为快速检测家畜衣原体,选取家畜衣原体ompA基因的高度保守区域,设计了针对家畜衣原体的引物和探针,优化反应条件,建立家畜衣原体荧光定量PCR检测方法.结果显示:该方法的最低检测限为1.68×101 copies/μL,在1.68×102～1.68×106 copies/μL范围内具有良好的线性关系;该方法可特异性检测家畜衣原体,与肺炎衣原体、沙眼衣原体、鹦鹉热衣原体、流产衣原体4种4个菌株无交叉反应,表明特异性好;其批间重复试验和批内重复试验的变异系数分别为0.14％～0.51％、1.09％～1.49％,表明重复性良好.本方法可用于家畜衣原体的早期检测和流行病学调查.

为了建立检测绵羊流产衣原体MOMP(main outer membrane protein)蛋白抗体的间接ELISA方法,试验将编码MOMP蛋白的基因序列经密码子优化后进行基因合成,并克隆和原核表达截短蛋白,以该蛋白为抗原建立绵羊流产衣原体抗体的间接ELISA方法,优化该方法的反应条件,同时验证其特异性、敏感性和重复性,最后用该方法对临床待检羊血清进行检测,并与间接血凝试验试剂盒的检测结果进行比较.结果表明:截短蛋白MOMP201～390的表达、纯化效果最好.经探索,优化后的间接ELISA方法最优反应条件:抗原包被浓度为1 mg/L,37℃包被2 h,待检血清最佳稀释度为1:100,血清和酶标二抗的最佳作用时间分别为45 min、60 min.建立的间接ELISA方法的敏感性较高,特异性和重复性较好,使用该方法从209份临床待检羊血清样本中检出47份阳性血清,与间接血凝试验试剂盒分别检测临床待检羊血清样本86份,总符合率为80.23％.说明基于截短蛋白MOMP201～390建立的绵羊流产衣原体抗体间接ELISA方法可用于绵羊流产衣原体临床血清抗体的检测及流行病学调查.

【Objective】In this study, monoclonal antibodies (MAbs) against the p30 protein of African swine fever virus (ASFV) were prepared and the linear epitopes on p30 was analyzed, which could lay the foundation for ASFV and its antibody detection as well as the study of p30 protein structure and function.【Method】BALB/c female mice aged 6 to 8 weeks were immunized with prokaryotic expression and purified recombinant p30 protein. The mice were immunized once every two weeks, with three times in total. First immunization was done with emulsification of antigen and equal volume Freund’s complete adjuvant, then, the mice were immunized with emulsification of antigen and equal volume of Freund’s incomplete adjuvant for the second and third immunization. After three immunizations, the tail was cut off and the blood was collected, and the serum antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titer were selected for enhanced immunization. Three days later, the mice spleen lymphocytes and SP2/0 myeloma cells were fused with PEG at a ratio of 4∶1. The positive hybridoma cells were screened by indirect ELISA by using the recombinant p30 protein as coated antigen. and the MAbs which could secrete antibodies steadily were cloned and purified by limited dilution method. ASFV was inoculated into porcine alveolar macrophages, and the indirect immunofluorescence assay (IFA) was performed with MAbs as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. The ASFV-infected and uninfected cells were precipitated and transferred to the nitrocellulose membrane by SDS-PAGE. p30 MAbs were obtained by Western blotting analysis of MAbs and identified positive by IFA, which was used as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. Primers were designed to amplify two truncated genes p30ab and p30bc, p30ab stands for truncated amino acid residues at position 86-153 and p30bc stands for truncated amino acid residues at position 120-153. The recombinant protein GST-p30ab and recombinant protein GST-p30bc were obtained by partial overlapping truncated p30 protein expression in prokaryotes. GST-p30ab and GST-p30bc fusion proteins were used as coated antigens, and the epitopes of p30 protein were preliminarily identified by indirect ELISA with 5 MAbs as primary antibodies, with rabbit anti-rat HRP-IgG as secondary antibodies.【Result】The purified recombinant protein was used as the coated antigen, and 25 hybridoma cell lines were screened by indirect ELISA, which could secrete anti-recombinant p30 protein. IFA results showed that 5 MAbs (8F4, 1D3, 1H2, 6C3 and 8E11) were positive for ASFV-infected cells. Western blotting results showed that all 5 strains of MAbs could react positively with ASFV-infected cells and negatively with uninfected cells. The recombinant p30 protein GST-p30ab was expressed in soluble and inclusion body forms, and GST-p30bc was expressed in inclusion body form. The truncated fusion proteins of the two groups were used as the coated antigens. Indirect ELISA showed that MAbs 8F4, 1H2 and 6C3 could effectively bind to the two recombinant proteins that means MAbs 8F4, 1H2 and 6C3 recognized the epitope amino acid 120-153; MAbs 8E11 and 1D3 could only bind to GST-p30ab protein, which meant they recognized the epitope amino acid 86-119.【Conclusion】In this study, the recombinant p30 protein with amino acid truncated at position 86-153 was expressed in soluble form. Five MAbs were prepared and two p30 protein epitopes were located. Combined with ELISA and IFA, it could be formed a very reliable method for the detection of ASFV and its antibody.

