禽呼肠孤病毒强毒株和弱毒疫苗株感染鸡肝癌细胞的转录组学分析

本研究旨在研究禽呼肠孤病毒(avian reovirus,ARV)强毒株S1133和弱毒疫苗株aS1133分别感染鸡肝癌(LMH)细胞后,LMH细胞基因组的转录水平变化。将1 MOI的ARV强毒株S1133和弱毒疫苗株aS1133分别感染LMH细胞,感染后6、12和24h,收集感染细胞和阴性对照细胞,制备总RNA样品,使用Illumina HiSeq~(TM)2000测序仪进行转录组学测序。以感染组之间样品中上下调转录差异≥2且P≤0.001的基因为差异基因,进行生物信息学分析并进行荧光定量PCR验证。分析结果表明,与ARV aS1133感染组相比,S1133感染组共有4 013个差异表达基因,其中6hpi时,58个上调表达差异基因,5个下调基因;12hpi时,1 429个上调基因,354个下调基因;24hpi时,1 680个上调基因,481个下调基因。K-平均值聚类分析结果说明这些差异基因的表达模式主要呈上调表达模式,并且有10种共表达趋势。GO功能注释和分析结果表明差异基因主要具有GTP酶调节活性、信号转导活性、蛋白激酶活性等生物功能;主要参与机体应激、细胞信号转导、细胞凋亡等生物过程。Pathway富集分析结果表明差异表达基因主要参与Toll样受体信号通路、TGF-β信号通路、病原体感染应答等细胞信号通路。随机选择部分差异表达基因进行荧光定量RT-PCR验证,其结果表明与转录组学测序结果基本一致。以上数据比较分析了ARV强毒株和弱毒疫苗株感染后LMH细胞的基因表达的变化差异,探讨了宿主细胞对ARV不同毒力毒株的可能不同应答机制,有利于进一步阐明ARV致病机制。     

基于转录组测序的新城疫疫苗免疫鸡胸腺差异表达基因分析

为研究新城疫(ND)疫苗接种的青脚麻鸡胸腺组织相关基因与调控通路变化,对无特定病原体(SPF)的青脚麻鸡人工接种ND疫苗48 h后,取胸腺组织采用Illumina HiSeq 2500高通量测序技术进行转录组测序,使用TopHat2软件对测序得到的reads序列与鸡(Gallus_gallus-5.0)的参考基因组比对,筛选出差异表达基因后,在GO和KEGG数据库中进行功能注释、富集分析和聚类分析。结果显示:接种ND疫苗后,共筛选到1 514个差异表达基因,其中1 016个基因上调,498个基因下调;GO功能分类注释到生物学过程、细胞组成和分子功能数据库中的差异表达基因分别有823、815和985个,注释到KEGG通路中差异表达基因1 274个,显著富集通路52个(P0.05);在差异基因中筛选出70个与鸡先天免疫相关的基因,并使用STRING构建蛋白质相互作用网络。研究提示:ND疫苗免疫接种后激活了青脚麻鸡先天免疫相关基因或通路,改善了体内的抗体水平,对探索鸡的宿主-病原体相互作用具有重要意义,为进一步探究青脚麻鸡接种ND疫苗后机体免疫应答反应提供了研究基础。     

北京油鸡和来航鸡脾脏差异表达基因的筛选与分析

为筛选北京油鸡和来航鸡脾脏组织中与抗病力差异相关的候选基因和信号通路,本研究采用高通量测序技术,构建了42日龄北京油鸡(抗病力较强)和来航鸡(抗病力相对较弱)脾脏组织的数字基因表达谱,对差异表达基因进行了GO功能分类、KEGG信号通路分析和STRING互作网络构建,并利用荧光定量PCR验证了部分差异表达基因。结果表明,以来航鸡为对照组,北京油鸡与之相比,差异倍数在2倍以上的共有1335个基因,其中693个上调表达,643个下调表达,包括多个与淋巴细胞的激活、分化和增殖、免疫器官发育等相关的基因,主要涉及到结合功能、细胞组成、细胞加工和生物学调节等功能。对通路显著性分析发现,与免疫相关生物学通路共有4个,其中BCR信号和TLR信号涉及脾脏B细胞介导的体液免疫应答反应,两条通路上的差异表达基因构成1个相互连接的互作网络,其中D79B、CD44、IL1B、SOCS1及TLR4等位于重要节点位置。为今后挖掘新基因和研究影响鸡抗病力的遗传因素等提供理论依据。     

