草鱼肠系膜脂肪组织高质量总RNA提取方法的研究

基于传统的酸性酚—异硫氰酸胍—氯仿一步提取法,比较分析多种优化操作步骤,摸索出一种高质量提取体质量为80~150 g草鱼肠系膜脂肪组织总RNA的改良方法。试验结果显示,相较于肝脏、脾脏、肠道等脏器组织,草鱼肠系膜脂肪组织RNA丰度低,且极易在样品前处理阶段出现顽固性降解问题。探索发现,将取样量增至约30 mg,可提升RNA产量以满足常规试验需求。针对降解难题,改良常规的样品前处理技术流程,采用鲜样液氮速冻,冻样直接放入TRIzol试剂中裂解,并即刻进行长时间机械匀浆,时长约3 min等核心操作步骤,可显著降低脂肪组织样品RNA的降解。Agilent生物分析仪检测结果显示,改良方法提取的草鱼肠系膜脂肪组织RNA完整度高,关键RIN值为8.7~9.0。研究推测,长时间机械匀浆所形成的持续剪切冲击力或许有助于TRIzol试剂中的异硫氰酸胍等成分突破油滴阻碍而有效抑制内源性RNA酶。本方法提取的草鱼脂肪组织总RNA质量可满足高通量转录组测序要求。     

Advance in study of batokines

Brown adipose tissue is a type of adipose tissue and is present in all mammals. It is not only the main site for adaptive thermogenesis in vivo, but also secretes and releases many cytokines in the form of endocrine to regulate a variety of metabolic processes and prevent and treat obesity and metabolic diseases. Therefore, identification of the types and effects of brown adipocytokines (batokines) may be crucial for the treatment of a variety of obesity-related metabolic diseases. Based on the latest research progress, batokines and their functions are reviewed.     

鸡脂肪组织TCF21基因启动子区DNA甲基化与其表达的关系

旨在研究鸡脂肪组织中TCF21基因启动子区DNA甲基化水平与其表达的关系。以东北农业大学高、低腹脂双向选择品系（简称高、低脂系）第24世代7周龄肉鸡为试验材料，利用RT-qPCR检测高、低脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平；利用生物信息学和双荧光素酶报告系统分析TCF21基因启动子的结构与功能；利用Sequenom MassARRAY飞行质谱检测高、低脂系肉鸡腹部脂肪组织中TCF21基因启动子区CpG位点的甲基化水平；利用CpG甲基转移酶处理TCF21启动子报告基因质粒，分析DNA甲基化对TCF21基因启动子活性的影响。结果显示，高脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平极显著高于低脂系（P<0.001）；TCF21基因的启动子区存在40个CpG位点，且在启动子的近端和远端均有分布，但不存在CpG岛；将TCF21基因的启动子划分为5个功能区域，分别为R1区域（-2 000~-1 500 bp）、R2区域（-1 500~-1 000 bp）、R3区域（-1 000~-500 bp）、R4区域（-500~-200 bp）和Core区域（-200~-100 bp）；高脂系R2、R3和R2+R3区域的DNA甲基化水平显著或极显著高于低脂系（P<0.05或P<0.001）；R2、R3、R2+R3区域的DNA甲基化水平与TCF21基因mRNA表达水平呈显著正相关（R2区域：r=0.438，P<0.05；R3区域：r=0.371，P<0.05；R2+R3区域：r=0.489，P<0.05）；R2区域的DNA甲基化显著抑制其转录活性（P<0.05）。综上所述，TCF21基因在高、低脂系肉鸡腹部脂肪组织中的表达水平主要与其启动子R2区域的DNA甲基化水平有关。     

Evidence for functional G-coupled protein receptors 43 and 120 in subcutaneous and intramuscular adipose tissue of Angus crossbred steers

We conducted 3 independent experiments to demonstrate functional G-coupled protein receptor 43 (GPR43) and GPR120 in bovine intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues. We hypothesized that media volatile fatty acids and long-chain fatty acids would affect cAMP-activated protein kinase-alpha (AMPKα) protein expression and cAMP concentrations differently in i.m. and s.c. adipose tissue. Experiment 1: oleic acid (18:1n-9) decreased phosphorylated AMPKα protein (p-AMPKα) and the p-AMPKα/AMPKα protein ratio in i.m. preadipocytes, increased the p-AMPKα/AMPKα protein ratio in bovine satellite cells, and had no effect in s.c. preadipocytes. Experment 2: ex vivo explants from the 5th to 8th longissimus thoracic rib muscle section of Angus crossbred steers were cultured 48 hr in media containing 0.25 µM ciglitizone, 5 mM glucose, and 5 mM acetate, in the absence or the presence of 100 µM oleic acid. Oleic acid increased acetate incorporation into fatty acids and GPR43 gene expression in i.m. adipose tissue (P < 0.05), but oleic acid had no effect on fatty acid synthesis or GPR43 expression in s.c. adipose tissue. Experiment 3: fresh s.c. and i.m. adipose tissue from the 5th to 8th longissimus thoracic rib muscle section of Angus crossbred steers was transferred immediately to 6-well culture plates containing 3 mL of KHB/Hepes/5 mM glucose. Samples were preincubated with 0.5 mM theophylline plus 10 μM forskolin for 30 min, after which increasing concentrations of acetate or propionate (0, 10⁻³, 10^−2.3, and 10⁻³ M) in the absence or the presence of 100 μM oleic acid or 100 µM palmitic acid (16:0) were added to the incubation media. Acetate had no effect on forskolin-stimulated cAMP production in s.c. adipose tissue but decreased cAMP in i.m. adipose tissue (P < 0.05); this indicates a functional GPR43 receptor in i.m. adipose tissue. The combination of 10⁻² M acetate and oleic acid decrease cAMP production in s.c. adipose tissue, consistent with GPR120 receptor activity, but oleic acid and palmitic acid attenuated the depression of cAMP production caused by acetate in i.m. adipose tissue. Palmitic acid depressed cAMP production in s.c. adipose tissue, and increased cAMP production in i.m. adipose tissue (P < 0.05). Propionate had no effect on cAMP production in s.c. or i.m. adipose tissue. These results provide evidence for functional GPR43 receptors in i.m. adipose tissue and GPR120 receptors in s.c. adipose tissue, both of which would suppress lipolysis.     