为获得非洲猪瘟病毒(African swine fever virus,ASFV)体外诱导表达的MGF505-3R蛋白.实验依据GenBank中报道的MGF505-3R基因序列设计引物,引入His标签,以ASFV基因全序列为模板扩增目的基因.将目的基因克隆至原核表达载体pGEX-6p-1,并转入大肠杆菌Transetta(DE3)中.用1 mmol/L异丙基 β-D-硫代半乳糖苷(IPTG)诱导表达菌体,分别取上清液和沉淀进行十二烷基硫酸钠-聚丙烯酰胺(SDS-PAGE)凝胶电泳分析.将表达的蛋白经过Ni-NTA琼脂糖亲和层析纯化.免疫印迹实验(Western-blot)分析蛋白的活性.克隆得到的MGF505-3R基因片段长度为843 bp,与原序列一致.蛋白在沉淀中以包涵体的形式表达,相对分子质量约为53 ku.纯化后可得到纯度较高的MGF505-3R重组蛋白.免疫印迹实验结果呈阳性.MGF505-3R蛋白可以在大肠杆菌Transetta(DE3)中表达并纯化,蛋白具有良好的表达活性.近几年,多基因家族(Multigene Families,MGFs)基因缺失疫苗被认为是目前ASF疫苗研制的主要方向,本研究获得的MGF505-3R重组蛋白将为ASFV MGF505-3R蛋白的研究提供基础,也为基因缺失疫苗相关免疫学方面的检测提供前期技术储备.

为了获得非洲猪瘟病毒(African swine fever virus,ASFV)体外诱导表达的MGF360-13L蛋白,试验依据GenBank中报道的MGF360-13L基因序列设计引物,引入His标签,以ASFV全序列为模板扩增目的片段.将目的基因克隆到原核表达载体pGEX-6p-1上,转化BL21(DE3)大肠杆菌感受态细胞中进行体外诱导表达,聚丙烯酰胺凝胶电泳(SDS-P AGE)分析蛋白质的表达情况,将表达的重组蛋白通过镍琼脂糖凝胶柱亲和层析纯化,蛋白质印迹(Western-blot)分析蛋白质的活性.结果表明:克隆得到的MGF360-13L基因片段长度为1 060 bp,获得的重组蛋白在沉淀中以包涵体的形式表达,分子质量约为60 ku,纯化后可获得纯度较高的MGF360-13L,用BCA法测定最高浓度为2.73 mg/mL.Western-blot检测结果显示在60 ku处有一条明显的特异性条带说明MGF360-13L蛋白可以在BL21(DE3)大肠杆菌感受态细胞中表达并纯化,纯化后可获得纯度较高的MGF360-13L,且具有良好的反应原性.

