环黄芪醇对猪供体成纤维细胞衰老、细胞骨架与核移植胚胎早期发育的影响

doi:10.3864/j.issn.0578-1752.2025.12.014

中国农业科学 ›› 2025, Vol. 58 ›› Issue (12): 2453-2474.doi: 10.3864/j.issn.0578-1752.2025.12.014

环黄芪醇对猪供体成纤维细胞衰老、细胞骨架与核移植胚胎早期发育的影响

周旗¹^,³(), 张亮¹^,²(), 潘雨¹^,², 涂志¹^,², 王峥³, 刘航航³, 鲜凌瑾⁴, 夏运红⁵, 潘红梅¹^,²(), 龙熙¹^,²()

¹ 重庆市畜牧科学院，重庆 402460
² 国家生猪技术创新中心，重庆 402460
³ 西北农林科技大学，陕西杨凌 712100
⁴ 乐山职业技术学院，四川乐山 614013
⁵ 四川省内江市农业科学院，四川内江 641000

收稿日期:2025-01-02 接受日期:2025-05-11 出版日期:2025-06-19 发布日期:2025-06-19
通信作者:
龙熙，E-mail：13618288075 @163.com。
潘红梅，E-mail：panhm@cqaa.cn
联系方式: 周旗，E-mail：zhouqi111@nwafu.edu.cn。张亮，E-mail：zhangl@cqaa.cn。周旗和张亮为同等贡献作者。
基金资助:
国家重点研发计划(2021YFD1200303); 重庆市自然科学基金(CSTB2024NSCQ-MSX0787); 重庆现代农业产业技术体系(CQMAITS202312); 重庆市人民政府与中国农业科学院战略合作资金项目(23310)

Effects of Cycloastragenol on Cellular Senescence of Pig Donor Fibroblast, Cytoskeletal Dynamic, and Early Developmental Stage of Nuclear Transfer Embryo

ZHOU Qi¹^,³(), ZHANG Liang¹^,²(), PAN Yu¹^,², TU Zhi¹^,², WANG Zheng³, LIU HangHang³, XIAN LingJin⁴, XIA YunHong⁵, PAN HongMei¹^,²(), LONG Xi¹^,²()

¹ Chongqing Academy of Animal Science and Veterinary Medicine, Chongqing 402460
² National Swine Technology Innovation Center, Chongqing 402460
³ Northwest A&F University, Yangling 712100, Shaanxi
⁴ Leshan Vocational and Technical College, Leshan 614013, Sichuan
⁵ Sichuan Neijiang Agricultural Academy, Neijiang 641000, Sichuan

Received:2025-01-02 Accepted:2025-05-11 Published:2025-06-19 Online:2025-06-19

摘要/Abstract

摘要：

【背景】种质资源是种业振兴的基础，对其保护和利用至关重要。然而，非洲猪瘟的爆发以及日益激烈的市场竞争导致中国本土猪群数量大幅下降，威胁到猪种质资源的遗传多样性，许多猪种已处于濒危状态。体细胞核移植（somatic cell nuclear transfer，SCNT）是一种有前途的恢复濒危猪种的策略，但供体细胞的衰老严重降低了猪SCNT的效率。环黄芪醇（cycloastragenol，CAG）在早期研究中展现出抗衰老作用。【目的】通过评估CAG对猪耳缘成纤维细胞（pig ear marginal fibroblasts，PEMFs）衰老相关表型的影响，探索CAG调控细胞衰老的分子机制，进一步探讨PEMFs作为SCNT供体细胞预处理的潜力。【方法】通过连续传代方法获得具有衰老表型的PEMFs，通过细胞增殖试验和流式细胞术检测衰老相关β-半乳糖苷酶（senescence-associated β-galactosidase，SA-β-Gal）以分析CAG对不同衰老程度PEMFs的处理浓度和时间。通过检测衰老蛋白p53表达水平和衰老相关分泌表型（senescence-associated secretory phenotype，SASP）的表达，结合细胞骨架免疫荧光染色，进一步评估CAG的抗衰老作用。结合表型分析与转录组学数据，揭示CAG延缓PEMFs衰老的潜在机制。通过统计SCNT胚胎的卵裂率与囊胚率，对SCNT囊胚总细胞数与滋养层细胞数量染色并统计，探究CAG预处理PEMFs对SCNT胚胎发育潜能的影响。【结果】SA-β-Gal检测试验和CCK-8细胞增殖试验表明，CAG的抗衰老作用与增殖抑制作用呈浓度与时间依赖性。经剂量-效应平衡分析，推荐使用100 μmol·L^-1 CAG处理P3/P6 PEMFs 24 h及P9 PEMFs 12 h。在该培养条件下，CAG能够显著降低PEMFs中SA-β-Gal活性、p53蛋白表达与部分SASP因子的分泌。此外，CAG处理可显著减少细胞表面积、黏着斑数量、应力纤维的厚度和数量，并重塑细胞骨架。转录组学分析及验证结果表明，CAG通过调节黏着斑和细胞外基质（extracellular matrix，ECM）信号等通路，进而影响细胞骨架的稳定性。在胚胎试验中，CAG处理的PEMFs的重构胚胎显示出更高的卵裂率、囊胚形成率及更高的囊胚总细胞数和滋养层细胞数，表明其早期胚胎发育的质量和潜力得到了显著改善。【结论】CAG能够有效降低供体细胞的衰老水平，重塑细胞骨架，显著提高SCNT效率和胚胎发育潜力。研究结果为解决猪种质资源保护和猪克隆效率低下等问题提供了新思路。

