国外已广泛应用于乳制品中，尤其婴儿配方奶粉中的使用较多。在免疫方面，乳铁蛋白可以促进巨噬细胞或中性白细胞的杀菌作用和吞噬作用，调节NK细胞的活性和淋巴细胞、中性白细胞的增殖。     

牛乳铁蛋白稳定性的研究

通过对牛乳铁蛋白在不同温度下处理一定的时间，研究牛乳铁蛋白热的稳定性，并模拟胃液环境研究不同胃蛋白酶浓度下，牛乳铁蛋白对胃蛋白酶消化的抵抗作用，研究其蛋白酶解稳定性。结果表明，牛乳铁蛋白在一定时间内，提高温度不会发生明显的降解，不同浓度的胃蛋白酶对牛乳铁蛋白的消化作用不同，牛乳铁蛋白对1μg／mL胃蛋白酶的抵抗能力最强。     

牛乳铁蛋白稳定性的研究

探讨牛乳铁蛋白在不同温度下的热稳定性，并研究其蛋白酶解稳定性性质。使牛乳铁蛋白在不同温度下处理一定时间，然后通过SDS—PAGE法检测其降解情况，并模拟胃液环境研究不同浓度胃蛋白酶对牛乳铁蛋白酶解的变化，根据电泳结果的灰度扫描计算不同时间消化后牛乳铁蛋白的残存率。结果表明，牛乳铁蛋白在一定时间内提高温度不会发生明显的降解，不同浓度的胃蛋白酶对牛乳铁蛋白的消化作用不同，牛乳铁蛋白对1μg／mL胃蛋白酶的抵抗能力最强，但是在被消化120min后牛乳铁蛋白残存率下降到1％左右。     

牛乳生物活性成分及其研究进展

牛乳除了能够提供人们日常所需的营养物质外,还含有种类众多的生物活性成分,如免疫球蛋白、乳铁蛋白、生长因子和具备多种生理功能的生物活性肽等。这些生物活性物质是牛乳的精华所在,对人体的新陈代谢和健康具有非常重要的影响。综述了牛乳中几种主要生物活性物质的生物学功能及其研究进展。     

乳铁蛋白的免疫调节作用及在动物生产中的应用

乳铁蛋白(lactoferrin,Lf)是一种非血红素铁结合性糖蛋白,在乳腺分泌物中含量丰富。近年的研究表明乳铁蛋白有着更广泛的生物学功能,文章就乳铁蛋白对机体免疫调节及其机理作一综述,随着乳铁蛋白免疫机制的进一步深入研究,其作为免疫调节剂将会越来越广泛地应用于动物生产实际中。     

乳铁蛋白的免疫调节作用及在动物生产中的应用

乳铁蛋白（1actoferrin，Lf）是一种非血红素铁结合性糖蛋白，在乳腺分泌物中含量丰富。近年的研究表明乳铁蛋白有着更广泛的生物学功能，文章就乳铁蛋白对机体免疫调节及其机理作一综述，随着乳铁蛋白免疫机制的进一步深入研究，其作为免疫调节剂将会越来越广泛地应用于动物生产实际中。     

乳铁蛋白及其在家畜生产中的应用

乳铁蛋白 (Lactoferrin ,LF)是一种天然的、具有免疫功能的糖蛋白 ,由转铁蛋白演变而来 ,具有多种生物活性 ,如促进铁吸收、抑菌抗病毒、促进细胞增殖、调节骨髓细胞生成、调节补体系统、刺激溶菌酶再生、防止脂质过氧化等功能。它广泛存在于人与哺乳家畜的分泌物中 ,如人乳、牛乳(Lonnerdal,1 994)。乳铁蛋白在母乳中蛋白质含量仅次于酪蛋白 ,占普通母乳蛋白质的 1 0 % ,人乳中含量为 2～ 4mg/mL ,牛乳中含量为 0 0 2～0 35mg/mL ,商品乳铁蛋白一般是从牛乳中提取的。除乳汁外 ,其他消化、呼吸和生殖系统的外分泌液中也含有一定量的乳…     

乳铁蛋白检测及应用研究进展

乳铁蛋白(lactoferrin,LF),也称为乳运铁蛋白(1actotransferrin),是牛乳中重要生物活性物质之一。本文综述了乳铁蛋白的含量、分布,分离提纯及定性定量检测方法,同时阐明了乳铁蛋白在食品、动物生产及其他领域的应用。     

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.     

