咖啡因、双丁酰环磷腺苷调控体外猪嗜中性粒细胞对精子吞噬性的研究

实验旨在为提高家畜人工授精效率,寻找调控人工授精诱导子宫局部免疫反应的方法。通过添加不同浓度的咖啡因、双丁酰环磷腺苷(dbcAMP)检测嗜中性粒细胞(PMN)对精子或荧光beads的吞噬率。结果表明:1 mmol/L及以上浓度的咖啡因或0.1mmol/L dbcAMP明显地抑制PMNs对精子或荧光beads的吞噬性。咖啡因和dbcAMP抑制PMNs的吞噬性,可以作为降低子宫免疫反应的精液稀释液成分,提高人工授精的效率。     

miRNA调控嗜中性粒细胞和T细胞在呼吸道疾病发生中的作用

正MicroRNA(miRNA)是一类长约19~24 nt的内源性、小型非编码RNA,广泛存在于各种生物体中,miRNA可以通过与靶基因的相互作用,以序列特异性方式调控靶基因的表达,参与多种生理、生化、免疫调控和病理过程[1]。文献显示,在呼吸道疾病的发生发展过程中,miRNA能够通过对免疫系统的调节在炎症反应中扮演重要角色。1 miRNA调控嗜中性粒细胞在呼吸道疾病发生中     

猪转移因子对犬中性粒细胞数量和活性的影响

按标准方法提取制备了猪的转移因子（transfer factor, TF）,用吞噬杀伤试验MTT法检测了供试杂种牧羊犬肌肉注射猪TF后外周血中性粒细胞吞噬杀伤活性的变化。试验摸索出MTT法测定犬外周血中性粒细胞吞噬杀伤大肠杆菌的最佳条件为：中性粒细胞浓度1.3×106个/ml、大肠杆菌浓度6×105个/ml 时,大肠杆菌和中性粒细胞混合培养2 h,加入MTT后继续培养4 h。体外试验结果表明,猪TF浓度在0.052～1.56 mg/ml范围内,能够明显促进中性粒细胞吞噬杀菌作用,当猪TF浓度为1.56 mg/ml时,对中性粒细胞杀菌活性的影响最大。体内试验结果表明,注射猪TF后第2 d,外周血中性粒细胞数量最高,中性粒细胞吞噬杀菌能力最强。     

猪转移因子对犬外周血中性粒细胞数量和杀菌活性的影响

无菌采犬外周血,肝素抗凝,制备不同浓度的粒细胞悬液,分别与不同浓度的大肠杆菌悬液共同培养,采用MTT法测定粒细胞的杀菌效果,确定粒细胞体外培养时杀伤细菌的最优化条件;把不同浓度的猪转移因子(Swine transfer factor,sTF)加入注射sTF前后不同时期分离的犬外周血中粒细胞与E.coli的培养系统中,测定粒细胞的杀菌能力。结果表明,当粒细胞浓度为1.3×106个/mL,E.coli浓度为6×105个/mL时,试验最能反映粒细胞的杀菌效果。当sTF浓度为1.56mg/mL时,对PMN杀菌活性的影响最大;在犬注射sTF后的第2d,中性粒细胞单独杀菌活性最好,并且此时外周血中性粒细胞的数量达到峰值。本研究结果表明,sTF不仅能够增强犬中性粒细胞的杀菌活性,还能增加犬外周血中性粒细胞的数量。     

活化卵白蛋白对兔嗜中性粒细胞杀菌功能的影响

将25只大耳白兔随机分为3组，分别以100mg/kg、250mg/kg、500mg/kg的剂量连续灌服活化卵白蛋白5d，并在用药前后分别采血，测定嗜中性粒细胞杀菌功能。结果表明，100mg/kg即可显著提高嗜中性粒细胞杀菌功能（P＜0．05），250mg/kg和500mg/kg可极显著提高嗜中性粒细胞杀菌功能（P＜0．01）。     

