腹腔注射GnIH对雄性小鼠采食、体重和血糖稳态的影响

目的 探究促性腺激素抑制激素（gonadotropin-inhibitory hormone,GnIH）对雄性小鼠采食、体重和血糖稳态的影响。方法 选取体重及日龄相近的雄性小鼠20只,随机分为2组,分别为对照组[腹腔注射生理盐水100 μL/（次·只）]和试验组[腹腔注射20 μg/100 μL GnIH,100 μL/（次·只）],每组10只,每天注射2次,连续注射21 d。观察和记录小鼠的采食情况;对小鼠的体重、空腹血糖、葡萄糖耐量、胰岛素耐量进行测定;利用实时荧光定量PCR（qPCR）方法对小鼠胰腺中胰岛素转录因子基因（NeuroD1）、胰岛素基因（Ins）、胰高血糖素基因（Gcg）、胰岛素转录调控因子基因（Pdx1）mRNA相对表达量进行检测。结果 与对照组相比,试验组小鼠平均日增重和平均日采食量极显著（P<0.01）升高;试验组小鼠空腹血糖曲线下面积极显著（P<0.01）增加,葡萄糖耐量的血糖曲线下面积极显著（P<0.01）增加,胰岛素耐量的血糖曲线下面积极显著（P<0.01）增加;试验组小鼠胰腺Gcg基因mRNA相对表达量显著（P<0.05）升高,Ins基因mRNA、Pdx1基因mRNA和NeuroD1基因mRNA相对表达量均显著（P<0.05）下降。结论 慢性腹腔注射GnIH会引起雄性小鼠采食量和体重增加,并引起机体血糖紊乱。     

IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪不同组织和细胞中的表达特征分析

白细胞介素1受体拮抗剂（interleukin 1 receptor antagonist,IL1RN）是一种重要的宿主免疫调节因子,是天然存在的IL-1拮抗剂。为了解IL1RN基因在断奶仔猪不同组织和细胞中表达的特征,利用Trizol法提取晋汾白猪仔猪和新山西黑猪仔猪的心脏、肝脏、脾脏、淋巴细胞、巨噬细胞等18种组织和细胞的总RNA,采用RT-PCR方法检测IL1RN基因在不同组织和细胞中的表达情况,应用SPSS 19.0软件分析IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪4种免疫组织和细胞（脾脏、淋巴结、巨噬细胞、白细胞）中的表达差异情况。结果显示：IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪的脂肪组织中均不表达,在其余各组织和细胞中均表达;IL1RN基因在晋汾白猪仔猪的巨噬细胞与淋巴结、脾脏、白细胞之间的表达差异极显著（P<0.01）,且在淋巴结与脾脏、白细胞之间的表达差异极显著（P<0.01）;IL1RN基因在新山西黑猪仔猪的脾脏、淋巴结、巨噬细胞、白细胞的表达两两之间均呈极显著差异（P<0.01）;IL1RN基因在2个猪种的脾脏、巨噬细胞、白细胞之间的表达呈极显著差异（P<0.01）,在淋巴结中的表达呈显著差异（P<0.05）。该研究结果为进一步揭示2个猪种的IL1RN基因在疾病抵抗方面的作用及相关机制提供了依据。     

17α-甲基睾酮对稀有鮈鲫精巢DNA甲基转移酶基因表达的影响

[目的] 以稀有鮈鲫雄鱼为研究对象,探讨17α-甲基睾酮（17α-methyltestosterone,MT）对稀有鮈鲫精巢甲基转移酶（DNMT）基因相对表达量的影响。[方法] 采用不同浓度MT（25、50和100 ng/L）处理稀有鮈鲫雄鱼7、14和21 d,处理结束时,每组随机解剖10条鱼,取精巢组织,Trizol一步法提取总RNA,实时荧光定量PCR（qRT-PCR）检测DNA甲基转移酶基因（dnmts）相对表达量。[结果] MT处理稀有鮈鲫雄鱼7 d,25 ng/L MT处理组（P<0.05）、50 ng/L MT处理组（P<0.01）和100 ng/L MT处理组（P<0.05）精巢dnmt3基因相对表达量显著（或极显著）降低;25 ng/L MT处理组和50 ng/L MT处理组精巢dnmt5基因相对表达量显著（P<0.05）降低;50 ng/L MT处理组精巢dnmt7基因相对表达量显著（P<0.05）降低。MT处理稀有鮈鲫雄鱼14 d,100 ng/L的MT处理组dnmt3、dnmt5、dnmt6、dnmt7基因相对表达量显著（P<0.05）升高;50 ng/L的MT处理组dnmt1基因相对表达量极显著（P<0.01）升高。MT处理稀有鮈鲫雄鱼21 d,50 ng/L MT处理组和100 ng/L MT处理组dnmt1基因相对表达量显著（P<0.05）升高;100 ng/L MT处理组dnmt8基因相对表达量极显著（P<0.01）升高。[结论] MT可以干扰稀有鮈鲫精巢DNA甲基转移酶基因dmmts的mRNA表达。MT对DNMTs酶活性的影响以及是否可以通过改变DNA甲基化水平对稀有鮈鲫精子的发生产生影响有待研究。     

