HAN Le,ZHANG Guomin,YANG Hua,et al.Goat testis sertoli cell culture and autophagy dual-fluorescence reporter system construction[J].Journal of Nanjing Agricultural University,2018,41(1):132-139.[doi:10.7685/jnau.201612007]





Goat testis sertoli cell culture and autophagy dual-fluorescence reporter system construction
韩乐1 张国敏2 杨花1 庞静1 万永杰1 姚晓磊1 王锋1 张艳丽1
1. 南京农业大学动物科技学院/江苏省家畜胚胎工程实验室, 江苏 南京 210095;
2. 南京农业大学动物医学院, 江苏 南京 210095
HAN Le1 ZHANG Guomin2 YANG Hua1 PANG Jing1 WAN Yongjie1 YAO Xiaolei1 WANG Feng1 ZHANG Yanli1
1. College of Animal Science and Technology/Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
autophagytestis sertoli cellmicrotuble-associated protein Ⅰ light chain 3(LC3)goat
[目的]本试验旨在建立一个实时追踪山羊细胞自噬发生状况的工具,以探讨细胞自噬对睾丸支持细胞(SC)和生殖细胞的影响。[方法]通过PCR法扩增mCherry-EGFP-LC3基因序列1 884 bp,将其导入真核表达载体pEX-3中,得到重组质粒pmCherry-EGFP-LC3;将pmCherry-EGFP-LC3及对照质粒pEX-3分别转染体外分离培养的山羊SC,饥饿处理12 h后用荧光显微镜观察融合蛋白在细胞中的表达,用RT-qPCR和Western blot检测细胞中自噬相关基因和蛋白表达变化,用透射电镜观察细胞自噬体的形成。[结果]成功构建了双荧光自噬报告载体,该载体能在山羊SC中表达,荧光显微镜下可见绿色荧光和红色荧光表达;饥饿处理后,该载体可以指示细胞中自噬泡的形成过程,且细胞自噬相关标记Beclin1 mRNA以及LC3 Ⅱ/LC3Ⅰ mRNA比值和蛋白水平均显著上调,并能观察到初始自噬体的形成。[结论]获得的山羊SC可以作为后续试验的细胞模型;构建的双荧光pmCherry-EGFP-LC3自噬报告载体可为进一步研究山羊SC的自噬发生提供有效的工具。
[Objectives]This study aims to establish a real-time tool to investigate the goat cell autophagy,and to further explore the cell autophagy effect on testis sertoli cells(SC) and germ cells. [Methods]mCherry-EGFP-LC3 gene sequence were amplified by PCR,which was 1 884 bp and inserted into pEX-3 eukaryotic expression vector,then a recombinant plasmid pmCherry-EGFP-LC3 was constructed.The pmCherry-EGFP-LC3 and control plasmid pEX-3 were transfected into in vitro isolated goat testis sertoli cells,respectively,and the cells were then starved for 12 h,fluorescent microscope was used to observe fusion protein expression in cells,RT-qPCR and Western blot were used to detect cell autophagy related gene and protein expression changes,and the formation of cell autophagy body was further detected by TEM. [Results]The double fluorescent autophagy report plasmid were successfully constructed,which can be expressed in goat testis sertoli cells,green and red fluorescence can be detected under fluorescence microscopy,after starvation,the construct could indicate the formation process of autophagy bubbles in the cell,and autophagy related markers Beclin1 and LC3Ⅱ/LC3Ⅰ ratio of mRNA and protein levels were all significantly higher than those of the non-starved group,and the initial formation of autophagy body was also observed. [Conclusions]In total,the double fluorescent autophagy reporter vector constructed in this study can provide an effective tool for the further study of autophagy of goat SC.


[1] Wang S,Xia P,Rehm M,et al. Autophagy and cell reprogramming[J]. Cellular and Molecular Life Sciences,2015,72(9):1699-1713.
