miR-127和miR-136在转基因克隆绵羊胎盘中的表达分析及其靶基因的鉴定

克隆动物胎盘发育缺陷是造成动物克隆效率低下的一个重要原因。目前认为克隆胎盘发育异常通常是由于一些基因表达的异常所致,与表观遗传修饰有关。microRNA是一种重要的表观遗传修饰方式,对动物胚胎发育和胎盘的形成有着重要的调控作用。为研究miR-127和miR-136在克隆动物胎盘中的表达情况及其与克隆动物发育缺陷的关系,本实验运用荧光定量PCR分析了死亡克隆绵羊胎盘和同期普通绵羊胎盘组织中miR-127和miR-136的相对表达量,并鉴定了miR-127和miR-136的靶基因及靶基因在胎盘中的表达情况。结果显示,miR-127和miR-136在克隆绵羊胎盘中的表达量分别增加了3.1和2.8倍。EGFP荧光敲除实验证实,胎盘发育相关基因Rtl1是miR-127和miR-136的靶基因,同时定量PCR分析发现Rtl1基因在克隆绵羊胎盘中的表达量降低了3/5。结果说明,miR-127和miR-136在克隆胎盘中的异常表达可导致Rtl1基因的低表达,这很可能是导致克隆动物死亡的一个重要原因。     

Calcium homeostasis and its relationship to superoxide production in blood and milk neutrophils of lactating goats

Polymorphonuclear neutrophils (PMN), which comprise over 70% of the somatic cells in goat milk, are a major cellular component of innate immunity in the goat mammary gland. However, the function of milk PMNs is modified after diapedesis compared to PMNs in blood. As many aspects of PMN activity depend directly on intracellular Ca²⁺ concentration ((Ca²⁺)_i), the present study aimed to determine the changes in Ca²⁺ homeostasis of milk PMNs from lactating goats compared to autologous blood PMNs, and to examine the significance of these variations to the immuno-competency of milk PMNs. The intracellular Ca²⁺ store of freshly prepared milk cells was estimated from the elevation of (Ca²⁺)_i after ionomycin treatment, which was found to be significantly less than blood PMNs. Replenishment of the intracellular Ca²⁺ store in milk cells after intracellular Ca²⁺ depletion by Bapta-AM followed by spiking with 2.5 mM Ca²⁺ for 20 min was also compared to that of blood PMNs, showing that after depletion/spiking the intracellular Ca²⁺ store in milk cells was much less than blood PMNs. The production of superoxide anion (O₂^?) in vitro in response to (Ca²⁺)_i-dependent or (Ca²⁺)_i-independent modulators was used to evaluate the relevance of altered Ca²⁺ homeostasis on the immuno-competency of milk cells compared to blood PMNs. The results indicated that milk cells produced similarly low levels of O₂^? as blood PMNs when treated with ionomycin. However, the amount of O₂^? produced by milk cells in response to phorbol 12-myristate 13-acetate (PMA) stimulation, although greater than ionomycin treatment, was significantly less than that of blood PMNs. The capacity for O₂^? production by both cell types in response to PMA reverted to the resting state with use of the protein kinase C (PKC) inhibitor, staurosporine. In conclusion, the current study demonstrated an irreversible shortage of intracellular Ca²⁺ in the milk PMNs of lactating goats compared to blood PMNs. It also showed that preliminary O₂^–production, primed by ionomycin treatment, remained unchanged in milk PMNs, despite the shortage in intracellular Ca²⁺, but decreased O₂^? production capacity, mediated via the PKC pathway, in milk PMN. It is suggested that the defects in Ca²⁺ homeostasis in milk PMNs of lactating goats is partially attributable for the post-diapedesis functionality modifications.     

不同饲用燕麦品种生产性能的综合评价

基于熵权赋权法,运用灰色系统理论中的灰色关联度法对26个燕麦品种的生产性能进行综合评价,结果表明,生产性能综合表现较好的是初岛燕麦、青永久409、青永久709及青永久316;青永久18综合评价值最低,生产性能最差,其余品种生产性能表现一般。经逐步回归分析,发现其中的绿叶数、叶长、茎叶比、鲜干比、生长速度、叶面积指数、相对生长速率及生长天数8个指标对饲用燕麦生产性能贡献显著。     

Effects of road transportation on metabolic and immunological responses in Holstein heifers

This study examined the effects of road transportation on metabolic and immunological responses in dairy heifers. Twenty Holstein heifers in early pregnancy were divided into non‐transported (NT; n = 7) and transported (T; n = 13) groups. Blood was collected before transportation (BT), immediately after transportation for 100 km (T1) and 200 km (T2), and 24 h after transportation (AT). The T heifers had higher (P < 0.05) blood cortisol and non‐esterified fatty acid concentrations after T1 and T2 than did NT heifers. By contrast, the T heifers had lower (P < 0.05) serum triglyceride concentrations after T1 and T2 than had the NT heifers. The serum cortisol and triglyceride concentrations returned (P > 0.05) to the BT concentrations at 24 h AT in the T heifers. The granulocyte‐to‐lymphocyte ratio and the percentage of monocytes were higher (P < 0.05) after T2 in the T heifers than in the NT heifers, suggesting that transportation stress increased the numbers of innate immune cells. T heifers had higher (P < 0.01) plasma haptoglobin concentrations than NT heifers 24 h AT. In conclusion, transportation increased cortisol secretion and was correlated with increased metabolic responses and up‐regulation of peripheral innate immune cells in dairy heifers.     