PTD-FNK蛋白对雄性小鼠生理功能的影响

[目的] 探究PTD-FNK蛋白对雄性小鼠生理功能的影响。[方法] 将16只8周龄健康雄性C57BL/6J小鼠随机分为对照组（n=8）和PTD-FNK蛋白组（n=8）,对照组腹腔注射生理盐水,PTD-FNK蛋白组腹腔注射300 μg/（kg·BW）PTD-FNK蛋白,每天腹腔注射给药1次,连续给药7 d;给药期间每天记录小鼠的体重、体温、血糖、采食量、饮水量,计算试验期间总采食量、总饮水量、体增重;于试验第1天和最后1天测量鼻肛距,计算试验前后Lee's指数;试验结束立即处死小鼠,称量心脏、肾脏、肝脏、脾脏和睾丸重量,计算脏器指数;分离小鼠附睾,制备精子悬液,测定精子活力和质膜完整率;利用qPCR法检测睾丸组织中细胞凋亡相关基因Bax和Caspase-3、氧化应激相关基因SOD和GPX1、内分泌相关基因STAR、17β-HSD的mRNA相对表达量。[结果] 与对照组相比,PTD-FNK蛋白组小鼠总采食量极显著（P<0.001）下降,总饮水量显著（P<0.05）下降;Lee's指数、体温、体重、血糖无显著（P>0.05）变化。肝脏指数显著（P<0.05）下降,其他脏器指数无显著（P>0.05）变化。精子活力极显著（P<0.01）升高,精子质膜完整率无显著（P>0.05）变化。睾丸组织中Bax基因mRNA相对表达量极显著（P<0.01）升高,Caspase-3基因mRNA相对表达量无显著（P>0.05）变化,SOD基因mRNA相对表达量极显著（P<0.01）升高,GPX1基因mRNA相对表达量无显著（P>0.05）变化,17β-HSD、STAR基因的mRNA相对表达量无显著（P>0.05）变化。[结论] PTD-FNK蛋白可以抑制雄性小鼠的采食、饮水及肝脏的生长发育,提高小鼠的精子质量。     

斑马鱼E3泛素连接酶基因的生物信息学分析和mRNA表达

目的对斑马鱼E3泛素连接酶基因进行生物信息学分析,并研究其mRNA表达情况。方法利用生物信息学方法分析斑马鱼E3泛素连接酶基因的cDNA序列、蛋白质二级结构、结构域、氨基酸序列同源性,并构建进化树;利用整胚原位杂交技术研究目的基因在斑马鱼体内的mRNA表达模式。结果在NCBI基因库中找出TRIM54、TRIM55a、TRIM55b、TRIM63a、TRIM63b和TRIM101 6个斑马鱼E3泛素连接酶基因。这些基因之间有较高的同源性,二级结构以α-螺旋和无规卷曲为主,编码的蛋白质均含有3个保守结构域：环锌指、B-box锌指和卷曲螺旋。进化树分析显示,所有基因聚为4组：各物种TRIM54基因聚为一组;各物种TRIM55基因聚为一组,但靠近于斑马鱼TRIM101基因分支;TRIM63基因分为一组。整胚原位杂交结果显示,这些TRIM基因的mRNA均在受精后24 h的斑马鱼的肌节中表达,且TRIM55a、TRIM63a和TRIM101基因表达量较高。这些TRIM基因在肌肉发育和肌肉功能中可能发挥作用。结论斑马鱼的6个E3泛素连接酶基因有较高的保守性,其mRNA特异性表达于肌节部位。结果对深入研究这些基因在鱼类肌肉中的作用和指导鱼类生产具有一定的意义。     

