氟化物对FRTL细胞甲状腺激素代谢相关基因表达的影响

为探讨氟对FRTL细胞甲状腺球蛋白(TG),甲状腺过氧化物酶(TPO)和钠碘转运体(NIS)基因表达的影响,传代培养FRTL细胞,在对数生长期用氟化钠处理,使培养液中的氟浓度分别为20、10、5、2.5、1.25 mg/L,同时设空白对照细胞.培养72 h后收集细胞;采用半定量RT-PCR分析FRTL细胞TG、TPO、NIS mRNA和β-actin表达水平.试验结果表明,与对照细胞相比,较低浓度氟化钠处理的FRTL细胞TG基因表达量代偿性升高,随氟质量浓度增加(5.0 mg/L以上),TG表达量显著降低(P<0.05);各个浓度氟化钠处理的FRTL细胞TPO、NIS基因表达量均下降或显著下降(P<0.05).由此可知,氟化物降低了甲状腺细胞TG、TPO、NIS基因表达水平,造成甲状腺摄取和利用碘、甲状腺激素合成、贮存、分泌功能障碍,进而导致甲状腺结构和功能异常.     

蒙古绵羊原始生殖细胞(PGCs)的分离培养与鉴定

本试验从绵羊胎儿的生殖嵴中分离出原始生殖细胞,经体外培养后进行了初步鉴定。为建立绵羊原始生殖细胞(primordial germ cells,PGCs)的培养体系,试验对比了不同培养条件对蒙古绵羊PGCs生长的影响,将得到的胚胎生殖细胞(EG细胞)作形态学、酶学和免疫荧光鉴定。结果表明:绵羊EG细胞集落隆起,边缘整齐,呈鸟巢状,细胞排列紧密;AKP染色为弱阳性;免疫荧光检测特异标志物Oct3/4呈弱阳性,SSEA-1、SSEA-3、SSEA-4、TRA-1-61、TRA-1-80呈强阳性。     

冷应激对雏鸡下丘脑-垂体-甲状腺轴的影响

越来越多的证据表明,应激能够激活下丘脑-垂体-甲状腺轴,进而影响促甲状腺激素释放激素(TRH)、促甲状腺激素(TSH)及甲状腺激素的合成与分泌。为了探明冷应激对雏鸡下丘脑-垂体-甲状腺轴的影响,以公雏鸡为实验动物,进行急性(0.25、1、3、6、12h与24h)与慢性(5、10d与20d)冷应激处理(12±1)℃,检测了雏鸡下丘脑TRH mRNA的表达水平,血清TSH、FT(3T3的游离形式)及FT(4T4的游离形式)的含量。结果表明:急性应激时,TRH mRNA的表达水平在各应激时间点均显著升高,TSH变化不明显,FT3开始无变化,在6h时突然降低,而后又显著升高,FT4开始变化不大,6h后显著升高;慢性应激时,TRH mRNA的表达水平与相应的对照组相比显著降低,TSH变化仍不明显,FT3呈上升趋势,而FT4呈下降趋势。这说明冷暴露可以使雏鸡下丘脑-垂体-甲状腺轴的激素分泌发生改变,而且不同程度的冷应激对同一激素也会产生不同的影响。     

氟对体外培养绵羊成骨细胞RANKL mRNA基因表达影响

为了探讨氟对体外培养的绵羊成骨细胞中核因子кB受体活化因子配体(RANKL)mRNA基因表达的影响,进行了绵羊成骨细胞体外培养,分别加入含浓度为0、10-6、10-5、10-4、5×10-4mol/L氟化钠(NaF)的培养液,并于第6天提取细胞总mRNA,用半定量RT-PCR方法分析RANKL mRNA表达水平的改变.结果表明,10-6、10-5、10-4、5×10-4 mol/L NaF均能刺激体外培养成骨细胞RANKL mRNA表达,当氟化钠浓度为10-5及5×10-4 mol/L时,可以明显促进RANKL mRNA表达(与对照组相比P＜0.01).这说明氟呈剂量依赖性地刺激成骨细胞RANKLmRNA的表达.     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