本研究旨在克隆小尾寒羊B细胞易位基因2(B-cell Translocation Gene 2,BTG2)基因编码CDs区,并对其进行生物信息学分析,同时检测该基因在羔羊期各组织中的mRNA表达情况.通过RT-PCR和TA克隆方法获得BTG2基因CDs区序列;利用在线软件对该基因进行生物信息分析;采用qPCR检测该基因在3、40日龄绵羊的各组织中的表达情况.结果 表明:成功获得小尾寒羊BTG2基因CDs区序列453 bp;绵羊与牛、猪、虎鲸、人、大鼠、小鼠同源性分别为98.9％、90.29％、94.04％、88.52％和85.65％、86.98％,且绵羊与牛的亲缘关系最近,与小鼠的亲缘关系最远.理化性质结果显示BTG2基因编码150个氨基酸,蛋白分子式为C748H1190N208O213S8,分子质量为17 ku,理论等电点(pI)为9.14,半衰期为30 h,属于不稳定亲水性蛋白,且不存在跨膜结构和信号肽序列,蛋白结构主要通过α-螺旋、无规则卷曲和β股组成,为混合型蛋白;qPCR检测到BTG2基因mRNA在不同日龄羔羊心脏、肝脏、脾脏、肺脏、肾脏、肠和肌肉均有表达,且均在肝脏中表达量最高;在心脏、肝脏、脾脏、肺脏组织中,40日龄BTG2表达量极显著高于3日龄,而在肾脏、肠和肌肉中,40日龄极显著低于3日龄.结果表明,BTG2广泛存在各组织中,且随着日龄增加,在各组织中的表达趋势不同,提示其功能的重要性和多重性,并且BTG2在肝组织中表达量最高,说明BTG2可能与脂代谢以及糖代谢等调控功能相关.

试验旨在分离绵羊骨骼肌卫星细胞(skeletal muscle satellite cells,SMSCs),建立绵羊SMSCs体外分离、培养及鉴定体系,为后续研究提供种子细胞.以新生健康绵羊为试验动物,采用胶原酶Ⅳ和胰酶两步酶消化法和差速贴壁法分离并纯化SMSCs.用RT-PCR和免疫荧光法鉴定SMSCs标记基因配对盒基因7(paired box 7,Pax7)、结蛋白(Desmin)和生肌调节因子1(myogenic regulatory factors 1,MyoD1)的表达情况;用血清撤离法诱导SMSCs向成肌细胞方向分化,成肌诱导后观察肌管的形成,免疫荧光法检测成肌分化特异性标志肌球蛋白重链(myosin heavy chain,MHC)的表达.RT-PCR结果显示,扩增条带与预期相符,所分离细胞表达SMSCs标记基因Pax 7、Desmin和MyoD 1;免疫荧光鉴定结果显示,所分离细胞表达SMSCs标记蛋白Pax7、Desmin和MyoD1;成肌诱导后镜下可见细胞相互融合形成多核的肌管,并表达成肌特异性标志MHC.本试验分离了绵羊SMSCs,建立了适用于绵羊SMSCs的体外培养体系,并成功进行了成肌诱导分化,为今后研究绵羊骨骼肌生长发育机制提供了试验材料和技术支撑.

Many local sheep breeds in China have poor meat quality. Increasing intramuscular fat (IMF) content can significantly improve the quality of mutton. However, the molecular mechanisms of intramuscular adipocyte formation and differentiation remain unclear. This study compared differences between preadipocytes and mature adipocytes by whole-transcriptome sequencing and constructed systematically regulatory networks according to the relationship predicted among the differentially expressed RNAs (DERs). Sequencing results showed that in this process, there were 1,196, 754, 100, and 17 differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), respectively. Gene Ontology analysis showed that most DERs enriched in Cell Part, Cellular Process, Biological Regulation, and Binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DERs primarily focused on Focal adhesion, phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR) signaling pathways. Forty (40) DERs were randomly selected from the core regulatory network to verify the accuracy of the sequence data. The results of qPCR showed that the DER expression trend was consistent with sequence data. Four novel promising candidate miRNAs (miR-336, miR-422, miR-578, and miR-722) played crucial roles in adipocyte differentiation, and they also participated in multiple and important regulatory networks. We verified the expression pattern of the miRNAs and related pathways’ members at five time points in the adipocyte differentiation process (0, 2, 4, 6, 8, 10 days) by qPCR, including miR-336/ACSL4/LncRNA-MSTRG71379/circRNA0002331, miR-422/FOXO4/LncRNA-MSTRG54995/circRNA0000520, miR-578/IGF1/LncRNA-MSTRG102235/circRNA0002971, and miR-722/PDK4/LncRNA-MSTRG107440/circ RNA0002909. In this study, our data provided plenty of valuable candidate DERs and regulatory networks for researching the molecular mechanisms of sheep adipocyte differentiation and will assist studies in improving the IMF.

