山羊睾丸支持细胞的培养与双荧光自噬报告载体的建立

Goat testis sertoli cell culture and autophagy dual-fluorescence reporter system construction

[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p EX-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LC3Ⅱ/LC3ⅠmRNA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 ceils, 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 Beclinl and LC311/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.