SFRP4基因对牛前体脂肪细胞分化作用的影响

doi:10.11843/j.issn.0366-6964.2025.02.013

摘要/Abstract

摘要：

旨在探究分泌型卷曲相关蛋白4(screted frizzled related protein 4，SFRP4)基因对于牛前体脂肪细胞分化的影响，本研究以秦川牛肾周前体脂肪细胞为试验对象，处理组和对照组进行了3个生物学重复。克隆秦川牛SFRP4基因完整的蛋白质编码区序列，并进行生物信息学分析；利用实时荧光定量PCR技术检测该基因在秦川牛各组织及肾周前体脂肪细胞各分化阶段中的表达；筛选有效的牛SFRP4基因siRNA序列，构建腺病毒过表达载体，分别转染至牛肾周前体脂肪细胞中诱导分化，通过油红O染色、qRT-PCR和蛋白质印迹等试验方法探究SFRP4基因对肾周前体脂肪细胞分化的影响。结果表明，秦川牛SFRP4基因CDS全长1 041 bp，氨基酸序列与绵羊和山羊具有高度同源性，与小鼠的亲缘关系较远；该蛋白分类为亲水性蛋白，共有24个磷酸化修饰位点；该基因在秦川牛各组织广泛表达，在肾脏和皮下脂肪相对表达量较高。时序表达谱显示前体脂肪细胞分化过程中SFRP4基因表达量呈现先升高后降低趋势。干扰SFRP4后，牛肾周前体脂肪细胞脂滴明显减少，脂质细胞成脂分化标志基因PPARγ、FABP4、PLIN2、SCD1表达水平显著下调(P < 0.05)，CEBPβ表达水平极显著下调(P < 0.01)，PPARγ(P < 0.05)和CEBPβ(P < 0.01)蛋白水平显著下调；过表达该基因后，显著上调PPARγ(P < 0.01)、CEBPβ(P < 0.01)、FABP4(P < 0.05)、PLIN2(P < 0.01)、SCD1(P < 0.01)基因表达水平，显著上调了PPARγ、CEBPβ、FABP4、SCD1蛋白水平(P < 0.05)。以上结果为进一步阐述SFRP4基因调控牛肾周前体脂肪细胞成脂分化和脂肪沉积提供了重要的依据。

关键词: 秦川牛, SFRP4, 肾周前体脂肪细胞, 成脂分化

Abstract:

This study aimed to explore the effects of the screted frizzled related protein 4 (SFRP4) gene on the differentiation of bovine preadipocytes, the Qinchuan cattle perirenal preadipocytes was used as the experimental subject. Both treatment and control groups had 3 biological replicates. The full-length protein-coding sequence of the SFRP4 gene was cloned and analyzed via bioinformatics. Quantitative real-time PCR assessed SFRP4 expression across various tissues and differentiation stages in perirenal preadipocytes. Effective bovine SFRP4 siRNA sequences were screened, and an adenoviral overexpression vector was constructed for transfection and differentiation induction in bovine preadipocytes. Oil Red O staining, qRT-PCR, and Western blotting were used to evaluate the function of SFRP4. Results demonstrated the CDS of SFRP4 gene was 1 041 bp; There were high homology between Qinchuan cattle SFRP4 amino acid sequences and those of sheep and goats, with more distant relationships to mice. This protein was classified as a hydrophilic protein and had a total of 24 phosphorylation modification sites. SFRP4 was broadly expressed across tissues, with higher expression in kidney and subcutaneous fat. Temporal expression patterns of SFRP4 during perirenal preadipocyte differentiation indicated an initial increase followed by a decrease. SFRP4 knockdown resulted in reduced lipid droplet accumulation in perirenal preadipocytes, with significant downregulation of adipogenic differentiation marker genes PPARγ, FABP4, PLIN2, and SCD1 (P < 0.05), and a highly significant reduction in CEBPβ expression (P < 0.01). PPARγ (P < 0.05) and CEBPβ (P < 0.01) protein levels were also downregulated. Conversely, SFRP4 overexpression led to significant upregulation of PPARγ (P < 0.01), CEBPβ (P < 0.01), FABP4 (P < 0.05), PLIN2 (P < 0.01), and SCD1 (P < 0.01) genes expression and increased PPARγ, CEBPβ, FABP4, and SCD1 protein levels (P < 0.05). These findings provide critical insights into the regulatory role of SFRP4 in bovine perirenal preadipocyte adipogenic differentiation and lipid deposition.

Key words: Qinchuan cattle, SFRP4, perirenal preadipocytes, adipogenic differentiation

中图分类号:

S823.2

赵刚奎, 高海旭, 尹思琦, 孙红红, 辛怡然, 昝林森, 赵春平. SFRP4基因对牛前体脂肪细胞分化作用的影响[J]. 畜牧兽医学报, 2025, 56(2): 611-620.

ZHAO Gangkui, GAO Haixu, YIN Siqi, SUN Honghong, XIN Yiran, ZAN Linsen, ZHAO Chunping. The Effects of the SFRP4 Gene on Bovine Preadipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 611-620.

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引物 Primer	引物序列(5'→3') Sequence	扩增片段大小/bp Fragment size
β-actin-F	TCTAGGCGGACTGTTAGC	82
β-actin-R	CCATGCCAATCTCATCTCG
SFRP4-F	GCGCTCACGGATGATGCTTCT	135
SFRP4-R	CCTGCTGTTCGCTTCTTGTCCTG
PPARγ-F	GAGATCACAGAGTACGCCAAG	216
PPARγ-R	GGGCTCCATAAAGTCACCAA
CEBPβ-F	TTCCTCTCCGACCTCTTCTC	79
CEBPβ-R	CCAGACTCACGTAGCCGTACT
FABP4-F	GCTGCACTTCTTTCTCACCTTG	194
FABP4-R	ACCACACCCCCATTCAAACT
SCD1-F	CCATCAACCCCCGAGAGA	76
SCD1-R	AAGGTGTGGTGGTAGTTGTGGAA
PLIN2-F	AGTGAACTTGCCAGGAAGAATG	120
PLIN2-R	TTCATCTGTATCATCGTAGCCG
CTNNB1-F	AATCAGCTGGCCTGGTTTGA	146
CTNNB1-R	GCTTGGTTAGTGTGTCAGGC
Wnt7A-F	CGTCATAGGAGAAGGCTCGC	115
Wnt7A-R	TTTGAGCTCCTTGCCGAAGA

类别 Sort	不同二级结构占比/% Percentage of different secondary structures
类别 Sort	α-螺旋 α-helix	延伸链 Extended strand	无规则卷曲 Random coil	模棱两可状态 The ambiguous state of being
DSC	24.86	7.51	67.63	0
MLRC	32.37	9.25	58.38	0
PHD	28.90	8.67	62.43	0
Sec.Cons.	28.32	7.80	61.85	2.02

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