褪黑素的添加减轻猪精原干细胞的氧化损伤

The Addition of Melatonin Reduces the Oxidative Damage of Pig Spermatogonial Stem Cells

旨在研究褪黑素(melatonin,MT)对体外培养猪精原干细胞(spermatogonial stem cells,SSCs)的作用机制。本研究采集3头7日龄健康大白公猪睾丸,利用差速贴壁法获得SSCs。后经形态学观察、碱性磷酸酶染色、标记基因检测及免疫荧光染色鉴定后以SSCs作为试验材料,设置MT浓度梯度(0、50、250、500、1000μmol·mL^(-1))组处理SSCs,每组设3个重复(n=3),空白对照组加入0.1%DMSO处理,分别检测添加MT后猪SSCs的细胞活力、活性氧(reactive oxygen species,ROS)水平、谷胱甘肽(glutathione,GSH)含量及凋亡基因表达变化。结果显示:1)分离的克隆团细胞具有SSCs的生长特性,可被碱性磷酸酶染色并表达干细胞标志基因OCT4、SOX2和SSCs标志基因NANOG、PLZF、UCHL1;2)50μmol·mL^(-1)以上的MT在处理48 h后可显著提高SSCs的细胞活力(P<0.05);3)MT可显著降低猪SSCs内ROS水平(P<0.05),极显著增加细胞内GSH含量(P<0.01);4)MT可显著抑制猪SSCs内凋亡蛋白Bax和Caspase3的表达(P<0.05)。MT具有清除猪SSCs中的ROS,提高总GSH含量,抑制凋亡基因表达进而提高细胞活力的作用,可为养殖过程中提高公猪繁殖性能提供参考。

The aim of this study was to explore the effect mechanism of melatonin(MT)on porcine spermatogonial stem cells(SSCs)in vitro culture.The SSCs in the testicles of 37-day-old healthy Large White pigs were isolated by differential adhesion method.After morphological observation,alkaline phosphatase staining,marker gene detection and immunofluorescence staining identification,the SSCs were used as experimental materials,the MT concentration gradient(0,50,250,500,1000μmol·mL^(-1))groups were set up to treat SSCs,each group had 3 replicates(n=3),and the blank control group was treated with 0.1%DMSO.The cell viability,reactive oxygen species(ROS),glutathione(GSH)content and apoptosis gene expression were detected after MT treatment.The results showed that:1)The isolated clonal colony cells had the growth characteristics of SSCs,which could be stained by alkaline phosphatase and the stem cell marker genes OCT4,SOX2 and SSCs marker genes NANOG,PLZF,UCHL1 were expressed;2)After treated with more than 50μmol·L^(-1) MT for 48 h,the cell viability of SSCs were significantly improved(P<0.05);3)MT could significantly reduce the level of ROS in pig SSCs(P<0.05),and extremely significantly increase the content of intracellular GSH(P<0.01);4)MT could significantly inhibit the expression of apoptosis proteins Bax and Caspase3 in pig SSCs(P<0.05).These results indicate that MT has the functions of removing ROS in pig SSCs,increasing GSH content,inhibiting the expression of apoptotic genes and improving cell viability,which can provide a reference for enhancing the reproductive performance of boars during breeding.