细胞因子在猪呼吸道疾病中的作用及存在的问题和研究价值

细胞因子，如干扰素-α(IFN-α)，肿瘤坏死因子-α(TNF-α)，白细胞介素-1、-6和-8(IL-1，-6，-8)，在多数感染的早期都会产生。这些细胞因子的活性已在体外和裸鼠实验中广泛研究，但有关细胞因子在肉用动物感染中的作用的体内实验研究却很少，本文综述细胞因子在猪的呼吸道感染中的作用，并探讨其对呼吸道病毒性感染的相关性。     

酒精性肝病的内毒素信号通路研究

酒精性肝病(ALD)发病的重要机制在于饮酒导致肝巨噬细胞对内毒素——脂多糖(LPS)的敏感性升高,因而细胞内毒素(LPS)诱导信号相关机制在酒精肝损伤的初期和发展过程中发挥着至关重要作用。LPS被枯否细胞上的受体识别,激活下游信号通路,最终激活核转录因子(NF-κB),导致肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)、白细胞介素-6(IL-6)和生长因子-β(TGF-β)等炎症细胞因子产生增加,炎症细胞因子又激活枯否细胞,产生更多细胞因子,加重肝脏损伤。     

荷斯坦泌乳牛4种炎性细胞因子随胎次变化规律的研究

本试验选择北京地区5个牛场不同胎次(1胎、3胎、5胎及以上)、无临床疾病记录的150头荷斯坦泌乳牛,于2017年7月至8月测定促炎细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和抑炎细胞因子白细胞介素-10(IL-10)、转化生长因子-β(TGF-β)4种血液炎性细胞因子浓度,利用SAS 9.4软件GLM过程分析血液炎性细胞因子浓度随胎次变化的规律,所用固定模型考虑了牛场、胎次、泌乳阶段等因素影响,同时进行各细胞因子间Pearson相关性分析;利用GLM过程分析炎性细胞因子对产奶性能的影响,产奶性能指标包括日产奶量、校正日产奶量、乳蛋白率、乳脂率、乳糖率,所用固定模型考虑了牛场、胎次、泌乳阶段、炎性细胞因子水平等因素的影响。结果显示:荷斯坦牛血液IL-6和TGF-β浓度随胎次升高显著降低;整体来说,IL-6和TGF-β显著正相关,IL-10和TGF-β显著负相关;5胎及以上胎次奶牛各细胞因子间相关关系均不显著;5胎及以上胎次奶牛日产奶量、校正日产奶量和乳糖率显著降低;高TNF-α组的奶牛日产奶量显著低于低TNF-α组,高IL-10组奶牛乳脂率和校正日产奶量显著高于低IL-10组。综上,高胎次奶牛生产性能的降低可能与炎性细胞因子的变化有关。     

PYY~(3-36)对LPS诱导的BV-2细胞中促炎酶类及促炎细胞因子的影响

为明确酪酪肽(peptide YY,PYY~(3-36))对脂多糖(lipopolysaccharide,LPS)诱导的BV-2细胞中促炎细胞因子及促炎蛋白酶类的影响,本试验用不同浓度的PYY~(3-36)预处理后,再用LPS刺激BV-2细胞,利用MTT法检测PYY~(3-36)的细胞毒作用;分别用荧光定量RT-PCR和Western blot方法检测促炎细胞因子(IL-1β、IL-6和TNF-α)及促炎蛋白酶类(iNOS和COX-2)基因水平和蛋白水平表达变化;分别利用NO检测试剂盒和ELISA试剂盒检测iNOS和COX-2的主要产物NO和PGE2;利用PCR方法检测PYY~(3-36)受体表达情况。结果显示,PYY~(3-36)(10-9~10-11 mol/L)呈浓度依赖性抑制LPS在BV-2细胞中诱导的促炎细胞因子和促炎蛋白酶类的基因和蛋白水平,并能浓度依赖性抑制NO和PGE2。PCR结果表明BV-2细胞中PYY~(3-36)受体Y1、Y2、Y5和Y6表达明显,并且LPS刺激后Y2R受体表达增加。表明PYY~(3-36)在BV-2细胞中能抑制LPS诱导的促炎细胞因子和促炎蛋白酶类的表达以及促炎酶类产物的生成,这个过程可能涉及到其受体Y2。     