齐兴肉兔转录组测序及肌肉生长发育相关基因的筛选

为了研究不同发育阶段齐兴肉兔肌肉生长发育相关的差异表达基因,试验选取胚胎期18日龄、26日龄的齐兴肉兔骨骼肌进行转录组测序,与兔基因组数据库进行比对,筛选差异表达基因并进行GO分析,进一步筛选与肌肉生长发育相关的差异表达基因,并通过qRT-PCR对筛选基因进行验证。结果表明:胚胎期26日龄共获得5 320个差异表达基因,其中上调基因2 607个、下调基因2 713个;KEGG功能注释发现差异基因显著富集到癌症通路、细胞骨架调控通路、pi3k-akt信号通路和RAP1信号通路;筛选获得的6个基因与肌肉生长发育关系较为密切,其中上调基因分别为TPM1、CASQ1、MYOZ1、CKM、MYL3基因,下调基因为IGF2BP1基因。     

鸡皮肤毛囊生长差异基因的生物信息学分析

【目的】 筛选鸡胚胎早期发育期间背部皮肤上皮和间充质中调控毛囊生长的关键基因、生物过程和信号通路,为了解鸡胚胎早期发育期间毛囊生长的分子机制提供参考。【方法】 从GEO数据库中下载基因芯片GSE62882数据集,采用R语言limma包对数据集进行差异分析,分别筛选出胚胎期第7和9天皮肤上皮和间充质差异基因以及皮肤上皮和间充质共同上下调基因,通过STRING在线数据库对差异基因和共同上下调基因进行蛋白互作网络分析,用Cytoscape 3.8.2软件进行可视化,运用R语言中的clusterProfiler程序包对差异基因和共同上下调基因进行GO功能和KEGG通路富集分析。【结果】 皮肤上皮在胚胎期第7和9天共筛选出626个差异基因,获得189条相互作用关系、10个生物过程和4条信号通路;皮肤间充质在胚胎期第7和9天共筛选出690个差异基因,获得326条相互作用关系、10个生物过程和3条信号通路;皮肤上皮和间充质在胚胎期第7和9天共同上调基因26个,共同下调基因48个,共同上下调基因存在34条相互作用关系,主要富集在Wnt信号通路上。【结论】 本研究从鸡胚胎期第7和9天皮肤上皮和间充质中筛选到共同上调基因26个,共同下调基因48个,这些基因主要富集在Wnt信号通路上,为探究上皮和间充质在毛囊生长发育中的作用机制提供理论参考。     

鸡毒支原体MG-HY株感染鸡气管的转录组学分析

旨在进一步探究鸡毒支原体（MG）感染SPF雏鸡后气管基因组转录水平的变化,筛选出MG感染后参与气管黏膜炎性损伤的差异表达基因和调控通路。使用MG-HY株菌液按0.2 mL·羽^-1经点眼滴鼻感染SPF雏鸡,感染后收集气管组织利用RNA-Seq技术进行测序分析。转录组分析结果显示,在MG感染期间RNA-seq共筛选出3 112个显著（P<0.01）差异表达基因（DEGs）,其中,1 646个上调基因,1 466个下调基因。GO功能分析显示,差异基因主要涉及生物调节、刺激的反应、多细胞生物过程、细胞成分组织或生物发生等生物过程。KEGG-Pathway分析发现差异基因参与黏膜免疫信号传导通路,例如：细胞因子-细胞因子受体相互作用,细胞黏附分子（CAMs）,细胞外基质（ECM）受体相互作用、紧密连接、PPAR信号通路和MAPK信号通路等,表明这些基因参与了MG诱导的雏鸡气管炎症反应和损伤。使用qRT-PCR验证与黏膜免疫相关的13个差异表达的基因,其结果与转录组分析一致。本研究为进一步阐明MG感染导致宿主气管黏膜上皮损伤和黏膜免疫机制提供了基础。     

不同滴度鸭肠炎病毒感染对鸭脾脏的转录组变化分析

为探讨不同滴度鸭肠炎病毒(duck enteritis virus,DEV)感染对鸭组织器官转录组的影响,本研究采用2个不同DELD50滴度的DEV,接种50日龄健康鸭90h后,采集脾脏样本,高通量测序技术进行转录组测序,筛选差异表达基因,并应用GO与KEGG数据库进行分析。结果显示,当接种量为100×DELD5 0时,鸭脾脏差异表达基因有685个,其中上调基因341个,主要参与免疫反应、核糖体结构和酶活性等生物学过程,并在核糖体、氧化磷酸化和吞噬体信号通路中显著富集;下调基因为344个,主要参与细胞分化正调控、β转化生长因子生成正调控、酶与相关受体活性、细胞结构等生物学过程,并在细胞黏附分子、内吞作用、肠道免疫网络IgA产生、ECM受体互作、缝隙连接和黏着信号通路上显著富集。当接种量为10-2×DELD50时,鸭脾脏差异表达基因有485个,其中上调基因297个,主要参与细胞功能、蛋白结合和蛋白复合物等生物学过程,并在细胞黏附分子、吞噬体、肠道免疫网络IgA产生、球系列鞘糖脂生物合成及N-糖链合成信号通路中显著富集;下调基因188个,主要参与补体激活、肌肉组织活动调节、受体复合物、酶与受体活性等生物学过程,并在基础转录因子、PPAR信号通路、α-亚麻酸代谢等代谢途径与信号通路上显著富集。这些结果表明不同滴度DEV感染对鸭脾脏转录组产生不同的影响,为深入探究DEV致病分子机制提供基础资料。     

