腺病毒载体介导Smad3基因在秦川牛前体脂肪细胞中的过表达和沉默  

The Overexpression and Interference of Smad3 Gene in Qinchuan Cattle Preadipocytes Mediated by Adenovirus Vector

作　　者：张乐[1] 宁越[1] 李佩韦 王洪宝[1,2] 昝林森[1,2] ZHANG Le;NING Yue;LI Pei-wei;WANG Hong-bao;ZAN Lin-sen(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;National Beef Cattle Improvement Center,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《畜牧兽医学报》2018年第9期1840-1850,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划(2015BAD03B04);国家肉牛牦牛产业技术体系(CARS-37)

摘　　要：旨在通过在秦川牛前体脂肪细胞中过表达和沉默Smad3基因,探讨其在秦川牛前体脂肪细胞分化过程中的生物学功能,从而为阐明TGF-β信号通路在牛脂肪组织生长发育过程中发挥的作用提供理论依据。本试验采用Ad5腺病毒改建的AdMaxTM重组腺病毒包装系统成功重组获得了过表达腺病毒pDC316-EGFP-bSMAD3和干扰腺病毒pDC316-EGFP-ShRNA-bSMAD3-1405。感染牛前体脂肪细胞后,利用实时荧光定量检测Smad3基因及脂肪形成相关基因mRNA的表达。结果表明,将获得的具有较高滴度的过表达腺病毒pDC316-EGFP-bSMAD3感染秦川牛前体脂肪细胞3、6、9d后,分别较对照组Smad3基因表达量显著上调了1 031、1 286、633倍(P<0.001)。与对照组相比,高滴度干扰腺病毒pDC316-EGFP-ShRNA-bSMAD3-1405感染牛前体脂肪细胞3、6、9d后,Smad3基因的表达量分别下调了70%、55%、57%(P<0.01)。油红O染色和实时荧光定量结果均证明,Smad3基因抑制了前体脂肪细胞脂质积累并促使标志基因PPARγ、FABP4、C/EBPα和C/EBPβ的表达显著下调。综上所述,腺病毒载体介导体外表达载体可以成功的实现Smad3基因在秦川牛前体脂肪细胞中的过表达和沉默,并对秦川牛前体脂肪细胞的成脂分化具有一定的调控作用。The purpose of this study was to investigate the biological function of Smad3 gene during Qinchuan cattle preadipocytes differentiation,and provide a theoretical basis for clarifying the role of TGF-βsignaling pathway in the growth and development of cattle adipose tissue by overexpressing and interfering Smad3 gene in Qinchuan cattle preadipocytes.In this study,overexpression adenovirus pDC316-EGFP-bSMAD3 and interference adenovirus pDC316-EGFP-ShRNA-bSMAD3-1405 were successfully obtained by using AdMaxTM recombinant adenovirus packaging system reconstructed with Ad5 adenovirus.Then we detected the mRNA expression of Smad3 gene and adipogenesis-related genes by real-time fluorescence quantitative PCR after the adenovirus infected bovine preadipocytes.The results showed that,compared to the control group,the expression of Smad 3 gene in Qinchuan cattle preadipocytes were significantly increased about 1 031,1 286,633 times,respectively(P<0.001)when bovine preadipocytes were infected by pDC316-EGFP-bSMAD3 for 3,6,9 d.Meanwhile,the interference group’s(pDC316-EGFP-ShRNA-bSMAD3-1405)expression of Smad 3 gene in cattle preadipocytes reduced by 70%,55%and 57%,respectively(P<0.01)compared with the control group.Moreover,both Oil Red O staining assay and qRT-PCR results demonstrated that Smad 3 gene suppressed the lipid accumulation in preadipocytes and markedly down-regulated the expression of adipogenesis-related gene PPARγ,FABP4,C/EBPαand C/EBPβ.In conclusion,Smad 3 gene is successfully overexpressed and interfered in Qinchuan cattle preadipocytes mediated by adenovirus,and plays an important role in adipogenic differentiation process in Qinchuan cattle preadipocytes.

关 键 词：秦川牛 SMAD3基因 前体脂肪细胞 腺病毒载体 

分 类 号：S823.2[农业科学—畜牧学]