绵羊季节性繁殖相关基因TSHR外显子多态性研究

旨在研究乌珠穆沁羊与湖羊这两种具有不同繁殖性能和发情周期绵羊的促甲状腺激素受体(Thyroid stimulating hormone receptor,TSHR)基因外显子区遗传变异特性及其构建的单倍型在品种间的分布和对mRNA和蛋白质二级结构的影响,为进一步揭示其对TSHR表达调控的影响及表型效应奠定基础。本研究采用直接测序的方法对中国蒙古系2个绵羊品种共172个个体TSHR基因的遗传变异情况进行分析。共发现了6个突变位点,独立性卡方检验发现SNP1、SNP3、SNP4和SNP5 4个多态位点的基因型在两个绵羊群体间的分布存在极显著差异(P0.01),其中SNP5为错义突变,其余为同义突变;对这4个位点进行连锁不平衡分析发现,SNP3和SNP4两个位点完全连锁,其余位点两两之间存在不同程度连锁关系;生物信息学分析发现不同的单倍型使TSHR基因的mRNA二级结构发生改变,SNP5使受体蛋白的二级结构发生改变。结果提示:(1)TSHR基因外显子区存在4个在两个绵羊品种中具有潜在生物学功能和表型效应的突变位点,可组成H1~H6共6种单倍型,其中H1(CGCC)和H4(TTTG)分别在乌珠穆沁羊和湖羊群体中为优势单倍型,这可能与这两种绵羊不同的发情周期有关。(2)外显子区SNP5位点突变影响mRNA和蛋白质二级结构的稳定性,可能是具有潜在表型效应的重要功能位点。     

酿酒酵母细胞壁诱导体外培养绵羊瘤胃上皮细胞表达SBD-1的研究

旨在探究酿酒酵母细胞壁对原代培养绵羊瘤胃上皮细胞β-防御素-1(Sheep beta-defensin-1,SBD-1)表达的影响。本试验首先提取酿酒酵母细胞壁,并进行鉴定;再用酿酒酵母细胞壁(0、25、50、100、200、400μg·mL~(-1))刺激健康绵羊的原代瘤胃上皮细胞(0、2、4、8、12、24h)后,进行RT-qPCR和ELISA试验,确定诱导SBD-1表达的最佳浓度和时间。结果表明:1)本试验提取的酿酒酵母细胞壁成分较纯,可用于后续试验;2)体外培养绵羊瘤胃上皮细胞呈鹅卵石铺路状,符合试验要求;3)使用200μg·mL~(-1)的酿酒酵母细胞壁去刺激瘤胃上皮细胞12h,SBD-1mRNA和蛋白表达量最大,与其他组相比均差异极显著(P0.001)。综上表明,酿酒酵母细胞壁能够促进绵羊瘤胃上皮细胞SBD-1的表达,且酵母细胞壁最佳浓度和刺激时间分别为200μg·mL~(-1)和12h。     

奶牛原代乳腺上皮细胞的培养及β酪蛋白mRNA的表达

本试验旨在建立原代乳腺上皮细胞系的体外培养方法,并进行β酪蛋白mRNA的表达验证。取新鲜泌乳期的乳腺组织,采用组织块法培养纯化原代乳腺上皮细胞,利用显微镜观察细胞形态并进行细胞生长计数,采用实时荧光定量PCR技术检测β酪蛋白mRNA表达。结果显示,纯化培养的原代乳腺上皮细胞集聚成岛屿状生长,具有典型的铺路石和鹅卵石形状,细胞生长曲线呈"S"形,符合一般细胞的生长规律,并成功表达β酪蛋白mRNA。综上所述,本研究采用组织块法成功培养出具有正常生理功能的奶牛原代乳腺上皮细胞,为后续的乳腺上皮细胞功能研究提供了良好的细胞试验模型。     

GnRH脉冲对体外原代培养大鼠GTH细胞中FSHβ mRNA表达水平的影响

为了从分子水平阐述不同脉冲频率促性腺激素释放激素(GnRH)对促性腺激素(GTH)分泌促卵泡素(FSH)的影响,采用细胞体外培养技术结合荧光定量PCR技术,研究体外培养大鼠原代垂体细胞在不同脉冲频率GnRH刺激下,细胞FSHβ mRNA的变化.在相同振幅条件下,低频刺激(120 min间隔)时,FSHβ mRNA表达水...     

