ADCY2基因表达对延边黄牛脂肪前体细胞成脂分化的影响

doi:10.16431/j.cnki.1671-7236.2020.11.021

摘要/Abstract

摘要： 本试验旨在研究环腺苷酸（3'，5'-cyclic adenosine monophosphote，cAMP）环化酶合成酶2（adenylyl cyclase 2，ADCY2）基因对延边黄牛脂肪前体细胞成脂分化的影响。选取3日龄的延边黄牛腹股沟皮下脂肪组织进行脂肪前体细胞的分离、培养和诱导分化；设计ADCY2基因的干扰片段（siADCY2-1、2、3），构建pEX4过表达载体；分别收集正常诱导（对照组）、干扰和过表达0、5和10 d的脂肪细胞。用实时荧光定量PCR检测过氧化物酶体增殖激活物受体（PPARγ）、CCAAT/增强子结合蛋白（C/EBPα）和ADCY2在细胞分化过程中mRNA的转录水平，并用Western blotting法检测其蛋白的表达；用油红O染色检测脂滴含量的变化；用甘油三酯试剂盒检测甘油三酯含量。试验结果表明，在成脂分化过程中，与未分化相比，ADCY2基因在分化的第5和10天表达量均极显著上升（P<0.01），而分化第10天与第5天相比水平极显著下降（P<0.01）；siADCY2-1干扰效率最高，过表达ADCY2基因使其mRNA水平升高；与对照组相比，过表达组细胞内ADCY2基因mRNA水平提高了约4 000 000倍，脂肪细胞内脂滴含量和甘油三酯的含量极显著提高（P<0.01），成脂关键基因C/EBPα和PPARγ表达极显著上调（P<0.01），而RNA干扰组细胞内ADCY2基因mRNA水平极显著降低（P<0.01），脂肪细胞内脂滴含量和甘油三酯的含量极显著下降，成脂关键基因C/EBPα和PPARγ表达极显著下调（P<0.01）。因此，ADCY2基因过表达能显著增加成熟脂肪细胞的脂滴含量及甘油三酯含量，促进成脂关键基因C/EBPα和PPARγ的表达，说明ADCY2基因对脂肪细胞分化具有正向调控作用。

关键词: ADCY2基因; 延边黄牛; 脂肪细胞分化

Abstract: The aim of the experiment was to study the effect of 3',5'-cyclic adenosine monophosphote (cAMP) adenylyl cyclase 2 (ADCY2) gene on adipogenic differentiation of preadipocytes in Yanbian Yellow cattle.The 3-day-old inguinal adipose tissue of Yanbian Yellow cattle was selected for the isolation,culture and induction differentiation of preadipocytes.The interference fragment of ADCY2 gene was designed and the pEX4 overexpression vector was constructed.Adipose cells of normal (Control),interference,and overexpression groups at 0,5,and 10 d were collected,respectively.The mRNA transcriptional levels of peroxisome proliferation activator receptor γ(PPARγ),CCAAT/enhancer binding protein (C/EBP) and ADCY2 in the process of cell differentiation were detected by Real-time quantitative PCR and the protein expression was detected by Western blotting.Oil red O staining was used to detect the change of lipid droplets,and triglyceride content was detected with triglyceride kit.The results showed that during adipogenic differentiation process,compared with the undifferentiated group,the expression of ADCY2 gene increased significantly at the 5th and 10th day (P<0.01),but decreased significantly at the 10th day compared with that of the 5th day (P<0.01).Compared with control group,the mRNA level of ADCY2 gene was increased about 4 000 000 folds in the overexpression group,the number of lipid droplets and the content of triglyceride in adipocytes was extremely increased (P<0.01),the expression of key lipid-forming genes C/EBPα and PPARγ were significantly up-regulated (P<0.01),but the mRNA levels of ADCY2 gene was significantly decreased in the RNA interference group,and the number of lipid droplets and the content of triglyceride in adipocytes decreased significantly(P<0.01),the expression of key lipid-forming genes C/EBPα and PPARγ were significantly down-regulated (P<0.01).Therefore,overexpression of ADCY2 gene could significantly increase the content of lipid droplets and triglyceride in mature adipocytes,and promote the expression of key lipid-forming genes C/EBPα and PPARγ,indicated that ADCY2 gene had a positive regulatory effect on adipocyte differentiation.

Key words: ADCY2 gene; Yanbian Yellow cattle; adipocyte differentiation

中图分类号:

Q952.6

罗晓彤, 金鑫, 严昌国, 朴政玉. ADCY2基因表达对延边黄牛脂肪前体细胞成脂分化的影响[J]. 中国畜牧兽医, 2020, 47(11): 3595-3601.

