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

doi:10.11843/j.issn.0366-6964.2018.09.005

摘要/Abstract

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、9 d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、9 d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。

Abstract:

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-β signaling 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 Smad3 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 Smad3 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 Smad3 gene suppressed the lipid accumulation in preadipocytes and markedly down-regulated the expression of adipogenesis-related gene PPARγ, FABP4, C/EBPα and C/EBPβ. In conclusion, Smad3 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.

中图分类号:

S823.2

张乐, 宁越, 李佩韦, 王洪宝, 昝林森. 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[J]. 畜牧兽医学报, 2018, 49(9): 1840-1850.

ZHANG Le, NING Yue, LI Pei-wei, WANG Hong-bao, ZAN Lin-sen. The Overexpression and Interference of Smad3 Gene in Qinchuan Cattle Preadipocytes Mediated by Adenovirus Vector[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1840-1850.

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