罗格列酮对非酯化脂肪酸体外诱导的犊牛原代肝细胞线粒体功能紊乱的影响

本试验旨在探讨非酯化脂肪酸(Nonestesterified fatty acid,NEFAs)对体外原代培养犊牛肝细胞线粒体功能以及罗格列酮对NEFAs诱导的线粒体功能紊乱的影响。体外添加NEFAs(1.8mmol/L)处理犊牛原代肝细胞不同时间,运用Western blot和实时荧光定量PCR(qRT-PCR)方法检测线粒体呼吸链复合体IV(COX IV)蛋白水平及mRNA水平的影响。另外选取细胞,罗格列酮(10nmol/L)预处理细胞24h后添加NEFAs(1.8mmol/L)处理9h。运用Western blot和qRT-PCR方法检测PGC-1α、Mfn-2的蛋白表达水平及COX IV mRNA表达水平。结果显示,随着NEFAs作用时间的增加,与0h组相比,NEFAs处理5h以上时COX IV蛋白表达水平极显著降低(P0.01),处理7~12h时COX IV mRNA表达水平呈显著降低(P0.05)。添加罗格列酮后,罗格列酮可极显著增加COX IV mRNA水平,PGC-1α、Mfn-2蛋白表达水平(P0.01)。结果表明,NEFAs可诱导犊牛原代肝细胞发生线粒体功能紊乱,罗格列酮可缓解由NEFAs诱导的犊牛原代肝细胞线粒体功能紊乱。     

Does closantel in therapeutic doses display thyroid hormone‐like activity in sheep?

The aim of this study was to define the thyroid hormone‐like activity of closantel in sheep by measuring some blood parameters that are known to be influenced by thyroid hormones triiodothyronine (T3) and thyroxine (T4). Our hypothesis was that, if closantel possesses thyroid hormone‐like activity, its use under in vivo conditions will result in changes similar to those in hyperthyroidism. The study was conducted in 20 Jezersko‐Solchava breed sheep. Blood sampling was performed before and 10 days after routine anthelmintic treatment with closantel. Complete blood count, plasma cholesterol, triglycerides, protein, and albumin levels, as well as those of serum T3 and T4, were compared before and 10 days after closantel administration. This routine anthelmintic treatment of sheep with closantel did not significantly influence hematological parameters, thyroid hormone levels, or most of the biochemical parameters. No evidence was found for thyroid hormone‐like activity of closantel in sheep. However, significantly (P < 0.01) elevated levels of plasma triglycerides were present 10 days after closantel administration.     

Identification of differentially expressed genes in ileum, intestinal lymph node and peripheral blood mononuclear cells of sheep infected with Mycobacterium avium subsp. paratuberculosis using differential display polymerase chain reaction

Discovery of differentially expressed genes aids in understanding molecular mechanisms underpinning normal and pathological states. When studying animals such as sheep where the entire genome has not been characterized, techniques that do not require knowledge of gene sequences are particularly advantageous. We used one such technique, differential display polymerase chain reaction (DD-PCR), to identify genes that had different degrees of expression in response to Mycobacterium avium subsp. paratuberculosis (M. ptb), the organism that causes Johne's disease in ruminants. Differentially expressed genes were validated by quantitative PCR using especially selected reference genes established in this study. Sheep (n = 47) were classified according to history of exposure to M. ptb and infection status by histology and faecal and tissue culture. Differences in levels of gene expression were analyzed using restricted maximum likelihood (REML) in a linear mixed model. Five genes from the ileum and 17 genes from lymph node were differentially expressed in ovine Johne's disease. Expression of seven of these genes was also significantly different in peripheral blood mononuclear cells. Genes identified in association with M. ptb infection had a wide range of functions in pathways including: antigen presentation, signal transduction and cell differentiation, TLR signaling, immune cell activation and chemokine functions, granulomatous inflammation, Th1 suppression and apoptosis.     

In vitro secretion of bovine immunoglobulins during pokeweed mitogen or pokeweed mitogen and antigen activation of lymphocytes

Activation of bovine peripheral blood mononuclear cells (PBM) towards immunoglobulin (Ig) synthesis and secretion was examined in vitro using pokeweed mitogen (PWM) or PWM plus sheep red blood cells (SRBC). Bovine PBM were composed of 7–30% B cells, 25–56% T cells and 1–4% macrophages. Cell density and PWM concentrations were critical parameters for obtaining reproducible maximum lymphocyte proliferation and polyclonal B cell activation. Inclusion of aminopterin, a folic acid antagonist, reduced cellular proliferation and viability but had no apparent effect on the time of appearance of peak proliferation. Co-culture of PBM with SRBC and PWM generated anti-SRBC specific antibodies.     