初生及成年牦牛大脑皮质的转录组分析

旨在分析初生和成年牦牛大脑皮质转录组表达谱,筛选影响大脑皮质发育的候选基因。本研究选取初生(1～7日龄(NYB),n=3)和成年(3～4岁(AYB),n=3)牦牛,放血处死,采集大脑皮质,利用Illumina HiSeq平台对转录组进行测序与分析,筛选得到差异表达基因,在此基础上对差异基因进行GO功能注释、KEGG富集分析和实时荧光定量PCR(qRT-PCR)方法验证。测序结果共分析得到了4 790个显著差异表达的基因(P<0.05),其中包括2 670个上调表达的差异基因,2 120个下调表达的差异基因。随机选取了9个差异基因进行qRT-PCR验证,表达趋势与转录组测序结果一致,表明测序结果可靠。GO功能注释发现其中占比较多的条目为与蛋白合成及ATP利用相关的转录调控、RNA聚合酶II正调控转录、蛋白结合及ATP结合;KEGG富集分析发现占比较多与大脑发育相关的通路为调控轴突发育及重塑的轴突导向通路,与神经胶质细胞增殖相关的MAPK信号通路,与突触传递相关的神经活性配体-受体相互作用、 PI3K-Akt、钙信号及磷脂酶D信号通路,与免疫防御相关的Toll样受体通路,与低氧适应调...     

基于RNA-Seq筛选鸭肠炎病毒感染鸭脾差异表达免疫相关基因

为挖掘鸭肠炎病毒(duck enteritis virus,DEV)感染鸭的差异表达免疫相关基因,本研究以DEV GZ株经腿部肌肉接种50d鸭后,于接种后66、90和114h采集鸭脾组织样本,提取总RNA,通过高通量RNA-seq技术测序,应用GO和KEGG数据库进行比对注释,并采用荧光定量PCR对部分差异表达基因进行验证,结果显示:DEV接种66h时鸭脾的差异表达基因有511个,参与免疫相关生物学过程的为70个,其中上调基因46个,下调基因24个;90h时差异表达基因有485个,参与免疫相关生物学过程的为64个,其中上调基因49个,下调基因15个;114h时差异表达基因有531个,参与免疫相关生物学过程的为66个,其中上调基因35个,下调基因31个。GO数据库分析显示,差异表达免疫相关基因主要涉及细胞黏附、抗原加工和呈递、补体激活等免疫反应过程;KEGG数据库分析显示,差异表达免疫相关基因主要富集在细胞黏附分子、ECM受体互作、PPAR等信号通路;荧光定量PCR对7个差异表达基因进行检测显示,差异表达基因相对表达量与RNA-Seq技术测序结果基本一致。这些结果为进一步阐明DEV感染机制和机体免疫应答机制奠定了基础。     

羊源D型多杀性巴氏杆菌感染小鼠脾脏转录组分析

试验旨在探索羊源D型多杀性巴氏杆菌入侵宿主脾脏组织中引发的免疫应答途径。首先利用羊源D型多杀性巴氏杆菌(HN01菌株)感染小鼠,建立羊源D型多杀性巴氏杆菌感染的动物模型;之后利用转录组测序技术获得感染小鼠与正常小鼠的脾脏转录组数据,并使用COG、KOG、eggNOG、GO、KEGG数据库对测序结果中的差异表达基因(differentially expressed genes,DEGs)进行功能注释与分析,同时对于显著富集到关键免疫通路的差异表达基因使用STRING软件和KEGG mapper进行蛋白互作分析,筛选出核心通路中起关键作用的基因;最后选取关键的10个基因进行实时荧光定量RT-PCR验证。转录组分析结果显示,与正常组相比,感染组中筛选出3 380个差异表达基因(P<0.01,log₂|FoldChange|≥0.5),其中1 691个基因上调,1 689个下调。基因功能富集分析结果表明,感染组脾脏中的差异表达基因主要发挥信号转导的功能,其主要参与的生物途径包括细胞因子与细胞因子受体互作通路、趋化因子信号通路、HIF-1信号通路、TNF信号通路。蛋白互作分析筛选出约28个核心差异表达基因,结合实时荧光定量RT-PCR验证后,其中9个基因的表达结果与测序一致,分别是C3、Cd4、Cxcl13、Lck、Gnai1、Grap2、IL-6、Cxcr6及Serping1基因。本研究初步证明了脾脏在抵抗多杀性巴氏杆菌入侵中参与了一系列免疫应答反应,为进一步研究羊源D型多杀性巴氏杆菌与宿主之间相互作用的分子机制奠定理论基础。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.