体外胚胎干细胞(embryonic stem cells,ESCs)向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型.试验旨在探讨视黄酸(retinoic acid,RA)诱导鸡胚胎干细胞(chicken embryonic stem cells,cESCs)向雄性生殖细胞(male germ cells,MGCs)分化的作用效果.利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维(chicken embryo fibroblast,CEF)细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶(alkaline phosphatase,AKP)染色和胚胎阶段特异性表面抗原(embryo specific surface antigen 1,SSEA-1)检测对获得的胚胎干细胞进行鉴定.结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性.采用10-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达.结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrinα6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆.本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性.10-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考.

肌钙蛋白Ⅰ(Troponin Ⅰ-TNNI)属于钙离子结合蛋白多基因家族成员,存在于不同类型的骨骼肌中.它受钙离子浓度的作用,控制肌肉的收缩与放松,影响肌肉的纤维直径、类型,进而影响家畜肌肉纤维的发育、肌间脂肪蓄积、肌肉含水率、剪切力、嫩度、熟肉率、风味等肉质性状.TNNI基因在鉴定肉制品掺假,监控肉制品生产过程安全问题等实际生产也有所应用.因此,本文对TNNI基因结构、功能以及对家畜肉质性状的影响、应用进行综述,为深入研究其影响动物肉质性状作用机制,加强在实际生产中的应用提供参考.

为了探究小尾寒羊脂肪细胞分化过程中相关基因的变化规律,试验采集2月龄小尾寒羊腹股沟白色脂肪组织,通过酶消化法体外分离小尾寒羊前体脂肪细胞.培养前体脂肪细胞布满细胞板后,分别用诱导Ⅰ液、诱导Ⅱ液对细胞进行诱导分化,使其成为成熟的脂肪细胞.利用油红O染色法验证成熟脂肪细胞并检测脂滴含量.分别在增殖期细胞增殖70％、90％及分化期诱导Ⅰ液处理48 h、诱导Ⅱ液处理48 h、完全培养液处理48 h时(2、4、6、8、10 d)提取细胞总RNA,反转录成cDNA.采用实时荧光定量PCR检测PPA Rγ、C/ EBPα、LPL、SREBP1、KLF5、KLF6、FAP4、STAT5、ACSS2、IGF1、ADD1、FOXO1、ACACA、DGAT1、CPT1A基因的表达规律.结果 表明,试验成功分离并诱导前体脂肪细胞变为成熟的脂肪细胞,细胞内部具有明显脂滴;实时荧光定量PCR结果表明,上述基因在细胞分化阶段具有明显波动,峰值出现的时间均不相同;C/EBPα、FOXO1基因表达峰值出现在第6天,可能在细胞分化早期发挥作用;PPARγ、LPL、SREBP1、KLF5、KLF6、FABP4、STAT5、ADD1、ACSS2基因表达峰值出现在第8天,但表达倍数与趋势均不相同;ACACA基因表达量出现上下波动;IGF1、DGAT1基因表达峰值出现在第10天;CPT1A基因表达量则一直下降;FABP4基因表达倍数显著高于其他基因.本研究全面检测了小尾寒羊前体脂肪细胞在分化过程中关键基因的表达规律,可为探究小尾寒羊脂肪分化过程分子机制、挖掘参与脂肪分化新的关键基因、提高小尾寒羊肌间脂肪含量等研究提供一定的理论参考.

