抑制猪支持细胞外泌体对精原干细胞自我更新及分化的影响  

Effects of inhibition of porcine Sertoli cells exosomes on self-renewal and differentiation of porcine spermatogonial stem cells

作　　者：田海瑞 汪晶晶[1] 李晓晓[1] 张丹琛 邹康 TIAN Hairui;WANG Jingjing;LI Xiaoxiao;ZHANG Danchen;ZOU Kang(Stem Cell Research and Translation Center/Germline Stem Cells and Microenvironment Laboratory,College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学干细胞研究与转化中心/动物科技学院生殖干细胞与微环境实验室,江苏南京210095

出　　处：《南京农业大学学报》2023年第5期898-906,共9页Journal of Nanjing Agricultural University

基　　金：2021江苏省特聘教授项目;江苏省种业振兴项目(JBGS〔2021〕024)。

摘　　要：[目的]本文利用体外模型检测猪支持细胞分泌外泌体的能力及其对猪精原干细胞(porcine spermatogonial stem cells,pSSC)自我更新及分化状态的影响。[方法]通过两步酶消化法分离猪支持细胞和pSSC并建立猪支持细胞-pSSC体外共培养模型,抑制猪支持细胞外泌体的分泌,检测pSSC自我更新及分化指标,研究支持细胞外泌体对pSSC命运的影响。[结果]分离得到的支持细胞的Wilm肿瘤蛋白1(WT1)阳性率为(87.1±0.02)%,整合素β-1蛋白(ITGB1)阳性率为(94.2±0.01)%,分离得到pSSC的早幼粒细胞白血病锌指蛋白(PLZF)阳性率为(80.6±0.02)%;通过Western blot分析、免疫荧光检测证实支持细胞外泌体的存在;证实20μmol·L^(-1)GW4869可以抑制外泌体分泌且不损害支持细胞;GW4869处理后的支持细胞与pSSC共培养发现试验组较对照组pSSC状态差异较为明显,且RT-PCR结果显示猪精原干细胞PLZF基因表达水平显著升高;肥大/干细胞生长因子受体基因(KIT)、胸腺细胞抗原1基因(THY1)、死盒解旋酶4基因(DDX4)表达水平显著降低;Western blot结果显示猪精原干细胞蛋白基因产物9.5(PGP9.5)蛋白表达水平升高,DDX4蛋白表达水平降低。[结论]体外培养时支持细胞分泌的外泌体对pSSC自我更新和分化均具有调控作用,暗示外泌体是支持细胞调控精原干细胞命运的一种重要方式,这为种公猪生殖力的改善提供了新思路。[Objectives]This study investigates the capability of exosome secretion by Sertoli cells and its influence on the self-renewal and differentiation status of porcine spermatogonial stem cells(pSSC)using an in vitro model.[Methods]The two-step enzyme digestion method was employed to isolate Sertoli cells and pSSC,and an in vitro co-culture model of Sertoli cells and pSSC was established.Suppression of exosome secretion by Sertoli cells was achieved to examine the effects on pSSC self-renewal and differentiation markers,thereby studying the impact of Sertoli cell-derived exosome on the fate of pSSC.[Results]The isolated Sertoli cells exhibited a Wilm tumor 1(WT1)positive rate of(87.1±0.02)%and an intorgrinβ-1(ITGB1)positive rate of(94.2±0.01)%.The isolated pSSC showed a promyelocytic lenkaemia zinc finger protein(PLZF)positive rate of(80.6±0.02)%.Presence of exosome secreted by Sertoli cells was confirmed through Western blot analysis and immunofluorescence assays.Treatment with 20μmol·L^(-1)GW4869 effectively inhibited exosome secretion without compromising Sertoli cells.Co-culture of pSSC with Sertoli cells treated with the inhibitor revealed significant differences in pSSC status compared to the control group.RT-PCR results showed a significant increase in the expression of the PLZF gene,while expression levels of receptor tyrosine kinase gene(KIT),thymus cell antigen 1 gene(THY1),and dead box 4 gene(DDX4)were significantly reduced.Western blot analysis indicated that levels of protein gene product 9.5(PGP9.5)protein expression elevated and DDX4 protein expression level decreased in pSSC co-cultured with inhibitor-treated Sertoli cells.[Conclusions]Exosomes secreted by Sertoli cells had a regulatory role in both self-renewal and differentiation of pSSC in vitro.This finding implied that exosome represented an important means through which Sertoli cells influence the fate of spermatogonial stem cells,offering novel insights for enhancing boar reproductive performance and fertility improvement.

关 键 词：猪支持细胞 精原干细胞 外泌体 

分 类 号：S828.9[农业科学—畜牧学]