Wnt/β-catenin信号通路上游基因在山羊绒周期性再生及着色过程中的表达分析

为初步探究Wnt/β-catenin信号通路是否参与山羊绒周期性再生及着色过程,本试验采用实时荧光定量技术对Wnt/β-catenin信号通路上游基因β-catenin、Lef1/Tcf3及该通路拮抗基因Dkk1mRNA在白色绒山羊绒毛生长不同时期及黑、白色绒山羊绒毛生长旺盛期时体侧部皮肤组织中的相对表达量进行研究。结果显示,上述通路中β-catenin、Lef1/Tcf3基因mRNA在白色绒山羊不同时期体侧部皮肤组织中的相对表达量具有明显变化规律即生长前期缓慢上调,旺盛期时表达量最高,生长后期及退行期逐渐下调,休止期表达量最低;Dkk1基因mRNA在白色绒山羊不同时期体侧部皮肤组织中的相对表达量则无明显规律性。上述各基因mRNA在黑、白色绒山羊生长旺盛期时体侧部皮肤组织中的相对表达量并无显著差异(P0.05)。结果提示,Wnt/β-catenin信号通路参与山羊绒周期性再生过程,但该通路并不涉及生长旺盛期时绒毛的着色过程。     

APAP急性肝损伤小鼠肝脏组织转录组RNA-seq及相关信号通路分析

[目的]筛选与对乙酰氨基酚（acetaminophen,APAP）诱导的急性肝损伤发生发展相关的基因和关键信号通路。[方法]建立C57BL/6J小鼠APAP急性肝损伤模型。构建正常小鼠肝脏组织样本和APAP急性肝损伤小鼠损伤后第2天肝脏组织样本的2个cDNA文库,进行转录组测序。对获得的转录组测序数据进行组装及功能注释。使用DESeq R包（1.10.0）分析具有生物学重复的差异基因的表达,利用KOBAS软件确定KEGG信号通路中差异表达基因的静态富集。[结果]APAP急性肝损伤小鼠损伤后第2天与正常小鼠肝脏组织转录组相比,共有7 270个DEGs,包括3 707个显著（P<0.05）上调表达基因和3 563个显著（P<0.05）下调表达基因。在所有显著上调表达基因中,表达量变化幅度较大的是Col1a1、Gsta1、S100a6基因;在所有显著下调表达基因中,差异表达量位于前3位的基因是Slc1a2、Glul、Acaa1b。共有2 515个DEGs在316个不同的KEGG通路中富集,显著上调表达基因富集的信号通路主要是趋化因子信号通路、B细胞受体信号通路、NOD样受体信号通路、ECM受体相互作用信号通路等免疫和炎症相关信号通路,显著下调表达基因富集的信号通路主要是氧化磷酸化、脂肪酸降解、脂肪酸代谢、碳代谢等合成代谢信号通路。[结论]在APAP急性肝损伤过程中涉及多个信号通路的参与,趋化因子信号通路和ECM受体交互作用信号通路可能是急性损伤期中发挥主要作用的信号通路。     

动物组织中GLUD1基因表达与GDH蛋白结构分析

[目的]研究动物不同组织中GLUD1基因的表达情况,比较不同物种中GDH蛋白的结构差异,明晰GLUD1基因的表达模式与GDH蛋白的功能。[方法]分别采集大鼠、绵羊、梅花鹿的肝脏、肾脏、皮肤、脂肪和肌肉5种新鲜组织样品。以β-actin为内参基因,采用荧光定量PCR法（qRT-PCR）分析3种动物不同组织中GLUD1基因的表达情况;利用无重复双因素分析方法表征GLUD1基因在不同组织中的表达量差异。比较人、小鼠、大鼠、山羊、绵羊、牛和猪7个物种GDH蛋白氨基酸序列,模拟这7个物种的GDH蛋白三维空间结构。[结果]GLUD1基因在大鼠、绵羊和梅花鹿的5个组织中均有表达,在肝脏中的表达量均高于其他组织;该基因在大鼠和梅花鹿肝脏组织中的表达量极显著（P<0.01）高于绵羊肝脏组织中的表达量,在大鼠肾脏组织中的表达量极显著（P<0.01）高于梅花鹿肾脏组织中的表达量。人GDH蛋白的氨基酸序列与猪、牛、山羊和绵羊的序列相似度较高,而大鼠与小鼠GDH蛋白的氨基酸序列相似度达到99.10%。大鼠、人、猪的GDH蛋白氨基酸序列中分别存在2个（第8位丙氨酸→缬氨酸、第40位丙氨酸→缬氨酸）、1个（第23位丙氨酸→丝氨酸）、2个（第33位丙氨酸→苏氨酸、第39位丙氨酸→苏氨酸）物种特异性突变位点。小鼠、大鼠、人的GDH蛋白预测为三聚体结构,山羊、绵羊、牛和猪的GDH蛋白预测为同源六聚体结构。[结论]GLUD1基因在大鼠、绵羊、梅花鹿不同组织中的表达量存在差异,在肝脏中都有较高水平的表达。不同物种GDH蛋白的氨基酸序列相似度较高,但依旧存在一定的序列差异性,并且存在数个对应物种特异性的氨基酸突变位点。     