关键词: 环黄芪醇, 猪耳缘纤维细胞, 细胞衰老, 细胞骨架, 体细胞核移植, 胚胎发育

Abstract:

【Background】 Germplasm resources are the foundation of the revitalization of the swine breeding industry, and their protection and utilization are critical. However, the outbreak of African swine fever and increasing market competition have caused a significant decline in the number of local Chinese pig breeds, therefore threatening the genetic diversity of pig germplasm resources, with many breeds now facing endangerment. Somatic cell nuclear transfer (SCNT) is a promising strategy for restoring endangered pig breeds, but the aging of donor cells severely limits the efficiency of pig SCNT. Cycloastragenol (CAG) has demonstrated anti-aging effects in earlier studies. 【Objective】 This study aimed to evaluate the impact of CAG on aging-related phenotypes of pig ear marginal fibroblasts (PEMFs) and to explore the molecular mechanisms by which CAG regulates cellular senescence using RNA sequencing. Additionally, the potential of CAG as a pre-treatment for SCNT donor cells was further investigated. 【Method】 PEMFs exhibiting aging phenotypes were obtained through continuous passaging. Cell proliferation assays and flow cytometry were performed to detect Senescence-associated β-galactosidase (SA-β-Gal) activity, in order to analyze the optimal concentrations and treatment times of CAG for PEMFs at different stages of aging. The anti-aging effects of CAG were further assessed by measuring p53 protein levels and the expression of Senescence-associated secretory phenotype (SASP) markers, coupled with immunofluorescence staining of the cytoskeleton. By integrating phenotypic analysis with transcriptomic data, the potential mechanisms by which CAG slowed down PEMF senescence were elucidated. Additionally, SCNT embryo cleavage and blastocyst rates were statistically analyzed, followed by staining and quantification of the total cell number and trophectoderm cell number in SCNT blastocysts to investigate the effects of CAG-pretreated PEMFs on SCNT embryo developmental potential. 【Result】 SA-β-Gal assay and CCK-8 cell proliferation assay demonstrated that the anti-aging and proliferation-inhibitory effects of CAG were concentration- and time-dependent. Based on dose-effect balance analysis, the recommended treatment conditions were 100 μmol·L^-1 CAG for 24 hours in P3/P6 PEMFs and 12 hours in P9 PEMFs. Under these conditions, CAG significantly reduced SA-β-Gal activity, p53 protein expression, and the secretion of certain SASP factors in PEMFs. Additionally, CAG treatment led to a significant reduction in cell surface area, focal adhesion number, and stress fiber thickness and quantity, while also reorganizing the cytoskeleton. Transcriptomic analysis and subsequent validation results revealed that CAG modulates pathways such as focal adhesion and extracellular matrix (ECM) signaling, thereby affecting the stability of the cytoskeleton. In embryo culture experiments, PEMFs treated with CAG exhibited higher cleavage and blastocyst formation rates, as well as greater blastocyst total cell numbers and trophoblast cell counts, indicating a significant improvement in the quality and developmental potential of early embryos.【Conclusion】 CAG effectively delayed donor cell senescence, reorganized the cytoskeleton, and significantly enhanced SCNT efficiency and embryo developmental potential. This discovery offered a novel approach to addressing the challenges of pig germplasm conservation and low cloning efficiency.