[2] Cheng C Y,Mruk D D. The biology of spermatogenesis:the past,present and future[J]. Philosophical Transactions of the Royal Society of London,2010,365(1546):1459-1463.
[3] Zheng Y,Zhang Y,Qu R,et al. Spermatogonial stem cells from domestic animals:progress and prospects[J]. Reproduction,2014,147(3):65-74.
[4] Rubinsztein D C,Mariño G,Kroemer G. Autophagy and aging[J]. Cell,2011,146(5):682-695.
[5] Eid N,Ito Y,Otsuki Y. Enhanced mitophagy in sertoli cells of ethanol-treated rats:morphological evidence and clinical relevance[J]. Journal of Molecular Histology,2012,43(1):71-80.
[6] Yefimova M G,Messaddeq N,Harnois T,et al. A chimerical phagocytosis model reveals the recruitment by sertoli cells of autophagy for the degradation of ingested illegitimate substrates[J]. Autophagy,2013,9(5):653-666.
[7] 马泰,孙国平,李家斌. 细胞自噬的研究方法[J]. 生物化学与生物物理进展,2012,39(3):204-209. Ma T,Sun G P,Li J B. Methods for autophagy detection[J]. Progress in Biochemistry and Biophysics,2012,39(3):204-209(in Chinese with English abstract).
[8] Kabeya Y,Mizushima N,Ueno T,et al. LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J]. EMBO Journal,2000,19(21):5720-5728.
[9] Kimura S,Fujita N,Noda T,et al. Chapter 1 monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J]. Methods in Enzymology,2009,452:1-12.
[10] 王婉,张庆,赵润鹏,等. 稳定表达RFP-GFP-LC3的RAW264.7细胞株的建立[J]. 细胞与分子免疫学杂志,2015,31(9):1175-1178. Wang W,Zhang Q,Zhao R P,et al. Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3[J]. Chiese Journal of Cellular and Molecular Immunology,2015,31(9):1175-1178(in Chinese with English abstract).
[11] Wang Y,Zheng W,Bian X,et al. Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells[J]. Toxicology Letters,2014,226(2):182-191.
[12] Zhang G,Wan Y,Zhang Y,et al. Expression of mitochondria-associated genes(PPARGC1A,NRF-1,BCL-2 and BAX) in follicular development and atresia of goat ovaries[J]. Reproduction in Domestic Animals,2015,50(3):465-473.
[13] 韩妲丽,王晓云,邢新,等. 睾丸支持细胞的基本生物学特性[J]. 生殖医学杂志,2008,17(5):361-365. Han D L,Wang X Y,Xing X,et al. Basic biological characteristics of sertoli cells[J]. Journal of Reproductive Medicine,2008,17(5):361-365(in Chinese with English abstract).
[14] Lahlou N,Fennoy I,Carel J C,et al. Inhibin B and anti-mullerian hormone,but not testosterone levels,are normal in infants with nonmosaic Klinefelter syndrome[J]. Journal of Clinical Endocrinology and Metabolism,2004,89(4):1864-1868.
[15] 黄东晖,赵虎,田永红,等. 大鼠睾丸支持细胞的分离、纯化和鉴定[J]. 解剖学报,2007,38(2):246-249. Huang D H,Zhao H,Tian Y H,et al. Isolation,purification and identification of sertoli cells from rat tesis[J]. Acta Anatomica Sinica,2007,38(2):246-249(in Chinese with English abstract).
[16] 刘欢欢,余树民,夏娟,等. 仔猪睾丸支持细胞的体外生物学特性研究[J]. 中国细胞生物学学报,2015,37(8):1122-1128. Liu H H,Yu S M,Xia J,et al. Characterization of sertoli cells from prepubertal porcine testis in vitro[J]. Chinese Journal of Cell Biology,2015,37(8):1122-1128(in Chinese with English abstract).
[17] Agarwal S,Tiwari S K,Seth B,et al. Activation of autophagic flux against xenoestrogen bisphenol-A-induced hippocampal neurodegeneration via AMP kinase(AMPK)/mammalian target of rapamycin(mTOR) pathways[J]. Journal of Biological Chemistry,2015,290(34):21163.