秦川牛CAP2基因CDS区克隆及表达谱分析

[目的]克隆秦川牛CAP2基因的编码区序列（CDS）,分析该基因在不同组织及原代脂肪细胞分化过程中的表达特征。[方法]采用RT-PCR方法扩增秦川牛CAP2基因的CDS区,运用ProtPram、TMpred、ProtFun 2.1 Server等在线网站进行CAP2蛋白的生物信息学分析;通过油红O染色和qPCR检测成脂标志基因PPARγ和FABP4基因的表达水平以构建牛原代脂肪细胞诱导分化体系;利用qPCR检测分析CAP2基因在秦川牛7种组织（心、肝、脾、肺、肾、肌肉和背脂）和原代脂肪细胞分化过程（0~10 d）中的表达。[结果]秦川牛CAP2基因CDS区长1 461 bp,编码486 个氨基酸,氨基酸序列主要由无规卷曲和α-螺旋构成。CAP2基因在脂肪组织中高表达,极显著（P<0.01）高于其他组织。牛原代脂肪细胞诱导分化过程中,PPARγ和FABP4基因的表达量逐渐上升,与第0天相比第10 天时表达量达到最大（P<0.01）;与对照组相比,诱导分化组脂滴积累明显增加;CAP2基因表达量也随时间推移逐渐上升,以0 d为对照,第10天表达量最高（P<0.001）。[结论]成功克隆了秦川牛CAP2基因全长1 461 bp的编码区;CAP2基因在牛脂肪组织中以较高水平表达,且CAP2基因可能参与脂肪生成与分化过程,预测CAP2基因可能是促进成脂分化的转录因子,对维持牛脂肪细胞状态发挥关键作用,可能作为秦川牛肉质性状的候选基因,该研究结果为进一步揭示牛CAP2基因的功能提供基础资料。     

沉默信息调节因子2相关酶3调控机制及其在疾病中的作用

沉默信息调节因子2相关酶3（silent information regulator 2-related enzyme 3,SIRT3）是烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD⁺）依赖性组蛋白去乙酰化酶家族成员,参与能量代谢、氧化应激和细胞凋亡等过程并起到关键作用,其调控作用与多种疾病的发生密切相关。综述了SIRT3下游转录因子腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）、线粒体通透性转换孔（mitochondrial permeability transition pore,mPTP）、核因子κB（nuclear factor-κB,NF-κB）等的调控机制,以及SIRT3在心血管疾病、肿瘤、糖尿病中的作用,以期为相关疾病的预防和治疗提供参考。     

Pathology of maternal genital tract, placenta, and fetus in equine viral arteritis

Six pregnant mares were given equine viral arteritis virus intravenously. Tissues from genital tracts, placentae, and fetuses were examined by light and electron microscopy to study the mechanism of abortion. Four mares which died with acute disease had diffuse vacuolation of endometrial epithelium and systemic necrotizing vasculitis. Two of these mares had dead fetuses and two had live fetuses; virus was isolated from tissues of one live fetus. Placentae of mares dying from acute disease did not have lesions attributable to infection; virus was isolated from two of these placentae. One of the two mares which recovered from clinical disease aborted a dead fetus eight days after inoculation. The mare had severe necrotizing myometritis and virus was isolated from maternal ovaries and from fetal tissues. The fetus did not have lesions attributable to arteritis virus. These results suggest that although fetal death may occur in utero during acute equine viral arteritis, abortion probably is due to lesions in the uterus of the mare.     

The role of Bcl-xL and nuclear factor-��B in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-_L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.     

