11个绵羊品种MSTN基因非翻译区的变异

利用PCR-RFLP技术对特克塞尔羊、夏洛莱羊、小尾寒羊、蒙古羊、乌珠穆沁羊、阿勒泰羊、呼伦贝尔羊、塔什库尔干羊、多浪羊、湖羊和岗巴羊11个品种的345个个体的肌肉生长抑制素(Myostatin, MSTN)基因非翻译区(UTR)的变异进行了多态性分析。结果表明大小为271 bp和1 003 bp的扩增片段经限制性内切酶MboⅡ和BsaⅠ酶切后表现多态, 经卡方检验所有品种在该基因座位均处于平衡状态(P>0.05), 3种基因型在11个绵羊品种中的分布差异极显著(P<0.01)。通过限制性内切酶HpyCH4Ⅳ 酶切实验, 证明我国9个地方绵羊品种不存在特克塞尔绵羊中发现的导致肌肉发达的SNP位点, 并在3′UTR区发现了个别碱基突变位点能够形成miRNA作用的靶基序, 测序表明3′UTR区的突变频率较高。     

转MSTN干扰载体细胞株的获得及外源基因整合情况的研究

转基因细胞株的建立能够为转基因体细胞克隆技术奠定重要基础。该实验利用MSTN干扰载体,转染鲁西黄牛胎儿成纤维细胞,获得5个相应的转基因单克隆细胞株。采用Realtime PCR和高效热不对称互交式PCR（hiTAIL-PCR）技术检测细胞克隆中MSTN表达载体的拷贝数及其在牛基因组中的整合位点。结果表明,荧光定量PCR有效检测到5个细胞克隆中质粒的拷贝数分别为2.26±0.32、1.52±0.25、25.68±1.02、8.43±0.73和6.72±0.10。hiTAIL-PCR对整合位点的检测结果表明,质粒片段在插入到基因组的过程中进行了重组,其与基因组的结点处有2或4个共同的碱基序列。该研究探索MSTN干扰载体在牛胎儿成纤维细胞中的整合机制,以期获得遗传背景清楚的转基因细胞作为体细胞核移植的重要材料,为高产转基因肉牛新品种的培育提供重要的理论和实验基础。     

急性失代偿性心力衰竭患者血清CST、CyPA、MSTN表达及其临床意义

摘要 目的：探讨急性失代偿性心力衰竭（ADHF）患者血清儿茶酚抑素（CST）、亲环素A（CyPA）、肌肉生长抑制素（MSTN）的表达及其临床意义。方法：选取2021年4月～2022年4月潍坊市人民医院收治的100例ADHF患者为ADHF组，根据预后情况将其分为预后不良组(32例)和预后良好组（68例），另选取50名健康体检志愿者为对照组。采用酶联免疫吸附法检测血清CST、CyPA、MSTN表达。采用多因素Logistic回归分析ADHF患者预后不良的因素，采用受试者工作特征（ROC）曲线分析血清CST、CyPA、MSTN表达对ADHF患者预后不良的评估价值。结果：ADHF组血清CST、CyPA、MSTN表达高于对照组（P＜0.05）。100例ADHF患者预后不良发生率为32.00%。多因素Logistic回归分析显示，纽约心脏协会（NYHA）心功能分级≥Ⅲ级、N末端前体B型钠尿肽（NT-proBNP）、CST、CyPA、MSTN升高为ADHF患者预后不良的独立危险因素，左室射血分数（LVEF）升高为独立保护因素（P＜0.05）。ROC曲线分析显示，血清CST、CyPA、MSTN表达联合评估ADHF患者预后不良的曲线下面积（AUC）大于CST、CyPA、MSTN表达单独评估。结论：血清CST、CyPA、MSTN高表达与ADHF患者预后不良有关，联合血清CST、CyPA、MSTN表达评估ADHF患者预后不良的价值较高。     

Different regulation role of myostatin in differentiating pig ADSCs and MSCs into adipocytes

