过表达和干扰PRKD1基因对牛成骨细胞分化的影响

doi:10.11843/j.issn.0366-6964.2025.07.014

摘要/Abstract

摘要：

旨在探究肉牛胴体胸深性状重要候选基因蛋白激酶D1(PRKD1)基因对牛成骨细胞分化的调控作用。本研究以妊娠3月龄华西牛胎儿肋骨来源成骨细胞为体外模型，根据PRKD1基因序列信息，分别构建合成了过表达PRKD1质粒(pPRKD1)和PRKD1 siRNA(siPRKD1)，并将其转染牛成骨细胞并对其进行诱导分化；在分化第4天时，采用RT-qPCR和免疫印迹试验检测成骨分化关键效应基因(RUNX2、SP7、SPP1、COL I)的表达量；在分化第7天时，检测ALP活性；在分化第21天时，用茜素红钙结节染色法检测成骨细胞成骨能力。以上所有试验均设立3个生物学重复，并于组内设立3次组内重复。结果表明，过表达PRKD1后，SP7和COL I的mRNA和蛋白表达量显著提高(P＜0.01)，RUNX2和SPP1则未产生显著性变化。过表达PRKD1引起成骨细胞ALP活性显著升高(P＜0.01)，矿化结节显著增多(P＜0.01)，而干扰PRKD1则反之。综上，PRKD1基因显著影响牛肋骨成骨细胞的分化和成骨功能，研究结果为进一步解析PRKD1基因调控牛胴体胸深性状的作用机制提供理论依据。

关键词: 牛, PRKD1, 成骨细胞, 成骨分化

Abstract:

The aim of this study was to investigate the regulatory effect of protein kinase D1 (PRKD1) gene, an important candidate gene for chest depth trait in beef carcass, on the osteogenic differentiation of bovine osteoblasts. In this study, osteoblasts derived from pregnancy Huaxi bovine fetal ribs with 3 months old were used as in vitro models. According to PRKD1 gene sequence, PRKD1 overexpressed plasmid (pPRKD1) and PRKD1 siRNA (siPRKD1) were constructed and synthesized, respectively, and transfected into bovine osteoblasts. On the 4 days of differentiation, RT-qPCR and Western blot assay were used to detect the expression levels of RUNX2, SP7, SPP1 and COL I genes. The activity of ALP was detected on the 7 days of differentiation. On the 21 days of differentiation, the osteogenic ability of osteoblasts was detected by Alizarin Red calcium nodule staining. All the above-mentioned experiments were conducted with 3 biological replicates each, and within each group, 3 intra-group replicates were set up. The results showed that after overexpression of PRKD1, mRNA and protein expressions of SP7 and COL I genes were significantly increased(P < 0.01), while RUNX2 and SPP1 genes were not significantly changed, PRKD1 overexpression also significantly increased the ALP activity and mineralized nodules of osteoblasts, while interference of PRKD1 showed the opposite effect. In conclusion, PRKD1 gene significantly affects the differentiation and osteogenic function of bovine rib osteoblasts, and the results provide theoretical basis for further analysis of the regulation mechanism of PRKD1 gene on bovine carcass chest depth traits.

Key words: bovine, PRKD1, osteoblasts, osteogenic differentiation

中图分类号:

S823.2

赵峂, 王亚慧, 吴天弋, 高晨, 高霄霄, 张路培, 高会江, 李俊雅. 过表达和干扰PRKD1基因对牛成骨细胞分化的影响[J]. 畜牧兽医学报, 2025, 56(7): 3188-3198.

ZHAO Tong, WANG Yahui, WU Tianyi, GAO Chen, GAO Xiaoxiao, ZHANG Lupei, GAO Huijiang, LI Junya. Effects of Overexpression and Interference of PRKD1 Gene on Osteogenic Differentiation of Bovine Osteoblasts[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3188-3198.

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siRNA名称Name	序列(5′→3′) Sequence
siPRKD1-1	CCGAAGAAACAGAAATGAA
siPRKD1-2	GCACATCAGCACAGTTTAT
siPRKD1-3	GCTGAATTACCACAAGAGA

基因Gene	上游引物(5′→3′) Forward primer sequence	下游引物(5′→3′) Reverse primer sequence
PRKD1	AGACTTTCAGATTCGCCCCC	TCAGCCCACATCCTTCACAT
RUNX2	CGGAGTGGAAGAGGCAAGAG	TGGATGGACGGAGGAGTCAT
COL I	AGGCCAAGAAGAAGACATCCC	GGGACTTTGGCGTTAGGACA
SPP1	GCTAAAGCCTGACCCATCTCA	GGACTTACTTGGGAGGGTATTTTG
SP7	TGGCACACGGGCGAGAGG	TGGAGCAGAGTAGGCAGGTGAAC
GAPDH	AGGTCGGAGTGAACGGATTC	CCAGCATCACCCCACTTGAT

细胞Cell	OCN水平/(ng·mL^-1) OCN level	ALP水平/(μmol·mL^-1) ALP level
成纤维细胞Fibroblasts	7.736±0.041 6	0.506±0.061 5
成骨细胞Osteoblasts	10.328±0.082 6^**	2.378±0.073 1^**

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