维生素D对低磷酸盐血症奶牛细胞因子的影响

试验选择安达市先锋乡奶牛场部分低血磷的奶牛为试验对象 ,经喂饲高磷牧草并添加维生素Ds,检测血清细胞因子 (IL - 1、IL - 6 )的动态变化 ,揭示维生素D对低磷酸盐血症奶牛细胞因子的影响。研究结果表明 :当奶牛发生低磷酸盐血症时IL - 1、IL - 6含量升高     

脂肪细胞因子对动物脂类代谢的调控机理

脂肪细胞是一种能分泌多种细胞因子的内分泌细胞,如脂联素(APN)、瘦素、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)及抵抗素等,这些细胞因子通过多种信号通路在机体脂类代谢中发挥着重要作用。本文主要综述了脂肪细胞因子对动物脂类代谢的调控机理,为通过饲粮途径调控动物的脂类代谢和改善肉品质提供参考依据。     

牦牛脾脏转移因子对小鼠脾淋巴细胞增殖及诱生细胞因子的影响

为了探究牦牛脾脏非特异性转移因子(specificity transfer factor,TF)对小鼠淋巴细胞增殖及诱生细胞因子的影响,应用ELISA测定了小鼠注射TF后不同时间相关细胞因子分泌量的变化。结果显示,牦牛TF对分泌细胞因子具有促进作用,且用ConA或用TF再次刺激鼠淋巴细胞后各细胞因子分泌量均高于鼠淋巴细胞单独增殖组。其中小鼠γ-干扰素(IFN-γ)含量表现为先增加后减少,峰值出现在第48 h,并持续分泌时间较长,与对照组相比,TF+ConA组鼠淋巴细胞分泌IFN-γ的量在48 h和72 h时显著高于TF组和细胞单独增殖组(P0.05);小鼠白细胞介素-1(IL-1)含量的变化随着时间的延长呈上升趋势;白细胞介素-12(IL-12)的动态变化全部表现出前期(12 h)和中期(48 h)两个分泌高峰,并且在48 h时,ConA协同TF组明显高于细胞单独增殖组与TF组(P0.05);白细胞介素-4(IL-4)的动态变化表现不明显;白细胞介素-2(IL-2)含量随着时间的延长,呈上升再下降的趋势,48 h达到峰值,并显著高于TF组和细胞单独增殖组(P0.05),而后下降。说明TF协同ConA刺激小鼠淋巴细胞后,均可提高各种细胞因子的分泌能力。     

PYY对LPS诱导小鼠腹腔巨噬细胞TNF-α、IL-6分泌的影响

为明确PYY对巨噬细胞炎性细胞因子分泌的调节作用,本试验分离培养健康小鼠腹腔巨噬细胞,不同浓度PYY预处理后,以LPS刺激。ELISA方法检测细胞培养上清中TNF-α、IL-6含量,半定量PCR方法检测细胞中TNF-α、IL-6mRNA表达变化。结果显示:高浓度的PYY1-36(10-9¹0-7 mol/L)和PYY3-36(10-810-7 mol/L)和PYY3-36(10-8¹0-7 mol/L)对LPS诱导小鼠腹腔巨噬细胞TNF-α分泌具有显著抑制作用(P<0.05);PYY1-36对LPS诱导小鼠腹腔巨噬细胞IL-6分泌无明显作用(P>0.05);不同浓度PYY3-36(10-1110-7 mol/L)对LPS诱导小鼠腹腔巨噬细胞TNF-α分泌具有显著抑制作用(P<0.05);PYY1-36对LPS诱导小鼠腹腔巨噬细胞IL-6分泌无明显作用(P>0.05);不同浓度PYY3-36(10-11¹0-7 mol/L)对LPS诱导小鼠腹腔巨噬细胞IL-6分泌均具有显著抑制作用(P<0.05)。表明PYY对LPS诱导小鼠腹腔巨噬细胞炎性细胞因子TNF-α及IL-6的分泌具有一定的抑制作用,提示PYY可能通过抑制炎性细胞因子的分泌而抑制炎症性疾病的发生发展。     