Lactoferrin against Staphylococcus aureus Mastitis. Lactoferrin alone or in combination with penicillin G on bovine polymorphonuclear function and mammary epithelial cells colonisation by Staphylococcus aureus

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

乳铁蛋白基因启动子元件调控效应分析

本试验先后进行2轮载体构建及转染试验,对牛乳铁蛋白基因启动子元件调控效应进行了分析。首先进行第1轮载体构建及转染试验：采用PCR方法,利用4对引物从牛乳铁蛋白基因5′调控区-1 799向3′端依次缺失500bp进行调控序列扩增,将获得的扩增片段替换pEGFP-N1的CMV启动子,构建了4个重组载体,将重组载体转染牛乳腺上皮细胞（BMEC）,经筛选获得稳定的单克隆细胞,测定并比较各组细胞的荧光强度。在以上试验基础上,为进一步精确确定牛乳铁蛋白基因启动子顺式调控元件序列,进行第2轮载体构建及转染试验,方法同第1轮。结果表明,牛乳铁蛋白基因上游调控序列-1 323～-1 372存在负调控元件,-1 372～-1 560存在正调控元件。     

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.     

乳铁素参与乳房链球菌对乳腺上皮细胞的粘附

免疫印迹试验表明，试验用的3株乳房链球菌（Streptococcus uberis)菌株均可与乳铁素（Lactoferrin)结合。培养基中加入乳铁素或乳清可以显著促进细菌与乳腺上皮细胞之间的粘附，抗乳铁素抗体可以特异性地抑制乳铁素或乳清预处理细菌与乳腺上皮细胞的粘附。乳铁素在细菌和细胞之间起着桥梁分子作用。有助于细菌与乳腺上皮细胞之间的粘附和乳腺感染的建立。     

Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal,ejaculated and frozen-thawed sperm

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p < .05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30 min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H₂O₂, the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.     

Effects of bovine lactoferrin on in vitro replication of feline herpesvirus

Objective To investigate the effects of bovine lactoferrin on in vitro replication of feline herpes virus (FHV‐1) and to determine at what points during viral replication these effects occur. Sample population Cultured Crandell‐Reese feline kidney (CRFK) cells and FHV‐1 strain 727. Procedure Five concentrations of bovine lactoferrin (0.5, 1, 2, 5, and 10 mg/mL) were added at one or more of three time points during conventional plaque reduction assays: (a) uninfected CRFK cells were incubated in lactoferrin‐containing medium for 30 min prior to viral adsorption; (b) virus was suspended in lactoferrin‐containing medium prior to and during adsorption, or (c) CRFK cells were incubated with lactoferrin‐containing medium for 48 h following viral adsorption. Plaques were counted and antiviral effect expressed as percent inhibition relative to control medium that contained no lactoferrin. Results Exposure of CRFK cells to lactoferrin prior to or during viral adsorption inhibited FHV‐1 replication by 87–96% (mean: 91%). Application of lactoferrin following viral adsorption had no appreciable effect on FHV‐1 replication. No additive or synergistic effects were noted when lactoferrin was added at multiple steps. These effects were similar at all concentrations of lactoferrin tested. Cytotoxic effects of lactoferrin on CRFK cells were not observed at any concentration tested. Conclusions and clinical relevance Bovine lactoferrin has a notable inhibitory effect on the in vitro replication of FHV‐1 prior to and during, but not following viral adsorption. These findings strongly suggest that lactoferrin inhibits FHV‐1 adsorption to the cell surface and/or penetration of the virus into the cell. Clinical effects of topical lactoferrin in acute or recrudescent herpetic episodes in cats warrant investigation.     

Reduction of Escherichia coli O157:H7 excretion in sheep by oral lactoferrin administration

Ruminants are an important reservoir of Escherichia coli O157:H7, therefore reducing E. coli O157:H7 excretion by these animals could play a key role in reducing human infections. The present study investigates the potential of bovine lactoferrin, a natural antimicrobial-immunomodulatory protein of milk, to prevent colonization and excretion of E. coli O157:H7 in sheep. The effect of two different doses of lactoferrin (1.5 g or 0.15 g per 12h) was evaluated on colonization of sheep intestine and faecal excretion of the NCTC12900 strain. Hereto, lactoferrin was orally administered to sheep during 30 consecutive days and sheep were experimentally infected with E. coli O157:H7 on the second day of the lactoferrin administration. Interestingly, both lactoferrin dosages significantly reduced the number of E. coli O157:H7 in faeces as well as the duration of faecal excretion. The high dose group showed a significantly higher antibody response against EspA and EspB, two structural proteins of the bacterial type III secretion system (TTSS), than the colonization control group. The results suggest that oral lactoferrin administration could be used to prevent persistent colonization of sheep with E. coli O157:H7.