Key words: cycloastragenol, porcine ear marginal fibroblasts, cell senescence, cytoskeleton, somatic cell nuclear transfer, embryonic development

周旗, 张亮, 潘雨, 涂志, 王峥, 刘航航, 鲜凌瑾, 夏运红, 潘红梅, 龙熙. 环黄芪醇对猪供体成纤维细胞衰老、细胞骨架与核移植胚胎早期发育的影响[J]. 中国农业科学, 2025, 58(12): 2453-2474.

ZHOU Qi, ZHANG Liang, PAN Yu, TU Zhi, WANG Zheng, LIU HangHang, XIAN LingJin, XIA YunHong, PAN HongMei, LONG Xi. Effects of Cycloastragenol on Cellular Senescence of Pig Donor Fibroblast, Cytoskeletal Dynamic, and Early Developmental Stage of Nuclear Transfer Embryo[J]. Scientia Agricultura Sinica, 2025, 58(12): 2453-2474.

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引物 Primer	引物序列 Primer sequence (5 ′-3 ′)	用途 Usage
β-actin F	GACCCAGATCATGTTCGAGACCT	SASP成分检测 SASP component detection
β-actin R	CGGAGTCCATCACGATGCCAG
p53 F	TCTGACTGTACCACCATCCACTA
p53 R	ACAAACACGCACCTCAAAGC
THBS1 F	CAAAGAGTTGGCCAGTGAGC
THBS1 R	ATGATGGGGCAGGACACTTT
IGFBP2 F	TCGTCTGGGCAGGGGTGCTACTG
IGFBP2 R	GCTGTGGTTTACTGCATCCG
IGFBP4 F	ACCGCAACGGCAACTTCCA
IGFBP4 R	TCCCGTCTTCCGGTCCACA
CXCL14 F	CGGCCAGCATGAGGCTCCTGAC
CXCL14 R	ACTTGCATTTGGACCCGTCCACGC
CXCL16 F	TCGCGGAGAATGTGGACGTGCTC
CXCL16 R	TCGTCTGGGCAGGGGTGCTACTG
MMP1 F	TGTTCTCACTCCAGGGAACC
MMP1 R	TTCCTCCAGGTCCATCAAAG
MMP2 F	TACACCTATACCAAGAACTTCCG
MMP2 R	TGTCCGCCAGATGAACCG
MMP3 F	GGCCTGCCCAAGTGGAGAAA
MMP3 R	GCGGAGTCACTTCCTCCCAG
TIMP2 F	GTAGTGATCAGGGCCAAAG
TIMP2 R	TTCTCTGTGACCCAGTCCAT
TIMP3 F	TCTGCAACTCCGACATCGTG
TIMP3 R	CGGATGCAGGCGTAGTGTT
PTGS2 F	AGACAGATCAGAAGCGAGGACC	转录组数据定量逆转录聚合酶联反应验证 Transcriptome data quantitative reverse transcription polymerase chain reaction validation
PTGS2 R	CATCATCAGACCAGGCACCA
SERPINB2 F	CAAACCAAAGGCAAAATCCC
SERPINB2 R	TTCATCTGGAAGCAACAGGAAC
FABP3 F	CACAAAGCACCTTCAAGAGCA
FABP3 R	ACAAGTTTGCCTCCATCCAGT
LGALS3 F	GCTGGATAATAACTGGGGAAGG
LGALS3 R	ACAAGTGAGCATCATTGACCG
COL6A3 F	GTATCCTTCCGCTTGGTTCAT
COL6A3 R	AGTTGGCTTCTGCCTCCCTA
GAPDH F	CAGCAATGCCTCCTGTACCA
GAPDH R	GACGTGGTGGGCGATGTTG
S100A2 F	AGGGGAGAAGGTAGATGAGGAA
S100A2 R	AGTGATGAGGGCCAGGAAAA
LUM F	TAGGCCCACTCCCCAAATC
LUM R	CTCTAAAAGCCGCTGAAACG
CHI3L1 F	GCCAAACTACAGGACACCACA
CHI3L1 R	CTCAGCACATAGCTCACAGCAT
CXCL8 F	TGCACTTACTCTTGCCAGAACTG
CXCL8 R	CAAACTGGCTGTTGCCTTCTT

环黄芪醇对猪供体成纤维细胞衰老、细胞骨架与核移植胚胎早期发育的影响

Effects of Cycloastragenol on Cellular Senescence of Pig Donor Fibroblast, Cytoskeletal Dynamic, and Early Developmental Stage of Nuclear Transfer Embryo

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