为了探究小尾寒羊富含半胱氨酸酸性分泌蛋白类似物1 (secreted protein acidic and rich in cysteine like 1,SPARCL1)基因结构及其各组织间表达差异,试验提取小尾寒羊肝脏组织总RNA,根据GenBank中公布的绵羊SPARCL1基因序列设计引物,应用PCR技术扩增SPARCL1基因编码区(CDS).将扩增产物连接到pMD18-T载体进行测序,获得小尾寒羊SPARCL1基因完整CDS区序列信息,应用生物信息学软件分析序列及蛋白结构.以小尾寒羊心脏、肝脏、肌肉、胃、十二指肠、小肠组织mRNA为模板,通过实时定量荧光PCR技术检测SPARCL1基因在小尾寒羊各组织间的表达差异.结果 显示,试验成功获得小尾寒羊SPARCL1基因CDS 1962 bp,编码653个氨基酸;分子质量为74.39 ku,理论等电点为4.64,为亲水性蛋白质;SPARCL1基因具有信息肽切割位点,为分泌蛋白;小尾寒羊SPARCL1基因序列与NCBI中绵羊序列同源性为99.80％,SPARCL1基因CDS区出现4处突变位点,但未引起氨基酸改变;SPARCL1蛋白存在77个蛋白磷酸化位点和3个糖基化位点;SPARCL1蛋白表达预测主要定位在细胞质;SPARCL1蛋白二级结构中α-螺旋、β-折叠、β-转角和无规则卷曲分别占31.9％、6.4％、28.7％和33.0％,三级结构预测结果与其一致.SPARCL1基因在小尾寒羊皮下脂肪组织中相对表达量明显高于其他组织.本研究成功克隆获得小尾寒羊SPARCL1基因CDS区完整序列,并对其序列、蛋白理化特性、结构及各组织间表达差异进行了详细分析,为研究小尾寒羊SPARCL1基因功能,探究其在小尾寒羊脂肪代谢过程中可能发挥的作用提供参考依据.

为促进畜禽育种工作的发展,采用测序法和群体遗传学方法进行数据分析,提取12月龄绵羊血液DNA,依据GenBank中羊MyoG基因序列扩增外显子1,研究小尾寒羊肌细胞生成素(Myogenin,MyoG)基因外显子在群体中的多态性.结果表明:在外显子1第211 bp位存在C>T的突变,共得到A A、AB、BB三种基因型.绵羊A A、AB、BB基因型频率分别为0.529,0.353,0.118;等位基因A和B的基因频率分别为0.706和0.294;其中AA基因型频率在0.5以上,为优势基因型.纯合度(Ho)为0.584;杂合度(He)为0.416;有效等位基因(Ne)为1.712;多态信息含量(PIC)为0.330,在0.25～0.50,为中度多态.DNAStar软件对片段同源性对比分析发现其与山羊的同源性为99％,与牦牛的同源性为97％,与猪的同源性为92％.

为探索胰高血糖素样肽-2受体(glucagon-like peptide-2 receptor,GLP2R)基因多态性与绵羊肉质性状的相关性,试验选用68只南非肉用美利奴羊×东北细毛羊和102只杜泊羊×小尾寒羊的杂交后代为研究对象,采用Sanger测序方法寻找GLP2R基因外显子11序列的突变位点,根据测序结果分析该突变位点的基因型频率和基因频率及卡方适合性检验、纯合度(Ho)、杂合度(He)和多态信息含量(PIC).结果 显示,绵羊GLR2R基因外显子11处存在C/T突变,存在3种基因型:CC、TC和TT,2种等位基因:C和T,其中在南非肉用美利奴羊×东北细毛羊群体中,TC基因型为优势基因型,在杜泊羊×小尾寒羊群体中,CC基因型为优势基因型;2个群体中C均为优势等位基因.卡方适合性检验结果显示,2个群体的C/T位点均处于Hardy-Weinberg平衡状态(P＞0.05);C/T突变位点在群体内变异较小且处于中度多态(0.25＜ PIC＜0.5),有一定遗传学意义.不同基因型肉质性状差异结果显示,南非肉用美利奴羊×东北细毛羊TC基因型剪切力显著高于CC和TT基因型,TT基因型失水率显著高于CC和TC基因型(P＜0.05);杜泊羊×小尾寒羊CC基因型剪切力显著高于TC和TT基因型,TT基因型失水率显著高于CC和TC基因型(P＜0.05).本研究在GLP2R基因外显子处发现C/T突变,可能是影响肉质性状的潜在位点,是否能够作为遗传标记还需要进一步探索.