大鼠外周血嗜中性粒细胞分离方法的优化改良

本试验旨在寻找一种高效、简便、经济及可靠的方法从大鼠外周血中分离出嗜中性粒细胞(PMNs),为后续抗菌药物筛选及机理研究提供基础。在常规的Percoll密度梯度离心法基础上加入淋巴细胞分离液以去除淋巴细胞,并对离心温度、Percoll浓度等进行改良,考察优化后方法提取的PMNs数量、活力及纯度。试验结果显示:(1)优化后提取的PMNs数量与常规方法相比差异显著(P0.05),约为常规提取方法的1.2倍;(2)优化后方法和常规方法提取的PMNs活力均为97%以上,无差异(P0.05);(3)优化后方法提取的PMNs纯度约为80%,常规方法提取纯度为20%,优化后方法与常规提取方法相比差异极显著(P0.01)。表明优化改良后的方法分离出的PMNs纯度高,活性好,数量多,是一种简单、高效的PMNs分离方法。     

C5a对类风湿关节炎小鼠嗜中性粒细胞的募集作用

通过使用双光子显微镜活体成像技术优化了K/B×N血清诱导的关节炎小鼠模型,并观察了嗜中性粒细胞的募集情况。皮下注射K/B×N血清可以快速诱导大量的嗜中性粒细胞募集到局部炎症关节。通过使用这种方法,发现阻断C5a活性会减少嗜中性粒细胞募集,说明C5a是关节炎期间嗜中性粒细胞募集的关键调节因子。因此,本试验将为补体相关生物制剂治疗类风湿关节炎提供新的靶点。     

肝素和钙离子载体诱导猪射出精子体外获能的研究

本文采用肝素和钙离子载体诱导猪射出精子体外获能,根据对去透明带仓鼠卵的穿透情况评价获能效果,并用台盼兰·姬姆萨染色观察精子的死活及顶体状态。结果表明,未经获能处理的猪精子不能穿透仓鼠卵,肝素和I-A均能诱导猪精子体外获能并引起顶体反应。经获能处理的精子,在授精后2小时穿透卵子,4小时开始形成雄原核,6小时形成发育良好的雄原核。肝素处理以100μg效果最好,穿透率达77.8%,有原核卵百分率达47.2%;I-A处理以0.3uM为宜,穿透率达66.7%,有原核卵百分率达42.4%。咖啡因与肝素和I-A具有协同作用,能提高精子的穿透能力。有顶体反应的活精子百分率与穿透率呈强正相关(肝素组:r=0.97,P<0.01;I-A组:r=0.94,P<0.01)。     

应注意应激导致的围产期奶牛嗜中性粒细胞增多症

围产期奶牛在很短的时间内所经历的变化对其本身而言是巨大的。首先,奶牛的泌乳突然停止,并在短时期内重新开始泌乳,打乱了奶牛正常的秩序。其次,在这个时期内奶牛经历了多达三种以上的日粮变化(泌乳后期日粮、干奶期日粮、围产前期日粮、围产后期日粮),影响奶牛瘤胃微生物群的数量和种类,由此影响饲料的消化;日粮变化影响瘤胃构造和其吸收能力,由此影响饲料的效率;在这个时期内奶牛从高精日粮到高粗日粮引起奶牛瘤胃乳头的退化,此时瘤胃吸收表面积减少50%,当喂给精料含量增加的围产期日粮时,乳头开始变长但需要4～6周的时间。这些就是在这…     

Phagocytosis and Related Phenomena in Polymorphonuclear Leukocytes from Cow's Milk

There were fewer efficient phagocytes among leukocytes collected from artificially irritated mammary glands than among the leukocytes from blood of the same animals. The milk polymorphonuclear (PMN) leukocytes adhered poorly to a column of siliconised glass beads when compared with the blood cells. However, investigations of the O₂ uptake and CO₂ production of the milk PMN leukocytes revealed that these cells appeared to utilize metabolic pathways similar to those used by human and guinea pig PMN leukocytes during phagocytosis. These pathways are associated with degranulation and the production of H₂O₂ following particle ingestion. It is therefore suggested that the milk PMN leukocytes appear not to have lost the ability to produce this bactericidal substance.     