Myostation (MSTN), which is primarily expressed in muscle, plays an important role in myogenic and adipogenic cells. However, there is little information about whether MSTN displays different roles between adipose-derived stem cells (ADSCs) and muscle satellite cells (MSCs). The two kinds of cells can both exist in the muscle and differentiate into adiposities. In this research, we isolated ADSCs and MSCs from porcine fat tissues and semitendinosus muscle, respectively, to investigate the effect of MSTN on the adipogenesis of those cells. ADSCs and MSCs were treated with recombinant human MSTN during the induction of adipogenesis or before the induction of differentiation. Then, we evaluated adipogenesis by Oil Red O staining and assessed the expression patterns of adipocyte-specific fatty acid binding protein (aP2) and peroxisome proliferator-activated receptor (PPAR) γ using real-time polymerase chain reaction methods. Our results indicated that the treatment with MSTN before or during the induction of differentiation in MSCs could both inhibit the adipogenesis. However, the treatment with MSTN only during the induction of differentiation in ADSCs could suppress the adipogenesis. Those results showed that MSTN had different roles in the adipogenesis of ADSCs and MSCs. It can shed new light on the origin of adipocyte located in muscle.     

大口黑鲈肌肉生长抑制素多克隆抗体的制备及其对仔鱼生长影响

肌肉生长抑制素(Myostatin,MSTN)是抑制肌肉生长和发育的生长调控因子.将大口黑鲈(Micropterus salmoides)MSTN成熟肽cDNA改造后克隆到pET32a(+)载体上,构建原核表达质粒,在大肠杆菌BL21中进行表达.SDS-PAGE检测表明重组表达的融合蛋白分子大小约28 kD,含量为菌体总蛋白的34%.用纯化后的表达蛋白免疫新西兰兔制备抗大口黑鲈MSTN多克隆抗体,Western blot和ELISA检测证实所获得的抗体可与抗原蛋白特异性结合,抗体的效价为1: 62 500.将获得的抗体显微注射到大口黑鲈的受精卵卵黄区,结果显示具有促进仔鱼生长的作用.     

猪肌肉生长抑制素基因侧翼区新微卫星标记的鉴定及分析

提取包含猪肌肉生长抑制素基因的BAC克隆,以EcoRⅠ进行酶切并回收其中大于4kh的酶切产物,连接到pGEM-3zf( )载体后得到了亚克隆。测序分析表明,插入片段不属于猪肌肉生长抑制素基因的一部分,但其中包含13拷贝的(TG)n重复。以该重复序列的侧翼设计引物对猪的基因组进行分析,得到了PCR扩增产物。对一个“双肌臀”大白猪家系进行PCR扩增及非变性聚丙烯酰胺凝胶分析的结果表明,该位点以并显性方式遗传,是一个新的微卫星标记。对莱芜猪、长白猪、大白猪、杜洛克、皮特兰、民猪和二花脸7个品种的381个无关个体的检测均显示该基因座只有两个等位基因,重复数分别为13和19,是一种多态性较低的微卫星标记。与包含该基因的序列(AY208121)比对分析表明,该微卫星属于肌肉生长抑制素基因(MSTN)侧翼区的一个分子标记,在猪肉用性状QTL的精细定位和MSTN功能分析中将发挥重要作用。     

肌肉生长抑制素对脂肪细胞生长发育和脂代谢的调控

肌肉生长抑制素(MSTN)对骨骼肌生长的抑制作用已得到证实,但其调控脂肪组织的作用还不是十分清楚。本文综述了MSTN调控脂肪细胞生长发育和脂代谢的相关机制和可能研究趋势。     

IDENTIFICATION OF THREE SNPs IN THE PORCINE MYOSTATIN GENE (MSTN)

ABSTRACT

Thirteen pairs of primers were designed for the entire porcine MSTN gene to enable PCR amplification for the detection of single nucleotide polymorphisms (SNPs) by a PCR-SSCP approach. Altogether 96.5% (1089/1128) of the encoding regions and 971?bp of the non-coding regions were screened. A total of three polymorphisms were identified with PCR-SSCP. They were located in the promoter, intron one and exon three regions of the gene. These polymorphisms were then confirmed to be point mutations (T→A transversion, G→A transition and C→T transition respectively) by sequencing. Allele frequencies were determined for all three SNPs in several different pig breed populations. The polymorphisms were found to be rare in Western breeds, but much more common in Chinese breeds. Whether they have any relationship with the marked difference in lean meat mass between Western and Chinese breeds requires further study.     

大黄鱼MSTN成熟肽区基因的原核表达

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。