Indel variants within the PRL and GHR genes associated with sheep litter size

Growth hormone and prolactin belong to the class of peptide hormones that have a wide range of regulatory functions. In this study, polymorphisms of growth hormone receptor (GHR) and prolactin (PRL) genes were analysed as candidate genes, which are responsible for the litter size in Australian White (AUW) sheep. According to the statistical analyses results, the polymorphism information content (PIC) values of the PRL-P1-ins-23 bp, GHR-P2-del-23 bp and GHR-P8-del-23 bp were 0.371, 0.366 and 0.375, respectively, which indicates the high genetic polymorphism in AUW sheep. Moreover, all indel loci are not conformed to the HWE (p < .05). Further, our findings revealed that the PRL-P1-ins-23 bp polymorphism in the ovine PRL gene was significantly related to the first parity litter size (p = .001) and the DD genotype displaying the highest genotypic mean. Meanwhile, the GHR-P2-del-23 bp and GHR-P8-23 bp indels in the ovine GHR gene were significantly correlated with first parity litter size (p < .05), and the individuals with the genotype II showed significantly higher litter size than others. Collectively, these results demonstrated that our findings could be useful for future sheep breeding strategies based on the molecular-assisted selection (MAS).     

二狼山白山羊遗传性甲状腺肿发病机理及诊断研究——Ⅱ.甲状腺组织中TG分布、TG带谱分析及血清TG含量检测

应用BA-免疫组化结合图像分析测定了遗传性甲状腺肿病山羊(以下简称IHO)甲状腺组织中甲状腺球蛋白(TG)的含量和分布,用梯度-PAGE结合免疫印迹法分析了甲状腺组织中异常TG相关抗原蛋白的带谱及分子量;用BA-ELISA法测定了血清中TG含量.结果表明:(1)IHG纯合子病山羊甲状腺组织中TG含量明显低于对照组山羊(P<0.01),且TG在胶质中多为弱阳性,在增生上皮细胞内几乎为阴性;(2)IHO纯合子病山羊甲状腺组织中异常TG相关抗原蛋白为331000、110000,而IHG杂合子病山羊异常TG相关抗原蛋白为110000;(3)IHG杂合子病山羊血清中TG含量明显低于对照组(P<0.05).提示:IHG病山羊甲状腺激素合成不足是由于TG合成障碍,且其具体发病环节可能是TG亚单位(110000、331000相关TG抗原蛋白)进一步聚合为1200000的TG过程缺陷的结果;异常TG相关抗原蛋白110000,可作为净化IHG杂合子病山羊的特异性生化指标,其血清中TG含量降低,亦可作为剔除IHG杂合子山羊的生化指标之一.     

Inositol and hepatic lipidosis. II. Effect of inositol supplementation and time from parturition on serum insulin, thyroxine and triiodothyronine and their relationship to serum and liver lipids in dairy cows

Percutaneous liver biopsies and blood samples were obtained from 80 dairy cows in nine Michigan herds over the peripartum period. Thirty-nine cows were fed 17 g of supplemental inositol and 41 were fed a placebo. Liver biopsies were assayed for total myoinositol and triglyceride (TG) concentrations. Blood samples were assayed for serum dextran precipitable cholesterol, nonesterified fatty acids (NEFA), insulin, thyroxine (T4), free (FT4), triiodothyronine (T3) and free T3 (FT3) concentrations. Serum concentrations of insulin and the thyroid hormones decreased near parturition, with lowest concentrations occurring in the immediate postpartum period. Concentrations of T3 correlated well with T4, and the concentrations of free thyroid hormones reflected concentrations of total thyroid hormones. The percentage of hormone in the free fraction remained constant over time. Serum insulin, T3 and T4 were negatively correlated with serum NEFA and liver TG concentrations. Thyroid hormone concentrations were positively correlated with serum dextran precipitable cholesterol concentrations. Inositol supplementation was associated with reduced circulating T3 and FT3 concentrations, but not T4 and FT4 concentrations. Changes in hormone concentrations at parturition and their relationship to liver TG and serum NEFA concentrations were consistent with a metabolic adaptation by the dairy cow to the negative energy balance of early lactation.     