不同饲养环境合作猪脂肪组织生脂基因 mRNA表达水平的比较研究

合作猪是生长在高寒草原以放牧为主的珍稀高原型猪种,为了探讨其品种特性的分子机制,分析测定了原产地合作猪及长期在不同环境和饲养条件下异地饲养合作猪脂肪组织脂肪生成的关键酶脂肪合成酶(FAS)、乙酰辅酶A羧化酶(ACC)1和转录因子固醇调控元件结合蛋白(SREBP)-1c、碳水化合物调控元件结合蛋白(ChREBP)mRNA表达水平,并与杜×大×长三元杂交猪进行了对比。试验结果发现,合作猪脂肪组织FAS、ACC1及ChREBP mRNA水平显著低于三元杂交猪(P<0.05),在长期异地饲养后,这些基因mRNA表达水平提高(P<0.05),而SREBP-1c mRNA表达水平在3个群体中没有明显变化(P>0.05)。结果提示,合作猪由于对低能量、低碳水化合物粗饲的长期适应,表现出脂肪组织生脂基因表达水平较低的生理学特性。     

动物脂肪组织的分化起源

动物脂肪组织是机体重要的器官,主要负责能量的储存和代谢,同时分泌多种脂肪细胞因子(adipokines)参与机体生理功能的调控。脂肪组织的功能紊乱与人类的肥胖病、糖尿病以及代谢综合症密切相关,多年以来动物脂肪组织的细胞分化起源一直是研究的热点。白色脂肪、棕色脂肪和肌肉组织都来源于机体的间充质干细胞(mesenchymal stem cells,MSCs),长期以来,人们一直以为白色脂肪和棕色脂肪的分化起源更加亲近,但随着对棕色脂肪分化机制的深入研究,发现白色脂肪和棕色脂肪在早期发育阶段有着不同的前体细胞,而棕色脂肪组织与肌肉组织的分化支系更加亲近。以往的脂肪分化研究往往利用基质血管组分(stromal vascular fractions,SVF)或肌卫星细胞等混合细胞群作为实验材料,随着流式细胞仪、转基因动物模型和干细胞表面标志抗原识别等技术的发展,使我们可以从这样的混合细胞群中分离得到纯的前体脂肪细胞系,从而提示我们之前的研究可能过高的估计了这样的混合细胞群体的活体分化潜能。进一步深入研究动物脂肪组织的分化起源,有助于我们理解机体脂肪沉积的具体机制,从而为治疗脂肪代谢相关疾病以及提高动物肉品品质提供理论依据。     

Dietary Fucoxanthin Increases Metabolic Rate and Upregulated mRNA Expressions of the PGC-1alpha Network,Mitochondrial Biogenesis and Fusion Genes in White Adipose Tissues of Mice

The mechanism for how fucoxanthin (FX) suppressed adipose accumulation is unclear. We aim to investigate the effects of FX on metabolic rate and expressions of genes related to thermogenesis, mitochondria biogenesis and homeostasis. Using a 2 × 2 factorial design, four groups of mice were respectively fed a high sucrose (50% sucrose) or a high-fat diet (23% butter + 7% soybean oil) supplemented with or without 0.2% FX. FX significantly increased oxygen consumption and carbon dioxide production and reduced white adipose tissue (WAT) mass. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), cell death-inducing DFFA-like effecter a (CIDEA), PPARα, PPARγ, estrogen-related receptor α (ERRα), β3-adrenergic receptor (β3-AR) and deiodinase 2 (Dio2) were significantly upregulated in inguinal WAT (iWAT) and epididymal WAT (eWAT) by FX. Mitochondrial biogenic genes, nuclear respiratory factor 1 (NRF1) and NRF2, were increased in eWAT by FX. Noticeably, FX upregulated genes of mitochondrial fusion, mitofusin 1 (Mfn1), Mfn2 and optic atrophy 1 (OPA1), but not mitochondrial fission, Fission 1, in both iWAT and eWAT. In conclusion, dietary FX enhanced the metabolic rate and lowered adipose mass irrespective of the diet. These were associated with upregulated genes of the PGC-1α network and mitochondrial fusion in eWAT and iWAT.     

蛹虫草与黄伞复合多糖抗氧化活性研究

[目的]研究蛹虫草与黄伞复合多糖的抗氧化活性。[方法]采用超声提取法分别提取蛹虫草多糖和黄伞多糖,并将二者按不同比例复合后,在不同浓度下进行羟基自由基的清除试验。[结果]在一定的剂量配比下,蛹虫草多糖与黄伞多糖表现出很好的复合效果,清除羟基自由基的能力较单独多糖有明显的提升。其中复合比例为2∶1,浓度为16 mg/ml时清除率较蛹虫草多糖提高了79.82%,是黄伞多糖的3.6倍;通过方差分析得出,复合多糖与单独多糖之间存在显著差异(P0.05),既表现出了较好的协同作用又呈现出很好的量效关系。[结论]复合后的多糖的活性提高,表现出了较好的清除羟基自由基清除效果。