Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响

为了探究miR-425-5p对小鼠3T3-L1前体脂肪细胞增殖、分化的影响效应,本试验采用实时荧光定量PCR检测miR-425-5p组织和细胞表达水平;运用CCK8、EdU、油红染色、甘油三酯含量分析等分别检测miR-425-5p对前体脂肪细胞增殖、分化的影响;利用生物信息学软件和双荧光素酶报告试验分别预测、验证miR-425-5p调控前体脂肪细胞分化的靶基因。结果表明,miR-425-5p在肥胖小鼠脂肪组织中低表达,在前体脂肪细胞增殖、分化过程中动态表达;与阴性对照相比,过表达miR-425-5p可促进前体脂肪细胞增殖,抑制脂肪细胞分化标志基因(PPARγ、C/EBPα、FAS等)表达,减少脂滴和甘油三酯积累;抑制miR-425-5p表达可抑制前体脂肪细胞增殖,阻止前体脂肪细胞诱导分化。在前体脂肪细胞分化过程中,过表达或抑制miR-425-5p可分别抑制或促进IGF1基因表达;与阴性对照相比,过表达miR-425-5p可抑制IGF1基因3′-UTR荧光活性,而突变miR-425-5p种子序列与IGF1基因3′-UTR的绑定位点可解除该抑制效果。综上所述,miR-425-5p可促进3T3-L1前体脂肪细胞增殖,并可直接靶向IGF1负向调控其分化。     

从江香猪肌内前体脂肪细胞分化过程中相关基因的表达研究

试验旨在探讨从江香猪肌内前体脂肪细胞分化过程中相关基因的表达。采集3日龄从江香猪背最长肌,采用Ⅱ型胶原酶消化法分离肌内前体脂肪细胞,进行原代和传代培养,并对其进行形态学观察。诱导培养后,利用油红O染色法对其进行鉴定。采用实时荧光定量PCR方法检测细胞诱导分化0、24、48、72和144 h时脂肪相关基因丙酮酸脱氢酶激4（PDK4）、成纤维细胞生长因子10（FGF10）、脂联素（ADIPOQ）、脂肪酸合成酶（FAS）、脂蛋白脂酶（LPL）、CCAAT增强子结合蛋白α（C/EBPα）、脂肪细胞脂肪酸结合蛋白4（FABP4）、蛋白激酶B（AKT2）的表达,选择诱导0 h作为对照组。结果显示,分离的肌内前体脂肪细胞5 h开始贴壁,贴壁的细胞呈圆形,胞体透明,经传代后,细胞形态均一,经诱导培养后,油红染色呈红色。实时荧光定量PCR结果显示,PDK4、ADIPOQ、C/EBPα、FAS、FABP4和AKT2基因mRNA表达水平在诱导48 h时均呈现较高表达,极显著高于其余各阶段（P<0.01）;FGF10基因mRNA表达水平在诱导24和48 h时均较高;LPL基因mRNA表达水平在诱导72 h时极显著高于对照组（P<0.01）,之后明显下降;PDK4、ADIPOQ和FGF10基因mRNA表达水平在诱导144 h时均极显著低于对照组（P<0.01）;C/EBPα基因mRNA表达水平在诱导144 h时显著高于对照组（P<0.05）;FAS基因mRNA表达水平在诱导144 h时显著低于对照组（P<0.05）;AKT2和LPL基因mRNA表达水平在诱导144 h时与对照组差异不显著（P>0.05）。本试验成功培养了从江香猪肌内前体脂肪细胞,并检测了不同诱导阶段脂肪相关基因的表达情况,为进一步研究从江香猪脂肪代谢和沉积提供参考依据。     

秦川牛CAP2基因CDS区克隆及表达谱分析

[目的]克隆秦川牛CAP2基因的编码区序列（CDS）,分析该基因在不同组织及原代脂肪细胞分化过程中的表达特征。[方法]采用RT-PCR方法扩增秦川牛CAP2基因的CDS区,运用ProtPram、TMpred、ProtFun 2.1 Server等在线网站进行CAP2蛋白的生物信息学分析;通过油红O染色和qPCR检测成脂标志基因PPARγ和FABP4基因的表达水平以构建牛原代脂肪细胞诱导分化体系;利用qPCR检测分析CAP2基因在秦川牛7种组织（心、肝、脾、肺、肾、肌肉和背脂）和原代脂肪细胞分化过程（0~10 d）中的表达。[结果]秦川牛CAP2基因CDS区长1 461 bp,编码486 个氨基酸,氨基酸序列主要由无规卷曲和α-螺旋构成。CAP2基因在脂肪组织中高表达,极显著（P<0.01）高于其他组织。牛原代脂肪细胞诱导分化过程中,PPARγ和FABP4基因的表达量逐渐上升,与第0天相比第10 天时表达量达到最大（P<0.01）;与对照组相比,诱导分化组脂滴积累明显增加;CAP2基因表达量也随时间推移逐渐上升,以0 d为对照,第10天表达量最高（P<0.001）。[结论]成功克隆了秦川牛CAP2基因全长1 461 bp的编码区;CAP2基因在牛脂肪组织中以较高水平表达,且CAP2基因可能参与脂肪生成与分化过程,预测CAP2基因可能是促进成脂分化的转录因子,对维持牛脂肪细胞状态发挥关键作用,可能作为秦川牛肉质性状的候选基因,该研究结果为进一步揭示牛CAP2基因的功能提供基础资料。     