妊娠期激素对Th1和Th2细胞因子的调控

妊娠是一个复杂的生理过程,受到神经、免疫和内分泌的共同调节。作为半自体抗原的胎儿不被母体排斥是子宫局部免疫水平调节的结果。其中激素对辅助性淋巴细胞(Th)发生、分化的调节作用愈来愈受到重视。辅助T淋巴细胞能分化为不同的亚型(Th1和Th2),孕酮可促进Th2型细胞因子(IL-4,IL-5)的产生,有利于妊娠的正常发生;松弛肽促进Th1型细胞因子(IFN-γ)的产生,对妊娠有害。在母胎界面存在激素-细胞因子网络,淋巴细胞产生的细胞因子还能够影响其他细胞因子的产生。由Th2样细胞产生的,对胚泡着床具有非常重要作用的白血病抑制因子(LIF),其表达受Th1细胞诱导因子的下调,如IL-12,IFN-α,IFN-γ,受IL-4和孕酮(诱导T淋巴细胞向Th2细胞分化)的上调。激素通过调控淋巴细胞分泌的细胞因子来调控免疫系统,保持一个动态的平衡,有利于维持妊娠。     

细胞因子对小尾寒羊胎盘成熟的影响

为了研究细胞因子对小尾寒羊胎盘成熟的影响 ,本实验采用液相竞争法和平衡法 ,对小尾寒羊空怀期 (n=5 )和妊娠期 (n=13)第 85天、10 5天、12 5天、14 0天和 15 0天 (足月 )时的血清白细胞介素 - 1β(IL - 1β)、白细胞介素 - 6 (IL - 6 )和肿瘤坏死因子(TNF)的含量分别进行检测。结果表明 ,IL - 1β和 IL - 6含量在 12 5天时与对照组间差异极显著 (P<0 .0 1) ,TNF含量在各时期与对照组差异均显著 (P<0 .0 1或 P<0 .0 5 )。随着妊娠过程的进展 ,三种细胞因子含量逐渐增加 ,12 5天时达到最高值 ,分别为 0 .390± 0 .196 ng/ ml,5 79.8± 15 2 .8pg/ ml和 2 .348± 0 .396 ng/ ml。而后逐渐下降 ,到足月前略有回升 ,但仍低于 12 5天时的最高值。由此看出小尾寒羊在妊娠期间 ,细胞因子与妊娠之间有着密切的关系 ,前者对于维持妊娠起到重要作用。由于 14 0天至足月3种细胞因子基本稳定 ,这表明胎盘于 14 0天左右达到成熟 ,IL- 1β和 IL- 6与启动分娩有关 ,而 TNF可能参与小尾寒羊分娩的启动。     

Assessment of meat quality defect genes in indigenous pigs of Bareilly region

This investigation was undertaken to assess the population of indigenous (Bareilly local) pigs for meat quality genes (RYR1, PRKAG3, HFABP, MYF-5, and MC4R). The results showed that indigenous pigs were monomorphic at RYR1locus (100% NN genotype), HFABP locus (100% HH genotype), and MYF-5 locus (100% DD genotype). Homozygote RR and heterozygote QR genotypes were observed at PRKAG3 (c.599 G>A) SNP locus with 89 and 11% frequency. The frequency of wild (R) and mutant (Q) allele at the said locus was 95 and 5%. The MC4R SNP had three genotypes; homozygote AA with 5% frequency, heterozygote AG with 53% frequency, and homozygote GG with 42% frequency. Corresponding frequency of A and G allele was 32 and 68%, respectively. Monomorphic status at RYR1locus for NN genotype, HFABP locus for HH genotype, and MYF-5 locus for DD genotype indicated that favorable genes for quality pork production have been fixed in the population. The higher frequency of RR genotype (89%) at PRKAG3 and GG genotype (42%) at MC4R locus further explained the existence of favorable genotypes in indigenous pigs.

     

Promoter region of the bovine growth hormone receptor gene: single nucleotide polymorphism discovery in cattle and association with performance in Brangus bulls

Expression of the GH receptor (GHR) gene and its binding with GH is essential for growth and fat metabolism. A GT microsatellite exists in the promoter of bovine GHR segregating short (11 bp) and long (16 to 20 bp) allele sequences. To detect SNP and complete an association study of genotype to phenotype, we resequenced a 1,195-bp fragment of DNA including the GT microsatellite and exon 1A. Resequencing was completed in 48 familialy unrelated Holstein, Jersey, Brown Swiss, Simmental, Angus, Brahman, and Brangus cattle. Nine SNP were identified. Phylogeny analyses revealed minor distance (i.e., <5%) in DNA sequence among the 5 Bos taurus breeds; however, sequence from Brahman cattle averaged 27.4 +/- 0.07% divergence from the Bos taurus breeds, whereas divergence of Brangus was intermediate. An association study of genotype to phenotype was completed with data from growing Brangus bulls (n = 553 from 96 sires) and data from 4 of the SNP flanking the GT microsatellite. These SNP were found to be in Hardy-Weinberg equilibrium and in phase based on linkage disequilibrium analyses (r(2) = 0.84 and D'= 0.92). An A/G tag SNP was identified (ss86273136) and was located in exon 1A, which began 88 bp downstream from the GT microsatellite. Minor allele frequency of the tag SNP was greater than 10%, and Mendelian segregation was verified in 3 generation pedigrees. The A allele was derived from Brahman, and the G allele was derived from Angus. This tag SNP genotype was a significant effect in analyses of rib fat data collected with ultrasound when bulls were ~365 d of age. Specifically, bulls of the GG genotype had 6.1% more (P = 0.0204) rib fat than bulls of the AA and AG genotypes, respectively. Tag SNP (ss86273136), located in the promoter of GHR, appears to be associated with a measure of corporal fat in Bos taurus x Bos indicus composite cattle.     