Role of polymorphonuclear neutrophils (PMNs) and NK cells in the protection conferred by different vaccines against Chlamydophila abortus infection

Ovine enzootic abortion (OEA) is caused by Chlamydophila abortus, an intracellular bacterium which acts by infecting the placenta, causing abortion in the last term of gestation. The main prevention strategy against OEA is the vaccination of flocks. An effective vaccine against C. abortus must induce a Th1-like specific immune response, which is characterized by the early production of IFN-gamma and the activation of CD8(+)T cells. Moreover, vaccine effectiveness could be modulated by the functioning of the innate immunity. The purpose of this study was to ascertain how polymorphonuclear neutrophils (PMNs) and NK cells might influence vaccine-induced protection. The live attenuated 1B vaccine and two inactivated experimental vaccines, adjuvated with aluminium hydroxide (AH) or QS-21 (QS), were used in PMN-depleted or NK cell-depleted mice. For PMN depletion, RB6-8C5 monoclonal antibody, which recognizes GR1(+) receptors (Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D. 2005. Recruitment of Gr-1(+) monocytes is essential for control of acute toxoplasmosis. The Journal of Experimental Medicine 201, 1761-1769.) was used, while for NK cell-depletion the anti-asialo GM1 polyclonal antibody was used. The depletion of PMNs caused 100% mortality in non-vaccinated mice (NV) and 60% mortality in the AH-vaccinated mice by day 10 p.i., while both groups showed a significant increase in their bacterial burden in the liver by day 4 p.i. The depletion of NK cells caused mortality only in the NV group (50% by day 10 p.i.), although this group and the 1B vaccinated mice showed an increased bacterial burden in the liver at day 4 p.i. Our results suggest that the importance of PMNs in inactivated vaccines depends on the adjuvant chosen. The results also demonstrated that the importance of NK cells is greater in live vaccines than in inactivated vaccines.     

In Vitro Effects of Acellular Milk on the Bactericidal Components of Caprine Polymorphonuclear Neutrophils

The effects of acellular milk on the activity of the microbicidal cationic enzymes of the polymorphonuclear cells of goats were studied in an attempt to explain the phenomenon by which PMN functions fail in mastitis. Assays were undertaken on the myeloperoxidase, lysozyme and elastase activities in a polymorphonuclear cell (PMN) lysate, both in the presence and absence of acellular milk from homologous species. There was a significant decrease (p<0.05) in the activity of lysozyme, myeloperoxidase and elastase in the presence of acellular milk. Superoxide and H₂O₂ production following activation of caprine PMNs by lipopolysaccharide (LPS) was significantly reduced (p<0.05) in the presence of acellular milk. Thus, the microbicidal function of PMNs is significantly impaired in the presence of acellular milk and this may contribute to the development of mastitis in dairy animals.     

肝素和钙离子载体诱导波尔山羊精子体外获能和体外受精

运用肝素、钙离子载体（IA）协同咖啡因诱导波尔山羊精子体外获能，利用去透明带金黄地鼠卵穿透试验检测了精子的获能效果。结果发现：精卵孵育4h和6h时的穿透率均高于2h时的穿透率；肝素和钙离子载体对精子获能有明显的促进作用，与未添加组相比差异极显著（P〈0．01）；20mg／L和50mg／L的肝素剂量组，去透明带金黄地鼠卵穿透率为64．7％、68．4％；钙离子载体的添加剂量在0．3μmol／L时，穿透率达到68．2％；咖啡因与肝素、钙离子载体具有明显的协同作用。将获能的精子与山羊卵母细胞进行体外受精孵育17h，也获得了60％以上的受精率。     

猪瘟活疫苗(传代细胞源)免疫猪的攻毒保护试验

为进一步证明猪瘟活疫苗(传代细胞源)在猪场实际使用的免疫效力,本试验将中试生产的5批疫苗在广东的广州、韶关地区各选一个猪场进行临床应用试验。按照20日龄首免,60日龄二免的免疫程序,并分别于二免后第28天和第90天从5批疫苗免疫猪中各抽取5头,连同条件相同的对照猪各5头,在广东永顺生物制药股份有限公司负压动物舍进行猪瘟强毒攻击,观察发病死亡情况,评估猪瘟活疫苗(传代细胞源)在猪场实际应用的免疫保护效果。试验结果显示,5批疫苗免疫猪均能有效抵抗猪瘟强毒的攻击,既无死亡,也无猪瘟临床症状。表明在临床条件下应用猪瘟活疫苗(传代细胞源)免疫猪能产生很好的免疫保护力。     

In vitro Studies of Group E Streptococci in Swine Leukocytes I. Phagocytic and Bactericidal Properties of Polymorphonuclear Leukocytes from Swine

Serum from both immune and nonimmune ten-week-old swine contained factors which promoted phagocytosis of group E Streptococci (GES). The factors in nonimmune serum, which were heat labile at 70°C for ten minutes, were less efficient than the factors present in immune serum.