Meat Science and Muscle Biology Symposium: the influence of extracellular matrix on intramuscular and extramuscular adipogenesis

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.     

Regulation of fibroblast growth factor 9 on the differentiation of goat intramuscular adipocytes

It has been found that fibroblast growth factor receptor (FGF-FGFR) signaling can regulate the expression of adipocyte differentiation genes. FGF9 is one of the members of FGFs that mainly binds receptors FGFR2 and FGFR3. FGF9 is highly expressed in the adipose tissue of humans and mice, but there are few reports on the role of FGF9 in goat intramuscular adipocyte differentiation. Therefore, this study explored the effect of FGF9 on adipocyte differentiation through cell culture, interference, and overexpression. The expression of receptors FGFR1–FGFR4 in adipocyte differentiation and their effects on differentiation were detected to screen receptor gene of FGF9. Finally, the interaction between FGF9 and the receptor was tested by cotransfection. Our results showed that FGF9 interacts with FGFR2 to inhibit goat intramuscular adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma (PPARγ) and preadipocyte factor 1 (Pref1), which is a data support for subsequent pathway research.     

Stimulatory effect of plasma samples from fattening cattle on adipogenesis‐related gene expression in preadipocyte cells

It is desirable to produce beef with high levels of monounsaturated fatty acids (MUFA), as this is related to fat softness and palatability. However, the physiology of MUFA synthesis in bovine fat during the fattening process remains to be established. In this study, in order to elucidate the relationship between plasma components and the fatty acid composition of intramuscular fat, we investigated the effect of plasma obtained from fattening cattle on the messenger RNA (mRNA) expressions of the adipogenesis‐related gene in a clonal bovine intramuscular preadipocyte line (BIP cells). The mRNA expressions of stearoyl‐CoA desaturase, adipocyte Protein 2, peroxisome proliferator‐activated receptor gamma and sterol regulatory element‐binding protein 1 in BIP cells were significantly higher following treatment with those plasma samples collected from the cattle with the highest diaphragmatic unsaturated fatty acids to saturated fatty acids ratio (US/S). Furthermore, the concentration of nonesterified fatty acid (NEFA) in the plasma samples had an inverse correlation with carcass diaphragmatic US/S. These results indicate that cattle with a low ratio of US/S in fat may be discriminated from the population of fattening cattle before slaughter by measuring the effect of their plasma on gene expression in BIP cells as well as their plasma concentration and composition of NEFA.     

Expression of lipogenic genes during porcine intramuscular preadipocyte differentiation

Intramuscular fat (IMF) content plays an important role in meat quality. Triglyceride (TG) metabolism in intramuscular adipocytes is strongly associated with the intramuscular fat deposition. To better understand the mechanisms leading to IMF deposition we compared the expression levels of genes related to preadipocyte differentiation and lipogenesis in the intramuscular preadipocytes isolated from the longissimus muscle of Wujin and Landrace pigs. The results showed that the intramuscular preadipocytes could differentiate into mature adipocytes in vitro. Triglyceride content in adipocytes isolated from Wujin pigs was higher than Landrace pigs during the middle and later phases of preadipocyte differentiation. The expression levels of genes related to preadipocyte differentiation such as PPARG and CEBPA showed differential expression between Wujin and Landrace porcine adipocytes during the early stage of differentiation. The expression levels of lipogenic genes such as FASN and SREBF1 were significantly higher in Wujin porcine intramuscular preadipocytes than in Landrace intramuscular preadipocytes at the middle and the later stages of differentiation. This suggests that preadipocyte differentiation and lipogenesis exhibited breed-related scheduling.     

