Studies for mechanisms of bovine marbling and developments of genetic markers

为了研究反刍动物肌肉内脂肪形成的相关基因，在我们之前对克隆牛肌肉内脂肪细胞(BIP)细胞系(Mizoguchi，2010)的SAGE研究中，选择了16个表达显著变化的靶基因(P&lt；10^&lt；-12&gt；)，参照3T3-L1细胞系作为单胃动物。在含全反式维甲酸(RA)的成脂刺激后0、3、6、9和12天分别收集BIP和3T3-L1细胞，用实时定量聚合酶链式反应(RT-PCR)检测其基因表达水平和甘油三酯(TG)的蓄积水平。SAGE研究证实了这16个基因中的14个基因在脂肪形成过程中的表达模式。在3T3-L1细胞和BIP细胞中，有11个基因的表达模式明显不同。同时，我们用视黄酸研究了靶基因的表达谱。在成脂刺激后第6天，维甲酸显著诱导BIP细胞TG蓄积(P&lt；0.05)，而完全抑制3T3-L1细胞的TG蓄积(P&lt；0.05)。在成脂后第3天，RA诱导BIP中6个基因的表达，并抑制其中8个基因的表达(P&lt；0.05)。将ADFP siRNA导入BIP细胞，观察ADFP下调对甘油三酯蓄积的影响。诱导后3d，ADFP-siRNA转染组ADFP的表达水平为SCR-siRNA转染组的21.4%(P&lt；0.05)。此外，诱导6天后，ADFP-siRNA转染组细胞中TG的积累量比对照组减少20%(P&lt；0.001)。我们对RA基因表达谱的发现将有助于我们对牛肌肉内脂肪生成的分子机制有更好和详细的了解。

To investigate genes involved in ruminants intramuscular adipogenesis, sixteen target genes(8 up and 8 down regulated genes) with dramatically changed expression(P<10^<-12>) were selected between differentiation and proliferation phases libraries in our previous SAGE studies of a clonal bovine intramuscular preadipocyte(BIP) cell line(Mizoguchi, 2010), with reference to 3T3-L1 cell lines as monogastric animals. We harvested the BIP and 3T3-L1 cells 0, 3, 6, 9 and 12 days after adipogenic stimulation containing all-trans retinoic acid(RA), which inhibits preadipocyte differentiation in non-ruminants and measured the gene expression levels by quantitative real-time PCR and the accumulation levels of triglyceride(TG). Fourteen genes expression pattern of the 16 genes during adipogenesis were confirmed the SAGE studies. The expression pattern of eleven genes clearly differed between 3T3-L1 and BIP cells. Simultaneously, we investigated the profiles of target genes by RA. TG accumulation was significantly induced(P<0.05) in BIP cells whereas completely inhibited(P<0.05) in 3T3-L1 cells by RA at day 6 after adipogenic stimulation. RA induced the expression levels of six genes and suppressed these of eight genes at day 3 after adipogenesis in BIPs(P<0.05 ; respectively). The effect of ADFP down-regulation on the accumulation of TG was examined by transfecting the BIP cells with ADFP siRNA. The expression level of ADFP in the ADFP-siRNA transfected cells was 21.4% of that in the scr-siRNA transfected cells in BIP 3 days after induction(P<0.05). Also, 6 days after induction, the accumulation of TG in the ADFP-siRNA transfected cells was 20% less than in the control cells(P<0.001). Our findings of gene expression profiles by RA will cause a favorable and detailed understanding in the molecular mechanisms of bovine intramuscular adipogenesis.