TGIF1 and SF1 polymorphisms are associated with litter size in Small Tail Han sheep

TGF-β induced factor homeobox 1 (TGIF1) and splicing factor 1 (SF1) are important for mammalian reproduction; however, the effects of these genes on litter size in sheep remain unexplored. In this study, we genotyped 768 ewes from seven sheep breeds at two loci: g.37871539C>T, a synonymous mutation of TGIF1; and g.42314637T>C, a 3′UTR variant of SF1. Our analysis of polymorphism revealed only two genotypes at locus g.37871539C>T in TGIF1, with most sheep populations being moderately polymorphic (0.25 < PIC < 0.5) at this site. In contrast, most breeds exhibited low polymorphism (PIC ≤0.25) at the SF1 locus g.42314637T>C. The association analysis revealed that a synonymous mutation at g.37871539C>T in TGIF1 was highly associated with litter size in Small Tail Han sheep, in which it causes a significant decrease in litter size. Conversely, while the SF1 3′UTR variant g.42314637T>C was also highly associated with litter size in sheep, it causes a significant increase in the number of litter size. Combined, these data provide valuable information regarding candidate genetic markers for sheep breeding programs.     

Hypothyroidism Affects Differentially the Cell Size of Epithelial Cells Among Oviductal Regions of Rabbits

Oviductal regions show particular histological characteristics and functions. Tubal pathologies and hypothyroidism are related to primary and secondary infertility. The impact of hypothyroidism on the histological characteristics of oviductal regions has been scarcely studied. Our aim was to analyse the histological characteristics of oviductal regions in control and hypothyroid rabbits. Hypothyroidism was induced by oral administration of methimazole (MMI) for 30 days. For both groups, serum concentrations of thyroid and gonadal hormones were determined. Sections of oviductal regions were stained with the Masson's trichrome technique to analyse both epithelial and smooth muscle layers. The percentage of proliferative epithelial cells (anti‐Ki67) in diverse oviductal regions was also quantified. Data were compared with Student t‐test, Mann–Whitney U‐test, or Fischer's test. In comparison with the control group, the hypothyroid group showed: (i) a low concentration of T3 and T4, but a high level of TSH; (ii) similar values of serum estradiol, progesterone and testosterone; (iii) a large size of ciliated cells in the ampulla (AMP), isthmus (IST) and utero‐tubal junction (UTJ); (iv) a large size of secretory cells in the IST region; (v) a low percentage of proliferative secretory cells in the fimbria‐infundibulum (FIM‐INF) region; and (vi) a similar thickness of the smooth muscle layer and the cross‐sectional area in the AMP and IST regions. Modifications in the size of the oviductal epithelium in hypothyroid rabbits could be related to changes in the cell metabolism that may impact on the reproductive functions achieved by oviduct.     

The metabolic profile of growing lambs fed diets rich in unsaturated fatty acids

The effect of two diets enriched with unsaturated fatty acids – one containing the addition of dried distillers grains with solubles (DGS) and the other the addition of false flax – Camelina sativa cake (CS) – on some metabolic parameters and hormone concentration in growing lambs was determined in this experiment. A total of 21 ram lambs of the Polish Whiteheaded mutton sheep were divided into three groups (the control, receiving DGS and CS). The diets were administered to animals for 6 weeks. During the experiment, peripheral blood was collected. Glucose (GL), total cholesterol (CH), triglycerides (TG), free fatty acids (FFA), insulin (IN), leptin (LE), triiodothyronine (T3) and thyroxine (T4) were assayed in serum. The age‐dependent reduction in CH and TG limited by both experimental diets were observed. A significant increase in FFA concentration was observed in samples collected in the last, that is, third, time period. This was most probably caused by a 12‐h pre‐slaughter fasting period. A significantly lower dynamic of FFA increase in that experimental period was found in animals receiving the experimental feed. Insulin concentration in DGS‐receiving lambs was increased, in contrast to the CS‐receiving lambs, in which it was lower when compared to the control. LE concentration was decreased by both experimental diets, more markedly in the DGS‐receiving animals. No differences between the experimental groups and the control were observed in T3 and T4 concentrations. The effect of 12‐h pre‐slaughter fasting was statistically highly significant for the levels of examined blood markers and hormones, except for TG and IN in the group of lambs receiving the experimental diet with CS.