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33
2025年4月5日 星期六
农业生物技术学报  2025, Vol. 33 Issue (2): 355-365    DOI: 10.3969/j.issn.1674-7968.2025.02.010
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
脂肪酸氧化参与鸡原始生殖细胞形成的功能研究
王哲, 武嵘峰, 耿晴晴, 赵梓多, 吴一凡, 程富富, 席一凡, 陈欣雨, 王春慧子, 牛英杰, 左其生, 张亚妮*
扬州大学 动物科学技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州 225009
Functional Studies on the Involvement of Fatty Acid Oxidation in the Formation of Chicken (Gallus gallus domesticus) Primordial Germ Cells
WANG Zhe, WU Rong-Feng, GENG Qing-Qing, ZHAO Zi-Duo, WU Yi-Fan, CHENG Fu-Fu, XI Yi-Fan, CHEN Xin-Yu, WANG Chun-Hui-Zi, Niu Ying-Jie, ZUO Qi-Sheng, ZHANG Ya-Ni*
Jiangsu Key Laboratory of Animal Genetics, Breeding and Molecular Design/College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
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摘要 禽类原始生殖细胞(primordial germ cells, PGCs)具有能够从血液迁移而定植于生殖嵴的特点,因此PGCs不仅能替代受精卵开展遗传修饰,而且在禽类的种质资源保护和濒危物种的复原方面都具有重要的应用前景。现有的PGCs体外培养体系对于支持雌雄PGCs生长之间存在差异,满足不了PGCs在种质资源保护中的需求。针对这一问题,本研究以鸡(Gallus gallus domesticus) PGCs为研究对象,探究脂肪酸氧化(fatty acid oxidation, FAO)在PGCs形成中的功能,旨在为后续建立完善的PGCs体外培养体系提供理论参考。基于转录组学分析FAO在胚胎干细胞(embryonic stem cells, ESCs)向PGCs分化过程中的作用,通过CCK8与EdU实验探究FAO激活剂BMS-687453 (BMS)和抑制剂马来酸哌克昔林(perhexiline maleate, Per)的最适添加浓度;分别通过观察细胞形态、qRT-PCR检测FAO关键基因和PGCs特异标记基因的表达、间接免疫荧光和流式细胞分析等检测FAO在ESCs向PGCs分化过程中的作用。转录组学分析显示,参与FAO的相关基因在ESCs与PGCs中差异表达,且富集于生殖细胞分化相关通路(MAPK signaling pathway, GnRH signaling pathway, PI3K-Akt signaling pathway);qRT-PCR结果表明,乙酰辅酶A酰基转移酶2 (acetyl-Coacyltransferase 2, ACAA2)和烯酰辅酶A水合酶1 (enoyl-CoA hydratase 1, ECH1)在PGCs高表达。CCK8与EdU实验确定了BMS和Per的最适添加浓度分别为80和160 nmol/L。细胞形态观察结果显示,BMS组在培养6 d的细胞中,类胚体数目显著高于Per组(P<0.05);BMS组中鸡核糖核酸同源蛋白(chicken VASA Homolog, CVH)和酪氨酸激酶受体蛋白(c-Kit proto-oncogene receptor tyrosine kinase, C-KIT)的表达量显著高于Per组(P<0.05);间接免疫荧光和流式细胞分析结果显示,BMS组的DDX4阳性细胞数显著高于Per组(P<0.05)。以上结果表明,高水平FAO能够促进PGCs的形成。本研究为建立完善的体外培养家禽PGCs体系提供理论依据,有助于从细胞能量代谢的角度系统地阐明PGCs的发生机理。
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作者相关文章
王哲
武嵘峰
耿晴晴
赵梓多
吴一凡
程富富
席一凡
陈欣雨
王春慧子
牛英杰
左其生
张亚妮
关键词 家禽脂肪酸氧化(FAO)转录组分析原始生殖细胞(PGCs)    
Abstract:Poultry primordial germ cells (PGCs) migrate from the blood and colonize the reproductive ridge. Therefore, PGCs can not only replace fertilized eggs for genetic modification, but also have important application prospects in germplasm resource conservation and the recovery of endangered species. In vitro culture systems for PGCs exhibit differences in supporting the growth of male and female PGCs. So it is difficult to meet the requirements of PGCs in germplasm resource conservation. In response to this problem, this study aims to explore the function of fatty acid oxidation (FAO) involved in the formation of PGCs, and provide theoretical guidance for the subsequent establishment of a perfect in vitro culture system of PGCs.The role of FAO in the differentiation of embryonic stem cells (ESCs) into PGCs was analyzed based on transcriptomics, and the optimal addition concentrations of FAO activator BMS-687453 (BMS) and inhibitor perhexiline maleate (Per) were investigated by CCK8 and EdU tests. Finally, the role of FAO in the differentiation process from ESCs to PGCs was detected by cell morphology, qRT-PCR, indirect immunofluorescence and flow cytometry.The results showed that the genes involved in FAO were differentially expressed in ESCs and PGCs by transcriptomic analysis. Moreover, it was enriched in germ cell differentiation related pathways (MAPK signaling pathway, GnRH signaling pathway, PI3K-Akt signaling pathway). qRT-PCR results showed that acetyl-Coacyltransferase 2 (ACAA2) and enoyl-CoA hydratase 1 (ECH1) were highly expressed in PGCs.CCK8 and EdU experiments determined that the optimal addition concentrations of BMS and Per were 80 and 160 nmol/L, respectively. The results of cell morphology showed that the number of blastoid bodies in BMS group was significantly higher than that in Per group at 6 d (P<0.05). In BMS group, the expression levels of chicken VASA homolog (CVH) and c-Kit proto-oncogene receptor tyrosine kinase (C-KIT) were significantly higher than those in Per group (P<0.05). Indirect immunofluorescence and flow cytometry results showed that the number of DDX4 positive cells in BMS group was significantly higher than that in Per group (P<0.05). The above results indicated that promoting FAO could promote the formation of PGCs. This study provides a theoretical basis for establishing a perfect PGCs system in vitro culture of poultry, and also helps to systematically clarify the genesis mechanism of PGCs from the perspective of cell energy metabolism.
Key wordsPoultry    Fatty acid oxidation (FAO)    Transcriptome analysis    Primordial germ cells (PGCs)
收稿日期: 2024-04-09     
中图分类号: S831.2
基金资助:国家自然科学基金(32272857); 国家重点研发计划(2021YFD1200305); 江苏省自然科学基金(BK20210813); 江苏省研究生科研与实践创新计划项目(SJCX24_2297)
通讯作者: * ynzhang@yzu.edu.cn   
引用本文:   
王哲, 武嵘峰, 耿晴晴, 赵梓多, 吴一凡, 程富富, 席一凡, 陈欣雨, 王春慧子, 牛英杰, 左其生, 张亚妮. 脂肪酸氧化参与鸡原始生殖细胞形成的功能研究[J]. 农业生物技术学报, 2025, 33(2): 355-365.
WANG Zhe, WU Rong-Feng, GENG Qing-Qing, ZHAO Zi-Duo, WU Yi-Fan, CHENG Fu-Fu, XI Yi-Fan, CHEN Xin-Yu, WANG Chun-Hui-Zi, Niu Ying-Jie, ZUO Qi-Sheng, ZHANG Ya-Ni. Functional Studies on the Involvement of Fatty Acid Oxidation in the Formation of Chicken (Gallus gallus domesticus) Primordial Germ Cells. 农业生物技术学报, 2025, 33(2): 355-365.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2025.02.010     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2025/V33/I2/355
 
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