[18] 李烈川,申明,宁彩波,等. 过氧化氢诱导猪卵巢颗粒细胞自噬及其对凋亡的影响[J]. 南京农业大学学报,2016,39(5):814-818. DOI:10.7685/jnau.201601001. Li L C,Shen M,Ning C B,et al. Induction of granulosa cell autophagy and effects in apoptosis by hydrogen peroxide in porcine ovaries[J]. Journal of Nangjing Agricultural University,2016,39(5):814-818(in Chinese with English abstract).
[19] Mauvezin C,Ayala C,Braden C R,et al. Assays to monitor autophagy in Drosophila[J]. Methods,2014,68(1):134-139.
[20] Singh N,Ojha S,Bhattacharya A,et al. Stable transfection and continuous expression of heterologous genes in Entamoeba invadens[J]. Molecular and Biochemical Parasitology,2012,184(1):9-12.
[21] Shpilka T,Weidberg H,Pietrokovski S,et al. Atg8:an autophagy-related ubiquitin-like protein family[J]. Genome Biology,2011,12(7):81-89.
[22] Joffre C,Dupont N,Hoa L,et al. The Pro-apoptotic STK38 kinase is a new beclin1 partner positively regulating autophagy[J]. Current Biology,2015,25(19):2479-2492.
[23] Kim D E,Kim Y,Cho D H,et al. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase[J]. Molecules and Cells,2015,38(2):138-144.
[24] Karim M R,Kawanago H,Kadowaki M. A quick signal of starvation induced autophagy:transcription versus post-translational modification of LC3[J]. Analytical Biochemistry,2014,465:28-34.
[25] 赵秀,朱小龙,何亚文,等. 酿酒酵母中磷脂合成相关基因突变对细胞自噬和液泡形态的影响[J]. 南京农业大学学报,2015,38(1):70-77. DOI.10.7685/j.issn.1000-2030.2015.01.011. Zhao X,Zhu X L,He Y W,et al. CDS1 is required for proper vacuole morphology but not for autophagy in Saccharomyces cerevisiae[J]. Journal of Nanjing Agricultural University,2015,38(1):70-77.(in Chinese with English abstract).


 LI Liechuan,SHEN Ming,NING Caibo,et al.Induction of granulosa cell autophagy and effects in apoptosis by hydrogen peroxide in porcine ovaries[J].Journal of Nanjing Agricultural University,2016,39(1):814.[doi:10.7685/jnau.201601001]
 XU Changmeng,WANG Zhijian,WANG Mi,et al.The study on autophagy induced by pseudorabies virus in PK-15 cells(PRV)[J].Journal of Nanjing Agricultural University,2018,41(1):701.[doi:10.7685/jnau.201801015]
 GAO Xiquan,RUAN Xinsen,MA Liang,et al.Regulation of programmed cell death responses in plant innate immunity[J].Journal of Nanjing Agricultural University,2018,41(1):971.[doi:10.7685/jnau.201803061]
 DUAN Yinping,XUE Ying,ZHOU Shuang,et al.Effect of Chemerin on apoptosis and autophagy in bovine granulosa cells[J].Journal of Nanjing Agricultural University,2019,42(1):145.[doi:10.7685/jnau.201801017]
 CHU Xiaoyan,ZHANG Yafei,ZHU Lei,et al.Effect of autophagy on piglet hippocampal nerve cells damaged by deoxynivalenol[J].Journal of Nanjing Agricultural University,2019,42(1):160.[doi:10.7685/jnau.201804028]
 FENG Nannan,WANG Bingjie,ZHU Qihang,et al.Effects of zearalenone on the autophagy and cell cycle of sertoli cells by PI3K/Akt/mTOR pathway[J].Journal of Nanjing Agricultural University,2018,41(1):708.[doi:10.7685/jnau.201712033]


更新日期/Last Update: 1900-01-01