山羊RPL26基因生物信息学分析及对肌内脂肪细胞分化的影响

旨在克隆山羊RPL26基因序列并对其在山羊各组织中的表达情况和对山羊脂肪细胞分化的调控作用进行探究.本研究以1周岁简州大耳羊公羊作为试验对象(健康生长状态良好,体重约50 kg,n=3),利用RT-PCR等方法克隆RPL26序列,对基因及蛋白质序列进行生物信息学分析;以山羊各组织cDNA为模板,利用qPCR方法构建组织...     

干扰Smad3促进山羊脂肪细胞分化

旨在克隆山羊Smad3的基础上，明确其组织和细胞表达谱，最终阐明干扰Smad3基因对山羊肌内和皮下脂肪细胞分化的影响。本研究选用5只体况良好的1周龄简州大耳羊，空腹24 h后屠宰并采集相应组织和细胞进行试验。利用RT-PCR技术克隆山羊Smad3基因cDNA区序列并进行生物信息学分析，利用实时荧光定量PCR（real-time quantitative PCR，qPCR）技术检测Smad3基因的组织和细胞时序表达水平；并且合成靶向Smad3的siRNA，采用油红O染色从形态学上明确干扰Smad3对山羊前体脂肪细胞成脂分化的影响，利用qPCR检测干扰Smad3对脂肪细胞分化标志基因C/EBPα、C/EBPβ、LPL、SREBP1、AP2、PPARγ、Pref-1、KLF3、KLF4、KLF6、KLF7、KLF8、KLF9、KLF10和KLF15以及Smads相关基因Smad2、Smad4、Smad7和TGF-β1基因mRNA表达水平的影响，探讨可能的作用机制。结果，获得山羊Smad3基因1 449 bp，其中CDS区序列为1 278 bp，编码425个氨基酸；Smad3在山羊各组织中具有广泛表达特性，且在肾脏中的表达水平最高（P<0.01）；Smad3均在山羊肌内和皮下两种脂肪细胞诱导分化的36 h表达量最低，极显著低于在未分化前体脂肪细胞中的表达丰度（P<0.01）；干扰Smad3后发现显著促进了山羊肌内和皮下脂肪细胞中脂滴的聚集，且脂肪细胞分化标志基因、KLF3、KLF4、KLF8、KLF9、KLF10和KLF15的表达水平显著上升（P<0.05），Pref-1的相对表达水平极显著下降（P<0.01），同时干扰Smad3基因下调了Smad2、Smad4和Smad7基因的相对表达水平（P<0.05）。研究结果指出，干扰Smad3促进山羊脂肪细胞分化，且可能通过调控脂肪细胞分化标志基因C/EBPα、C/EBPβ、LPL、SREBP1、AP2、Pref-1、KLF3、KLF4、KLF8、KLF9、KLF10和KLF15等及协同Smad2、Smad4和Smad7的表达来实现的。     

ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究

旨在探究缺失、小的、同源异形1（absent，small，or homeotic 1-like，ASH1L）甲基转移酶在牛卵丘细胞中的表达与功能。本研究通过免疫荧光染色在健康母牛卵丘细胞中对ASH1L甲基转移酶进行定位，并分析细胞的组蛋白H3第36位赖氨酸（histone H3 lysine36，H3K36）甲基化修饰模式；合成靶向Ash1L基因的siRNA，对siRNA-1、siRNA-2、siRNA-3及对照组进行荧光定量PCR和蛋白质免疫印迹，筛选有效siRNA；采用荧光定量PCR分析干扰Ash1L表达对处理组及对照组中凋亡相关基因及多梳抑制复合体（polycomb repressive complex 2，PRC2）组成基因的表达水平的影响。结果显示，ASH1L甲基转移酶位于牛卵丘细胞的细胞核中，呈点状分布。成功筛选到能有效干扰牛Ash1L基因的siRNA-2，其干扰效率为60%~70%。将siRNA-2转染卵丘细胞后，该干扰组细胞中H3K36的单甲基化、二甲基化及三甲基化3种甲基化水平均显著低于对照组（P<0.05）；干扰Ash1L导致凋亡相关基因Bax、Bcl-2及caspase-3表达水平显著上调，凋亡基因Bax和caspase-3表达量高于抗凋亡相关基因Bcl-2（1.311和1.179 vs 1.146）；同时，干扰Ash1L基因表达也引起PRC2蛋白亚基EZH2和Suz12基因的mRNA表达量显著升高（P<0.05）。综上所述，本研究探讨了ASH1L甲基转移酶在牛卵丘细胞中的表达和功能，ASH1L在牛卵丘细胞中呈点状分布，且Ash1L基因的抑制导致H3K36me1/2/3水平均显著下降及凋亡基因和PRC2蛋白相关亚基EZH2和Suz12基因表达的升高，为进一步研究其对家畜胚胎的调控作用提供技术和理论基础。