Metformin acts to suppress β-hydroxybutyric acid-mediated inflammatory responses through activation of AMPK signaling in bovine hepatocytes

The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes, such as prevention of laminitis and hyper-triglyceridemic. AMPK maintains energy homeostasis and is the intracellular target of metformin action. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signaling. The hepatocytes were isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). Treatments were conducted as follows: treated with PBS for 18 h (control); pretreated with PBS for 12 h followed by treatment of 1.2 mM BHBA for 6 h (BHBA); pretreated with 1.5 mM or 3 mM metformin for 12 h followed by the BHBA treatment (1.2 mM) for 6 h (M(1.5)+B; M(3)+B). The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses. RT-qPCR were applied for the mRNA expression while Western-blots and immunofluorescence were conducted for the target proteins expression. Among dose-dependent assays for BHBA, the concentration of BHBA at 1.2 mM activated NF-κB signaling by upregulating the expression of phosphorylated NF-κB and pro-inflammatory cytokines compared with the control cells (P < 0.05). Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signaling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, BHBA inhibited cells staining intensity in EdU assay were increased by pretreatment with metformin. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared with BHBA treated cells, the protein expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The bound of NF-κB onto IL1B promoter displayed higher in BHBA group and this was suppressed by pretreatment with metformin (P < 0.05). Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signaling in bovine hepatocytes.     

Maternal obesity upregulates fatty acid and glucose transporters and increases expression of enzymes mediating fatty acid biosynthesis in fetal adipose tissue depots

Maternal nutrient restriction leads to alteration in fetal adipose tissue, and offspring from obese mothers have an increased risk of developing obesity. We hypothesized that maternal obesity increases fetal adipogenesis. Multiparous ewes (Columbia/Rambouillet cross 3 to 5 yr of age) carrying twins were assigned to a diet of 100% (Control; CON; n = 4) or 150% (Obese; OB, n = 7) of NRC maintenance requirements from 60 d before conception until necropsy on d 135 of gestation. Maternal and fetal plasma were collected and stored at -80°C for glucose and hormone analyses. Fetal measurements were made at necropsy, and perirenal, pericardial, and subcutaneous adipose tissues were collected from 7 male twin fetuses per group and snap frozen at -80°C. Protein and mRNA expression of fatty acid translocase [cluster of differentiation (CD) 36], fatty acid transport proteins (FATP) 1 and 4, insulin-sensitive glucose transporter (GLUT-4), fatty acid synthase (FASN), and acetyl-coA carboxylase (ACC) was evaluated. Fetal weight was similar, but fetal carcass weight (FCW) was reduced (P < 0.05) in OB versus CON fetuses. Pericardial and perirenal adipose tissue weights were increased (P < 0.05) as a percentage of FCW in OB versus CON fetuses, as was subcutaneous fat thickness (P < 0.001). Average adipocyte diameter was greater (P < 0.01) in the perirenal fat and the pericardial fat (P = 0.06) in OB fetuses compared with CON fetuses. Maternal plasma showed no difference (P > 0.05) in glucose or other hormones, fetal plasma glucose was similar (P = 0.42), and cortisol, IGF-1, and thyroxine were reduced (P ≤ 0.05) in OB fetuses compared with CON fetuses. Protein and mRNA expression of CD 36, FATP 1 and 4, and GLUT-4 were increased (P ≤ 0.05) in all fetal adipose depots in OB versus CON fetuses. The mRNA expression of FASN and ACC was increased (P < 0.05) in OB vs. CON fetuses in all 3 fetal adipose tissue depots. Fatty acid concentrations were increased (P = 0.01) in the perirenal depot of OB versus CON fetuses, and specific fatty acid concentrations were altered (P < 0.05) in subcutaneous and pericardial adipose tissue because of maternal obesity. In conclusion, maternal obesity was associated with increased fetal adiposity, increased fatty acid and glucose transporters, and increased expression of enzymes mediating fatty acid biosynthesis in adipose depots. These alterations, if maintained into the postnatal period, could predispose the offspring to later obesity and metabolic disease.     

p65 Homodimer activity in distal airway cells determines lung dysfunction in equine heaves

Nuclear factor-κB (NF-κB) activity, which is a key regulator of inflammatory gene expression, is increased in bronchial epithelial cells from horses suffering from heaves (a hypersensitivity-associated inflammatory condition of the lung). To determine whether this increased activity extends to distal airways and to other pulmonary cells, cells recovered by broncho-alveolar lavage (BAL) in healthy and heaves-affected horses were assessed for NF-κB activity. NF-κB activity was much higher in BAL cells from heaves-affected horses, especially during crisis (disease exacerbation), than in cells from healthy horses. Moreover, the level of NF-κB activity found in BAL cells was positively correlated to total lung resistance and to the proportion of neutrophils present in BAL fluid. Finally, prototypical p65–p50 NF-κB heterodimers were absent from BAL cells, which mostly contained p65 homodimers. These results (1) show that increased NF-κB activity is a general feature of heaves lung; (2) demonstrate the importance of p65 homodimers in neutrophilic inflammation; and (3) suggest that the use of specific NF-κB inhibitors could improve lung function in heaves-affected horses.