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2025年8月12日 星期二
农业生物技术学报  2021, Vol. 29 Issue (4): 631-640    DOI: 10.3969/j.issn.1674-7968.2021.04.002
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
AMPK对饥饿诱导的绵羊睾丸间质细胞自噬和凋亡的作用研究
汪棋*, 庞静*, 蔡玉, 王锋, 张艳丽**
南京农业大学 动物科技学院/江苏省家畜胚胎工程实验室,南京 210095
Effect of AMPK on Starvation-induced Autophagy and Apoptosis of Sheep (Ovis aries) Leydig Cells
WANG Qi*, PANG Jing*, CAI Yu, WANG Feng, ZHANG Yan-Li**
College of Animal Science and Technology/Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
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摘要 雄性动物的繁殖能力受营养和能量代谢的影响,而AMP激活的蛋白激酶(AMP-activated protein kinase, AMPK)作为能量的感受器分子,在其生殖功能中起着重要作用,本实验旨在研究AMPK对饥饿诱导的绵羊(Ovis aries)睾丸间质细胞(leydig cells, LCs)自噬和凋亡的作用。采用绵羊LCs为实验材料,培养至第4代,分别用无血清培养液培养0、3、6、12、24 h后,利用Cell Counting Kit-8 (CCK8)试剂盒检测细胞活力,筛选合适的处理时间。饥饿12 h,通过透射电镜和流式细胞术观察LCs的自噬和凋亡,qRT-PCR和蛋白免疫印迹(Western blot, WB)方法分别检测自噬及凋亡相关的基因和蛋白表达变化。结果表明,与对照组相比,饥饿诱导的LCs中自噬体增多,细胞凋亡率显著升高(P<0.05),并且自噬相关基因程序性死亡受体-1 (programmed cell death-1, Beclin1)和微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain 3, LC3)等基因表达量显著升高(P<0.05),Beclin1蛋白表达和LC3Ⅱ/LC3Ⅰ比率显著升高(P<0.05),促凋亡相关基因Bcl-2相关X蛋白(Bcl-2 associated X protein, BAX)等基因表达显著升高(P<0.05),抗凋亡相关基因B淋巴细胞瘤-2 (B-cell lymphoma-2, Bcl-2)基因表达极显著降低(P<0.01),BAX/Bcl-2蛋白表达比率升高;AMP依赖的蛋白激酶(AMP-activated protein kinase, AMPK)/哺乳动物雷帕霉素蛋白激酶(mammalian target of rapamycin, mTOR)/UNC-51样激酶1 (UNC-51-like kinase 1, ULK1)通路的关键基因的表达均极显著上升(P<0.01)。综上,饥饿诱导绵羊LCs,能够激活AMPK/mTOR/ULK1通路并促进细胞自噬和凋亡的发生。本研究可为进一步探究AMPK对睾丸发育的影响提供理论参考。
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汪棋
庞静
蔡玉
王锋
张艳丽
关键词 饥饿绵羊睾丸间质细胞细胞自噬细胞凋亡AMPK/mTOR/ULK1通路    
Abstract:Nutrition and energy metabolism affect the reproductive ability of male animals, and AMP-activated protein kinase (AMPK) plays an important role in its reproductive function as an energy receptor molecule. This study aims to study the effect of AMPK on autophagy and apoptosis of sheep (Ovis aries) leydig cells (LCs) induced by starvation. Sheep LCs were used as experimental materials and cultivated to the fourth generation. After 0, 3, 6, 12, and 24 h in serum-free medium, used cell counting kit-8 to detect cell viability and selected appropriate treatment time. After starving for 12 h, the autophagy and apoptosis of LC were observed by transmission electron microscopy and flow cytometry, qRT-PCR and Western blot were used to detect the changes in gene and protein expression related to autophagy and apoptosis. The results showed that compared with the control group, there were more autophagosomes in starvation-induced LCs, and the apoptosis rate was significantly higher (P<0.05), programmed cell death 1 (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3) and other genes expression level were significantly increased (P<0.05), Becline1 protein expression and LC3Ⅱ/LC3Ⅰ ratio were significantly increased (P<0.05), pro-apoptosis related genes Bcl-2 related X protein (BAX) and other genes expression level were significantly increased (P<0.05), anti-apoptosis related genes B-cell lymphoma 2 (Bcl-2) gene expression level was significantly reduced (P<0.01), and the ratio of BAX/Bcl-2 protein was increased; AMPK/mammalian target of rapamycin (mTOR)/UNC-51-like kinase 1 (ULK1) pathway key genes expression level were increased extremely significantly (P<0.01). In summary, starvation-induced sheep LCs could activate the AMPK/mTOR/ULK1 pathway and promote the occurrence of autophagy and apoptosis. This research provides a theoretical reference for further exploring the impact of AMPK on testicular development.
Key wordsStarvation    Sheep leydig cells    Autophagy    Apoptosis    AMPK/mTOR/ULK1 pathway
收稿日期: 2020-06-24      出版日期: 2021-04-01
ZTFLH:  S8  
基金资助:国家自然科学基金(31872359); 江苏省肉羊产业技术体系建设专项(JATS[2020]427)
通讯作者: ** zhangyanli@njau.edu.cn   
作者简介: *同等贡献作者
引用本文:   
汪棋, 庞静, 蔡玉, 王锋, 张艳丽. AMPK对饥饿诱导的绵羊睾丸间质细胞自噬和凋亡的作用研究[J]. 农业生物技术学报, 2021, 29(4): 631-640.
WANG Qi, PANG Jing, CAI Yu, WANG Feng, ZHANG Yan-Li. Effect of AMPK on Starvation-induced Autophagy and Apoptosis of Sheep (Ovis aries) Leydig Cells. 农业生物技术学报, 2021, 29(4): 631-640.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.04.002     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I4/631
 
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