LUO Xiaotong, JIN Xing, YAN Changguo, PIAO Zhengyu. Effect of ADCY2 Gene Expression on Preadipocyte Differentiation in Yanbian Yellow Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3595-3601.

参考文献

[1] DANIAL P B,WALKER W H,HABNER J F.Cyclic AMP signaling and gene regulation[J].Annual Review of Nutrition,1998,18(1):353-383.
[2] HANOUNE J,DEFER N.Regulation and role of adenylyl cyclase isoforms[J].Annual Review Pharmacol Toxicol,2001,41(41):145-174.
[3] OMORI K,KOTERA J.Overview of PDEs and their regulation[J].Circulation Research,2007,100(3):309-327.
[4] HORNER K,LIVERA G,HINCKLEY M,et al.Rodent oocytes express an active adenylyl cyclase required for meiotic arrest[J].Developmental Biology,2003,258(2):385-396.
[5] BEAZELY M A,WATTS V J.Regulatory properties of adenylate cyclases type 5 and 6:A progress report[J].European Journal of Pharmacology,2006,535(1-3):1-12.
[6] CHARLES T M,ZHANG A G,CHEN G L,et al.Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity[J].Brain Reserch,2005,1045(1-2):45-56.
[7] PRONKOS P,SABA L M,HOFFMAN P L,et al.Type 7 adenylyl cyclase-mediated hypothalamic-pituitary-adrenal axis responsiveness:Influence of ethanol and sex[J].The Journal of Pharmacology & Experimental Therapeutics,2010,334(1):44-52.
[8] YUAN W,ANDRES L B.Cyclic AMP signalling pathways in the regulation of uterine relaxation[J].BMC Pregnancy & Childbirth,2007,7 Suppl 1(Suppl 1):S10.
[9] HE D,LIU L,WANG Y,et al.A novel genes signature associated with the progression of polycystic ovary syndrome[J].Pathology Oncology Reserch, 2019,26(1):575-582
[10] TANNOURLOUET M,HAN S,CORBETT S T,et al.Identification of de novo copy number variants associated with human disorders of sexual development[J].PLoS One,2010,26:5(10):e15392.
[11] JAJODIA A,KAUR H,KUMARI K,et al.Evaluation of genetic association of neurodevelopment and neuroimmunological genes with antipsychotic treatment response in schizophrenia in Indian populations[J].Molecular Genetics & Genomic Medicine,2015,4(1):18-27.
[12] MÜHLEISEN T W,LEBER M,SCHULZE T G,et al.Genome-wide association study reveals two new risk loci for bipolar disorder[J].Nature Communications,2014,5:3339.
[13] DANIELA O P,LAURA M S.HENRIETTE W,et al.Genetic markers of co-morbid depression and alcoholism in women[J].Alcoholism Clinical and Experimental Reserch,2013,37(6):896-904.
[14] PRATA D P,COSTA-NEVES B,COSME G,et al.Unravelling the genetic basis of schizophrenia and bipolar disorder with GWAS:A systematic review[J].Journal of Pyschiatric,2019,114:178-207.
[15] KAUR I,KAUR J,SOORAJ K,et al.Comparative evaluation of the aqueous humor proteome of primary angle closure and primary open angle glaucomas and age-related cataract eyes[J].International Ophthalmol,2019,39(1):69-104.
[16] DAILY J W,YANG H J,LIU M,et al.Subcutaneous fat mass is associated with genetic risk scores related to proinflammatory cytokine signaling and interact with physical activity in middle-aged obese adults[J].Nutrition & Metabolism(Lond),2019,16:75.
[17] CHEN G,SU Y,CAI Y,et al.Comparative transcriptomic analysis reveals beneficial effect of dietary mulberry leaves on the muscle quality of finishing pigs[J].Veterinary Medicine and Science,2019,5(4):526-535.
[18] WANG Y,DING X,TAN Z,et al.Genome-wide association study for reproductive traits in a Large White pig population[J].Animal Genetic,2018,49(2):127-131.
[19] LEE H L,QADIA A S,PARK H J,et al.cAMP/protein kinase A signaling inhibits Dlx5 expression via activation of CREB and subsequent C/EBPβ induction in 3T3-L1 preadipocytes[J].International Journal of Molecular Science,2018,19(10):3161.
[20] LI Y X,JIN H G,YAN C G,et al.Molecular cloning,sequence identification,and gene expression analysis of bovine ADCY2 gene[J].Molecular Biology Reports,2014,41(6):3561-3568.