马鹿特异性SNP分子标记的验证

试验旨在对基于基因分型测序（genotyping by sequencing,GBS）技术筛选出的马鹿特异性SNPs位点的准确性进行验证,为梅花鹿、马鹿及其杂交后代的鉴别提供可靠的分子遗传标记。随机选取30个马鹿特异性SNPs位点,根据SNPs位点前后各200 bp的序列,利用Primer Premier 6.0软件设计特异性引物,以随机选取的验证样本DNA作为模板进行PCR扩增,并进行Sanger测序,对测序结果利用BioEdit软件进行峰图的观察,利用Mega 6.0软件对测序得到的序列进行比对分析并观察每个特异性SNP位点在不同验证群体中的基因型,对每一个马鹿特异性位点的峰图和比对信息进行统计分析。结果表明,30个马鹿特异性位点中有28个和前期研究结果一致,其中1个SNP位点（SNP3）中G等位基因在梅花鹿中的基因频率为0.05,而G等位基因在马鹿中的基因频率为1,G等位基因在马鹿个体中的基因频率比在梅花鹿个体中高,同时,利用SPSS 22.0进行统计分析发现,该位点在马鹿和梅花鹿中基因型分布表现出显著性差异（P<0.05）;另外1个SNP位点（SNP5）中T等位基因在梅花鹿的基因频率为0.15,而在马鹿中的基因频率为1,且这个SNP位点在梅花鹿和马鹿中基因型分布差异显著（P<0.05）,所以这2个位点仍然可以作为马鹿的特异性SNPs位点。研究结果说明了GBS测序筛选出的马鹿特异性SNPs可以作为鉴定的分子标记,对梅花鹿、马鹿及其杂交后代的鉴别奠定了理论基础。     

Association Analysis on MAP3K5 Gene Polymorphism Site with Growth and Feed Efficiency Related Traits in Duroc Pigs

This study was aimed to analyze the association of four SNPs of MAP3K5 gene with growth and feed efficiency related traits in Duroc populations. Four SNPs(Ssc1:30769583 A>C;Ssc1:30781169 A>G;Ssc1:30940839 A>G;Ssc1:30962276 G>A)of MAP3K5 gene in Duroc populations were obtained by porcine SNP60 BeadChip（Illumina). The genotype frequency and allele frequency of four SNPs of MAP3K5 gene were analyzed,the result showed that four SNPs were the low-moderate mutation SNPs.The polymorphism of MAP3K5 gene and its association with feed efficiency related traits in Duroc population were analyzed. The results indicated that CC genotype individuals in Ssc1:30769583 A>C had significant lower RFI（133.08 g/d）than AA genotype individuals（P<0.05);GG genotype individuals in Ssc1:30781169 A>G had significant lower RFI（116.18 g/d）and ADFI（0.23 kg/d）than AG genotype individuals（P<0.05);GG genotype individuals in Ssc1:30940839 A>G had significant lower FCR（0.10%）than AG genotype individuals（P<0.05);AA genotype individual in Ssc1:30962276 G>A had significant lower ADG（0.04 kg/d）than GG genotype individuals（P<0.05). All in all,the results suggested that four SNPs polymorphisms of MAP3K5 gene had the significant impact on RFI, ADFI, FCR and ADG traits in Duroc pigs.     