In recent years, Yanhuang cattle, one of the Chinese five major breeds of cattle, has been received more and more attention because of its excellent meat quality, unique taste, and typical characteristics of the marble pattern. The aim of this study was to establish the preadipocyte in vitro cultures system and explore the biological characteristics and potential mechanism of Yanhuang cattle adipocyte at the cellular and molecular level, and lay a foundation for improvement of beef quality. The subcutaneous adipose tissue of bovine was isolated by collagenase digestion method, and the morphological characteristics were observed; the growth curve was drawn by MTS method; the lipid accumulation was detected by oil red O staining; the triglyceride method was used to detected intracellular triglyceride content; andreal-time fluorescent quantitative RT-PCR was used to detect the expression of adipogenic marker genes including peroxisome proliferator activated receptor (PPARγ) and CCAAT /enhancer binding protein α (C/EBPα) mRNA expression during the differentiation of buffalo preadipocytes. The results showed that the isolated adipocytes of primary adipocytes from Yanhuang cattle presented monolayer growth, the components of preadipocytes were uniform with the shape of fibroblasts after passage. The cells began to logarithmic growth phase from the second day, and reached the plateau phase after the fourth day, with obviously slow proliferation rate. The cell growth curve was approximately "S" shape, which was accorded with the normal law of cell growth. The differentiated cells could be stained with Oil Red O, showing the typical round lipid drop lets with the typical characteristics of mature adipocytes. The content of triglyceride changed slowly from Day 0 to Day 4 and significantly increased after Day 4. The adipogenic marker gene PPARγ, C/EBPαsignificantly increased in the early stage of differentiation as the differentiation progressed, and decreased slightly with the end of the differentiation process. In summary, the present study has successfully established the method of isolation and culture of Yanhuang cattle preadipocytes, which lays a foundation for further study on the mechanism of fat deposition in Yanhuang cattle and the improvement of beef quality.

为了提高蓝舌病病毒16型(BTV-16)的效价,试验将实验室长期冻存的BTV-16接种96孔仓鼠肾细胞(BHK-21)培养板,测定细胞毒半数感染量(TCID50)最初值,然后选取7日龄培养于隔离器中的SPF鸡胚,将BTV-16分别接种到鸡胚尿囊腔和卵黄囊中,接毒后24～ 72 h观察鸡胚状况,第72小时收毒.将收获的病毒接种到BHK-21细胞中繁殖传代,测定TCID50.结果表明:尿囊腔和卵黄囊接种BTV-16可感染部分鸡胚,收获的病毒接种到BHK-21可使病毒效价从原来的2.4提升到4.5～7.6,卵黄囊接毒的方式可以获得更好的效果,病毒效价可达到7以上.BTV对BHK-21细胞的适应性和敏感性较高,BTV先经过鸡胚尿囊腔和卵黄囊接种适应鸡胚后再进行BHK-21细胞传代,大大提高了BTV-16的细胞毒效价,高效价的BTV-16为血清型鉴定和血清分析提供了材料储备.

