山羊GPR35基因表达特性分析及对皮下脂肪细胞分化作用的研究

Goats GPR 35 Gene Expression Characteristics Analysis and the Study of Effect on Subcutaneous Fat Cell Differentiation

旨在克隆山羊GPR35基因序列,探究其表达特性并初步阐明该基因对山羊皮下前体脂肪细胞分化的影响以及可能的作用机制。本研究以1周岁的健康雄性(n=4)简州大耳羊为试验对象,利用RT-PCR技术(reverse transcription-polymerase chain reaction, RT-PCR)克隆山羊GPR35基因完整的蛋白质编码区(sequence coding for amino acids in protein, CDS)序列,并对其进行生物信息学分析;利用实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)技术检测GPR35基因在山羊各组织及不同分化阶段皮下脂肪细胞中的表达情况;随后将GPR35过表达载体(pEGFP-N1-GPR35)及其干扰序列(Si-GPR35)分别转染至山羊皮下前体脂肪细胞中并使用油酸诱导分化,通过油红O和Bodipy染色对其脂滴的变化进行形态学观察并对其甘油三酯含量变化进行检测,最后通过qRT-PCR技术来检测脂肪细胞分化、甘油三酯合成和分解相关标志基因的表达量变化(所有样本均设置3个生物学样本重复和3个技术重复)。结果表明,克隆获得的山羊GPR35基因序列全长为1 167 bp,开放阅读框长906 bp,共编码301个氨基酸残基。GPR35基因在山羊背最长肌中的表达量显著高于其它肌肉组织(P<0.01);在皮下脂肪中的表达量显著高于其它脂肪组织(P<0.01);在脾脏中显著高于山羊的其它内脏组织(P<0.01),此外该基因在诱导分化120 h后的皮下脂肪细胞中表达量最高(P<0.01)。过表达和干扰GPR35后分别抑制和促进皮下脂肪细胞中的脂滴含量和聚集程度,甘油三酯相对含量分别显著下降和上升;过表达该基因后C/EBPα、C/EBPβ、PPARγ、AP2、SREBP1基因的表达量极显著下调(P<0.01),HSL极显著上调(P<0.01);而干扰该基因后C/EBPα、C/EBPβ、AP2、SREBP1、DGAT1基因的表达量极显著上调(P<0.01),FASN极显著下调(P<0.01)。综合以上结果表明,GPR35基因可能是山羊皮下前体脂肪细胞分化过程中的一个负向的调控因子,这种作用可能是通过调控C/EBPα、C/EBPβ、AP2、SREBP1的表达或与CXCL17蛋白相互作用来实现的。

The purpose of this study was to clone the GPR 35 gene sequence of goat,explore its expression characteristics and elucidate its effect on the differentiation of goat subcutaneous preadipocytes and its possible mechanism.In this study,the complete sequence coding for amino acids in protein(CDS)of GPR 35 gene in one-year-old healthy male Jianzhou Big ear goats(n=4)were cloned by RT-PCR and analyzed by bioinformatics.Quantitative real-time PCR(qRT-PCR)was used to detect the expression level of GPR 35 gene in goat various tissues and goat subcutaneous adipocytes at different differentiation stages.Then GPR 35 overexpression vector(pEGFP-N1-GPR35)and its interference sequence(Si-GPR35)were transfected into goat subcutaneous adipocytes and induced by oleic acid.The changes of lipid droplets were observed by Oil Red O staining and Bodipy staining,and the changes of triglyceride content were detected.Finally,qRT-PCR was used to detect the changes in the expression of marker genes related to adipocyte differentiation,triglyceride synthesis and decomposition(all samples have 3 biological sample replicates and 3 technical replicates).The results showed that the cloned goat GPR 35 gene sequence was 1167 bp and the open reading frame was 906 bp,encoding 301 amino acid residues.The expression of GPR 35 gene in longissimus dorsi was significantly higher than that in other muscle tissues of goats(P<0.01);Its expression in subcutaneous fat content was significantly higher than that in other adipose tissues(P<0.01);It was significantly higher in spleen than that in other internal organs of goats(P<0.01).In addition to that,the expression of GPR 35 gene was the highest in subcutaneous adipose cells at 120 h after induction(P<0.01).Overexpression and interference of GPR 35 inhibited and promoted the content and aggregation of lipid droplets in subcutaneous adipocytes,respectively,and the relative content of triglyceride was significantly down-regulated decreased and up-regulated increased,respectively.The expression of C/EBPα,C/EBPβ,PPARγ,AP 2 and SREBP 1 genes was significantly down-regulated(P<0.01),and the expression of HSL was significantly up-regulated(P<0.01)after overexpressing GPR 35 gene.However,the expression of C/EBPα,C/EBPβ,AP2,SREBP 1 and DGAT 1 genes was significantly up-regulated(P<0.01),and the expression of FASN was significantly down-regulated(P<0.01)after silencing GPR 35 gene.These results suggest that GPR 35 gene may be a negative regulator during the differentiation of goat subcutaneous preadipocytes,which may be achieved by regulating the expression of C/EBPα,C/EBPβ,AP2,SREBP 1 or interacting with CXCL17 protein.