蒙山牛诱导多能干细胞的建立

doi:10.11843/j.issn.0366-6964.2025.04.022

摘要/Abstract

摘要：

旨在建立稳定传代并符合多能性标准的蒙山牛诱导多能干细胞。本研究首先用酶消化法建立了成年蒙山牛公牛耳缘成纤维细胞，并进行细胞存活率、生长曲线测定。同时采用了基于DOX调控的非病毒重编程外源基因表达系统，使用在3i/LIF和NBFR培养体系诱导蒙山牛成纤维细胞重编程，并对获得的蒙山牛诱导多能干细胞进行多能性检测。结果表明，本研究获得的蒙山牛耳缘成纤维细胞增殖正常、状态良好，蒙山牛诱导多能干细胞克隆形态呈现边界圆滑的圆顶型、碱性磷酸酶表达呈阳性、标志多能性基因表达显著高于成纤维细胞(P < 0.001)，OCT4、SOX2、NANOG多能性蛋白的免疫荧光染色呈阳性，展现出与之前研究中报道的牛胚胎干细胞和诱导多能干细胞相似的多能性状态。这一成果为牛诱导多能干细胞的研究和应用提供了新的方向，并有助于进一步探索其在农业生物技术领域中的潜力。为体外配子生产进而复原濒危、灭绝的牛地方品种提供了材料基础。

关键词: 蒙山牛, 多能性, 体细胞重编程, 诱导多能干细胞

Abstract:

The purpose of this study was to establish Mengshan cattle induced pluripotent stem cells that can be stably passaged and meet the criteria for pluripotency. Initially, fibroblasts were isolated from the ear skin of adult Mengshan cattle bulls using enzyme digestion, and cell viability and growth curves were assessed. Meanwhile, a doxycycline-inducible, non-viral reprogramming system was used to induce the reprogramming of Mengshan cattle fibroblasts in 3i/LIF and NBFR culture systems. The pluripotency of the resulting induced pluripotent stem cells was subsequently evaluated. The results showed that the Mengshan cattle ear fibroblasts obtained in this study exhibited normal proliferation and good condition. The induced pluripotent stem cells from Mengshan cattle displayed a smooth-edged, dome-shaped colony morphology. Alkaline phosphatase staining was positive, and the expression of pluripotency marker genes was significantly higher than that of fibroblasts (P < 0.001). Immunofluorescence staining for pluripotency proteins OCT4, SOX2, and NANOG was positive, demonstrating a pluripotent state similar to that observed in bovine embryonic stem cells and induced pluripotent stem cells reported in previous studies. This achievement provides a new direction for the research and application of bovine induced pluripotent stem cells and helps to further explore their potential in the field of agricultural biotechnology. It lays a material foundation for in vitro gamete production and the restoration of endangered or extinct local cattle breeds.

Key words: Mengshan cattle, pluripotency, somatic cell reprogramming, induced pluripotent stem cells

中图分类号:

S823.2

赵文轩, 高雪, 余大为, 高晨, 李俊雅. 蒙山牛诱导多能干细胞的建立[J]. 畜牧兽医学报, 2025, 56(4): 1731-1743.

ZHAO Wenxuan, GAO Xue, YU Dawei, GAO Chen, LI Junya. Establishment and Pluripotency Analysis of Induced Pluripotent Stem Cells from Mengshan Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1731-1743.

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基因 Gene	上游引物(5′→3′) Upstream primer	下游引物(5′→3′) Downstream primer
GAPDH	CCTGCCCGTTCGACAGATA	GGCGACGATGTCCACTTTG
OCT4	AACGAGAATCTGCAGGAGATATG	TCTCACTCGGTTCTCGATACT
KLF4	TCAGGAGACACAAGGAAACCA	CCCCTTGGCGTTTTGTAAGT
SOX2	CATTAACGGCACACTGCCCC	TGAAAATGTCTCCCCCGCCC
NANOG	CAGCTACAAGCAGGTGAAGA	CTATTCCTCGGCCAGTTGTT

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