为了研究葛根素(puerarin)对3T3-L1前体脂肪细胞分化的影响,探索其在成脂分化过程中的潜在作用机制,试验在脂肪形成过程中将0、10、50 μmol/L葛根素加入到诱导分化培养基中诱导分化,分别通过油红O染色法及甘油三酯酶法检测葛根素对3T3 L1脂肪细胞的脂滴积累、甘油三酯的影响;采用实时荧光定量PCR检测脂肪细胞中CCAAT-增强子结合蛋白α(C/EBPα)和过氧化物酶体增殖物激活受体γ(PPARγ)的mRNA表达量,Western blotting检测脂肪形成相关转录因子及Akt信号通路的蛋白水平的表达量.结果表明,10 μmol/L葛根素极显著增加了成熟脂肪细胞中脂滴和甘油三酯(TG)的积聚,极显著促进了脂肪形成相关转录因子C/EBPα和PPARγ的mRNA和蛋白水平的表达量(P＜0.01).进一步研究发现,与对照组相比,葛根素的刺激可增强Akt信号通路Ser473蛋白的磷酸化表达水平,表明葛根素对成脂分化过程的促进作用很大程度上是通过Akt信号通路的磷酸化来实现的.综上所述,葛根素能够促进3T3-L1前体脂肪细胞的分化,改善胰岛素敏感性,其作用机制与激活Akt信号通路Ser473位点的磷酸化水平有关.本试验结果可为研究胰岛素的效应机制提供新见解,为胰岛素抵抗相关疾病的治疗提供新思路.

We characterised wool traits, and skin gene expression profiles of fine wool Super Merino (SM) and coarse wool Small Tail Han (STH) sheep. SM sheep had a significantly higher total density of wool follicles, heavier fleeces, finer fibre diameter, and increased crimp frequency, staple length and wool grease (lanolin) production. We found 435 genes were expressed at significantly different levels in the skin of the two breeds (127 genes more highly in SM and 308 genes more highly in STH sheep). Classification of the genes more highly expressed in SM sheep revealed numerous lipid metabolic genes as well as genes encoding keratins, keratin-associated proteins, and wool follicle stem cell markers. In contrast, mammalian epidermal development complex genes and other genes associated with skin cornification and muscle function were more highly expressed in STH sheep. Genes identified in this study may be further evaluated for inclusion in breeding programs, or as targets for therapeutic or genetic interventions, aimed at altering wool quality or yield. Expression of the lipid metabolic genes in the skin of sheep may be used as a novel trait with the potential to alter the content or properties of lanolin or the fleece.

为了研究鸡白介素-6(Interleukin-6,IL-6)抗病基因外显子4在群体中的多态性,试验采用测序法和群体遗传学方法进行数据分析,提取150日龄吉林高脚芦花鸡、吉林矮脚芦花鸡、吉林黑鸡肝脏DNA,依据GenBank中鸡IL-6基因序列扩增外显子4.结果表明:外显子4第76位氨基酸有A＞G的突变,共得到AA、AB、BB三种基因型.纯合度(Ho)分别为0.535,0.671,0.556,杂合度(He)分别为0.465,0.329,0.444,有效等位基因数(Ne)分别为1.869,1.490,1.799,多态信息含量(PIC)分别为0.345,0.275,0.346.

为探讨寒冷季节不同遗传背景猪热休克蛋白70,90的分子行为,在寒冷季节(1月份)采集长白山野杂猪、民猪和大白猪外周血及肝脏组织样品,分别用ELISA和QRT-PCR方法检测HSP70,90的表达量.结果显示民猪群体外周血HSP70含量最高,野杂猪次之,大白猪最低,但三个群体中HSP70含量不存在显著差异(p>0.05).外周血HSP90含量检测发现野杂猪最高,民猪次之,大白猪最低,其中野杂猪与大白猪群体间HSP90含量呈现极显著的差异性(p<0.01).肝脏mRNA表达检测发现HSP70基因mRNA在野杂猪中高表达,表达量约为大白猪的1.5倍,民猪次之,表达量约为大白猪的1.2倍.HSP90基因mRNA同样在野杂猪中最高,约为大白猪的3.5倍,民猪约为大白猪的1.7倍.HSP70和90外周血含量及肝脏mRNA表达表现出一定的趋同性.本研究结果表明不同遗传背景猪中HSP70和90血清含量及肝脏组织表达量存在差异.