摘要
旨在探究circADAMTS16在牛不同组织和前体脂肪细胞中的表达水平及其对牛脂肪细胞分化的影响,为进一步研究circRNA在细胞分化和脂肪组织发育过程中的调控作用提供依据。采用组织块法分离培养牛原代前体脂肪细胞并诱导分化,模拟牛脂肪组织生长发育过程;通过qRT-PCR检测circADAMTS16在牛不同组织(心、肝、脾、肺、肾、肌肉、脂肪)和脂肪细胞分化不同阶段(0 d、2 d、4 d、8 d、10 d)的表达水平;构建circADAMTS16的过表达载体(pCD2.1-circADAMTS16),设计合成circADAMTS16的小干扰RNA(si-circADAMTS16),采用(Lipofectamine3000)转染试剂将pCD2.1-circADAMTS16和si-circADAMTS16转染牛前体脂肪细胞,利用qRT-PCR法检测转染效果,同时检测脂肪分化标志基因PPARγ、C/EBPα和C/EBPβ的表达量变化,并进行油红O染色,综合分析干扰和过表达circADAMTS16对牛脂肪细胞分化的影响。结果如下:(1)qRT-PCR检测结果表明circADAMTS16在心脏中表达量最高,肝脏次之,脾最低;(2)在牛原代前体脂肪细胞分化过程中,circADAMTS16在第2天显著高表达,随后逐渐降低,在第8天时达到最低。(3)在牛前体脂肪细胞中过表达circADAMTST16后,脂肪分化标志基因PPARγ、C/EBPα、C/EBPβ的表达量显著下降;油红O染色结果显示,过表达后脂滴形成数量明显少于对照组;而干扰circADAMTS16表达后,脂肪分化标志基因PPARγ和C/EBPα表达量显著上升。综上所述,circADAMTS16对牛原代前体脂肪细胞的分化过程具有显著的调控作用,过表达circADAMTST16可抑制牛原代前体脂肪细胞分化进程,而干扰circADAMTS16表达可以促进牛原代前体脂肪细胞分化进程,探明circADAMTS16对牛原代前体脂肪细胞分化的具体调控作用。
The expression level of circADAMTS16 in different tissues and precursors of bovine adipose cells and its effects on the differentiation of bovine adipose cells were studied in this paper,so as to provide a basisfor the further study of the regulatory role of circRNA in cell differentiation and adipose tissue development.Tissue block method was used to simulate the growth and development of bovine adipose tissue.The expression levels of circADAMTS16 in different tissues(heart,liver,spleen,lung,kidney,muscle and fat)and adipocyte differentiation in cattle at different stages(0,2,4,8 and 10 day)was detected by qRT-PCR.Then,the overexpression vector of circADAMTS16(pCD2.1-cir-cADAMTS16)was constructed,and the small interfering RNA of circADAMTS16(si-circAD-AMTS16)was designed and synthesized.The bovine precursor adipocytes were transfected with pCD2.1-circADAMTS16 and si-circADAMTS16 by using(Lipofectamine3000)transfection reagent.The transfection effect was detected by qRT-PCR,and the expression changes of adipose differentia-tion marker genes PPARγ,C/EBPαand C/EBPβwere detected at the same time.Oil red O staining was performed to comprehensively analyze the effects of interference and overexpression of circAD-AMTS16 on the differentiation of bovine adipocytes.The results showed that:①the results of qRT-PCR assay showed that the expression level of circADAMTS16 was the highest in the heart,followed by the liver,and the lowest in the spleen.②During the differentiation of bovine primary precursor adipocytes,the expression of circADAMTS16 was significantly higher on day 2(P<0.01),then gradually decreased and was the lowest on day 8.③After overexpression of circADAMTST16 in bo-vine precursor adipocytes,the expression levels of PPARγ,C/EBPαand C/EBPβdecreased signifi-cantly(P<0.01).Oil red O staining showed that the number of lipid droplets formed after overex-pression was significantly less than that in the control group.The expression levels of PPARγand C/EBPαsignificantly increased after circADAMTS16 was interfered with(P<0.01).In summary,circ-ADAMTS16 has a significant regulatory effect on the differentiation of primary bovine preadipocytes.Overexpression of circADAMTS16 inhibits the differentiation of primary bovine precursor adipocytes,while interfering with the expression of circADAMTS16 promotes the differentiation of primary bo-vine precursor adipocytes.The specific regulation of circADAMTS16 on the differentiation of bovine primary precursor adipocytes was proved.
作者
户春丽
雷召雄
汪书哲
盛辉
高玉红
马燕芬
马云
HU Chunli;LEI Zhaoxiong;WANG Shuzhe;SHENG Hui;GAO Yuhong;MA Yanfen;MA Yun(Key Laboratory of Ruminant Molecular Cell Breeding of Ningxia Hui Autonomous Region,School of Agriculture,Ningxia University,Yinchuan 750021,China)
出处
《西北农业学报》
CAS
CSCD
北大核心
2024年第2期208-217,共10页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金(U22A20506,32072720)
宁夏回族自治区重点研发项目(2021BEF01002,2021NXZD1)
自治区科技创新团队计划(03010360052)
自治区科技创新领军人才培养项目(2020GKLRLX02)。