寄生虫细胞因子免疫的研究进展（一）

寄生虫在入侵机体时必须具备逃避机体免疫,破坏宿主免疫屏障才能感染,这种入侵过程必然和宿主机体的免疫系统发生联系,不但使宿主的先天免疫细胞功能缺失,而且获得性免疫也受到抑制。寄生虫感染均不同程度地伴随免疫损伤,宿主在感染寄生虫后产生的免疫应答中,细胞因子作为免疫过程中的介质起到关键作用,主要是调节机体细胞免疫抗虫,保护机体不被感染,其中巨噬细胞是主要功能的承担者。本文将阐述细胞因子在寄生虫感染过程中所发挥的免疫作用,为解决寄生虫感染的防控问题奠定基础,同时为寄生虫的研究提供新的思路。     

棘孢木霉葡聚糖酶基因Glu1的克隆及原核表达

从棘孢木霉(Trichoderma asperellum)ACCC30536中克隆获得一个葡聚糖酶基因Glu1,其cDNA全长984bp,编码327个氨基酸。该葡聚糖酶属于Glyco-hydro-12家族,推测为β-1,4-葡聚糖酶,与深绿木霉IMI 206040的Glycohydro-12家族蛋白(XP_013940397.1)有91%相似性,且亲缘关系较近。运用qRT-PCR技术检测9种诱导条件下棘孢木霉Glu1基因的表达水平,结果表明,Glu1基因能参与棘孢木霉对山新杨(Populus davidiana×P.alba var.pyramidlis)或杨树病原菌的识别。构建原核表达载体并获得重组蛋白rGlu1。酶活特性分析结果表明,该酶最适pH为4.5,最适温度为45℃,且酶活性随诱导时间延长逐渐升高,在5h达到稳定。     

糖原合成酶激酶3β对奶牛乳腺上皮细胞增殖及细胞周期的影响

本试验通过向纯化的奶牛乳腺上皮细胞中添加不同浓度（0（对照组）、10、20、40 mmol/L）的糖原合成酶激酶3β（glycogen synthase kinase 3β,GSK3β）特异性蛋白抑制剂氯化锂（Licl）,作用细胞24 h,研究其对奶牛乳腺上皮细胞增殖及细胞周期的影响,并利用qRT-PCR和Western blotting分别检测不同浓度Licl对奶牛乳腺上皮细胞中GSK3β、细胞周期蛋白D1（Cyclin D1） mRNA水平及GSK3β、磷酸化GSK3β （p-GSK3β）、Cyclin D1蛋白水平表达的影响。结果显示,Licl能促进奶牛乳腺上皮细胞的增殖活性,Licl抑制GSK3β后促进奶牛乳腺上皮细胞增殖的最佳浓度为20 mmol/L。与对照组相比,添加Licl后GSK3β蛋白表达受到抑制,p-GSK3β蛋白表达上调,同时提高了Cyclin D1蛋白表达。表明GSK3β对于奶牛乳腺上皮细胞增殖的能力是负调控因子,失活的GSK3β通过Cyclin D1途径促进细胞周期的进行。     

中西医结合治疗牛青杠叶中毒

牛青杠叶中毒是由于牛采食大量壳斗科栎属植物青杠树叶（特别是细嫩叶芽）,其有毒成分栎单宁及其降解产物吸收入血而引起的以便秘或下痢、水肿、胃肠炎及肾损伤为特征的中毒性疾病。多发于春季（特别是4月中下旬,青杠树发芽开花盛期）,尤其是春季干旱,牧草生长发芽迟缓年份,发病率较高。     

Pyruvate kinase is necessary for Brucella abortus full virulence in BALB/c mouse

Brucellosis, caused by a facultative intracellular pathogen Brucella, is one of the most prevalent zoonosis worldwide. Host infection relies on several uncanonical virulence factors. A recent research hotpot is the links between carbon metabolism and bacterial virulence. In this study, we found that a carbon metabolism-related pyruvate kinase (Pyk) encoded by pyk gene (locus tag BAB_RS24320) was associated with Brucella virulence. Determination of bacterial growth curves and resistance to environmental stress factors showed that Pyk plays an important role in B. abortus growth, especially under the conditions of nutrition deprivation, and resistance to oxidative stress. Additionally, cell infection assay showed that Pyk is necessary for B. abortus survival and evading fusion with lysosomes within RAW264.7 cells. Moreover, animal experiments exhibited that the Pyk deletion significantly reduced B. abortus virulence in a mouse infection model. Our results elucidated the role of the Pyk in B. abortus virulence and provided information for further investigation of Brucella virulence associated carbon metabolism.