Bactericidal activity of the polymorphonuclear (PMN) leukocytes against GES was observed with serum from both immune and nonimmune ten-week-old swine, as well as with serum from normal sows and piglets. However, the bactericidal activity of PMN leukocytes in serum from either normal sows or immune ten-week-old swine was greater than the bactericidal activity of PMN leukocytes in either piglet serum or serum from nonimmune ten-week-old swine. When the serum was either heated to 70°C for ten minutes or treated with 2-mercaptoethanol, bactericidal activity of PMN leukocytes against GES was only observed in the presence of immune serum.

     

肝素浓度对辽宁绒山羊精子体外获能的影响

在TALP液中添加不同浓度(25、50、100μg/mL)的肝素对辽宁绒山羊精子进行获能处理,在显微镜下检测处理1、2、3、4、5h后的精子活力,用考马斯亮蓝染色法检测获能处理0.5、1、2、4h后的精子获能状况,探讨肝素浓度对绒山羊精子活力、存活时间、获能率的影响。结果发现,在38.5℃、5%CO2、饱和湿度条件下,精子活力随肝素浓度升高而下降,3 h以后25μg/mL肝素组精子活力显著高于其它肝素组(P<0.05)。添加肝素组获能率显著高于对照组(P<0.05),各肝素组精子获能率差异不显著(P>0.05)。对照组精子存活时间最长,25μg/mL肝素组精子存活时间为10.12 h,显著高于其它两组(P<0.05)。表明25μg/mL肝素处理辽宁绒山羊精子体外获能较为适宜。     

犬瘟热患犬中性粒细胞cAMP-PDE活性变化及中药作用研究

研究犬瘟热患犬中性粒细胞(PMN)中环磷酸腺苷-磷酸二酯酶(cAMP-PDE)的活性变化及中药对其活性的体外调节作用,为抗炎药研究和病毒性炎症的中药治疗积累资料。提取犬瘟热患犬(n=9)和正常犬(n=6)的中性粒细胞制备cAMP-PDE样品,通过高效液相色谱(HPLC)法测定其活性及中药对其活性的影响。结果显示,犬瘟热患犬组中性粒细胞cAMP-PDE活性(18.55%±2.66%)显著(P<0.05)高于正常组(11.54%±5.16%)。解表药等5类114味中药中芦根(77.27%)、胡黄连(70.41%)、女贞子(65.50%)、香薷(65.16%)、贝母(61.43%)、黄芩(60.67%)、淫羊藿(60.34%)、黄芪(59.13%)、青蒿(56.93%)、秦皮(55.55%)、辛夷(55.03%)、甘草(54.67%)、牛蒡子(53.40%)、麻黄(53.32%)14味对cAMP-PDE活性具有明显抑制作用。结果表明,中性粒细胞cAMP-PDE活性在犬瘟热患犬炎症过程中具有重要作用,对cAMP-PDE活性有明显抑制作用的中药对其炎症可能具有疗效,同时,中药抑制中性粒细胞中cAMP-PDE活性可能是其抗炎作用机理之一。     

培养液中不同浓度肝素对蓝狐精子体外获能的影响

采用上游法分离优化精子,以mTyrod's液作为基础培养液检测培养液中不同质量浓度肝素对蓝狐精子体外获能的影响,试验设4个肝素质量浓度:0,10,20,50 μg/mL,38.5℃、5%C02培养箱进行孵育,获能培养时间为6h.利用考马斯亮蓝染色法检测精子顶体反应率,伊红-苯胺黑染色法检测活精子比率,观测法检测精子活力...     

Viability and Ultrastructural Changes in Group E Streptococci After in vitro Phagocytosis by Swine Macrophages

The loss of viability and ultrastructural changes were studied in group E Streptococci (GES) after in vitro phagocytosis in immune swine macrophages. There was a 50% reduction in the viability of intracellular GES during the first 60 minutes of incubation and a total loss of viability by 300 minutes as compared to the control tubes where the GES increased.

Loss of viability in phagocytized group E Streptococci was associated with the appearance of degenerative changes in the bacterial cytoplasm. This was followed by disruption of the bacterial cell membrane and its separation from the bacterial cell wall. No definite evidence of cell wall degeneration could be found. Unphagocytized organisms incubated for similar periods and fixed in the same manner did not lose viability nor have any degenerative changes.