猪肉质性状功能基因在野猪群体内的变异研究

本试验旨在研究猪肉质性状功能基因突变位点在野猪群体里面的遗传变异规律。以MC4R基因(D298N G>A)、RYR1基因(c.1843 C>T)、PRKAG3基因(1849 G>A)和IGF2基因(3072 G>A)突变位点为基础,通过PCR-RFLP、PCR-SSCP技术,对72头圈养野猪群体进行突变位点的多态性检测和群体遗传变异分析。结果表明,RYR1基因在c.1843 C>T突变位点的CC基因型为野猪群体内的优势基因型,等位基因C是野猪群体内的优势基因,没有TT基因型,该突变位点处于不平衡状态(P<0.01和P<0.05)。在MC4R基因D298N G>A突变位点上,GG基因型为野猪群体内的优势基因型,等位基因G是野猪群体内的优势基因。而PRKAG3基因(1849 G>A)的突变位点检测结果是GG基因型为野猪群体内的优势基因型,等位基因G是野猪群体内的优势基因,没有发现AA基因型。IGF2基因3072 G>A在野猪群体内没有发现多态性,经测序验证全部为GG基因型。这3个基因突变都处于Hardy-Weinberg平衡状态(P>0.05)。本研究得出该野猪种群体遗传多样性和变异较小,符合其品种特性。     

The Correlation Analysis between Chinese Merino Sheep OLA-DQB1 Gene Exon 2 Genetic Polymorphism and Brucellosis Susceptibility

The single nucleotide polymorphisms (SNPs) of ovine lymphocyte antigen DQB1 (OLA-DQB1) gene exon 2 was amplified by PCR-SSCP method from 148 healthy and 60 infected with Brucella Chinese Merino sheep and then PCR products of different alleles were sequenced to determine the polymorphism loci of the gene.The differences in gene frequency and genotype frequency of each SNP loci were analyzed statistically to analyze its correlation with brucellosis susceptibility.The sequencing result showed that 43 SNPs were detected in 270 bp DNA sequence,the gene frequencies of G196A allele had extremely significant difference in case and control samples (P< 0.01),and its genotype frequencies presented significant difference (P< 0.05).Similarly,C211T allele was significantly different in case and control samples (P< 0.05).The results showed that the polymorphism of OLA-DQB1 gene exon 2 might be a significant association gene with brucellosis susceptibility.     

Homozygosity of single nucleotide polymorphisms in the 3′ region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40‐kb region at the 3′ end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3′ end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14–17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15–17). A common haplotype block was identified in an approximately 20‐kb region encompassing four SNPs (SNPs # 14–17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3′ end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.     

The Polymorphisms of Chinese Merino Sheep eNOS Gene exon8 and its Association with Brucellosis Susceptibility

The association of Chinese Merino sheep eNOS gene exon8 polymorphisms with brucellosis susceptibility was studied in this research.The sequences of human and Chinese Merino sheep eNOS gene exon8 were aligned by the bioinformatics methods,and the polymorphisms of human eNOS gene in the NCBI SNP database were statistically analyzed.The polymorphisms of 101 Chinese Merino sheep negative samples and 61 Chinese Merino sheep positive samples were detected by PCR-SSCP method,and then the PCR products of different alleles were sequenced,aiming to determine exon8's polymorphism loci,and the allele frequency and genotype frequency of SNP loci were statistically analyzed.A novel SNP locus (ss974768653:A142G) was detected at the 142 bp of eNOS gene exon8,and the locus had no difference in allele frequency and each genotype between negative and positive groups (P >0.05).There might be no correlation between the A142G polymorphism locus of Chinese Merino sheep eNOS gene exon8 and brucellosis susceptibility.     

中国美利奴羊eNOS基因exon8多态性及其与布鲁氏菌病易感性相关性分析

本试验旨在研究内皮型一氧化氮合酶(eNOS)基因exon8多态性与中国美利奴羊布鲁氏菌病易感性的相关性。利用生物信息学方法对人和中国美利奴羊eNOS基因exon8序列进行比对,并对NCBI SNP数据库上人eNOS基因exon8多态性进行了统计分析。通过PCR-SSCP对101只中国美利奴羊阴性样本和61只中国美利奴羊阳性样本eNOS基因exon8的多态性进行检测,然后对不同等位基因进行PCR产物测序,旨在确定该基因exon8多态性位点,并对SNP位点的等位基因频率、基因型频率进行统计分析。在eNOS基因exon8序列142 bp处检测到一个新的SNP位点(ss974768653:A142G),该位点在病例组和对照组之间的等位基因频率及各基因型间不存在显著差异性(P >0.05)。eNOS基因exon8 A142G多态性位点与中国美利奴羊布鲁氏菌病易感性可能无相关性。