山羊睾丸类器官的培养与鉴定  

Culture and Characterization of Testicular Organoid in Goats(Capra hircus)

作　　者：徐文静 吴文萍 杨玉梅 李娜[1,2] 华进联[1,2] XU Wen-Jing;WU Wen-Ping;YANG Yu-Mei;LI Na;HUA Jin-Lian(College of Veterinary Medicine,Northwest A&F University/Shaanxi Stem Cell Engineering Technology Center,Yangling 712100,China;Key Laboratory of Livestock Biology,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物医学院/陕西省干细胞工程技术中心,杨凌712100 [2]西北农林科技大学家畜生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2024年第9期2173-2180,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32372970,32072806);国家重点研发计划(2023YFF100904);陕西省农业核心技术攻关(2024NYGG005-6,20230978);内蒙古自治区揭榜挂帅项目(2022JBGUS0025)。

摘　　要：类器官是一种在结构和功能上与机体原器官相似的组织替代品。目前一些类器官培养体系已经比较完善,但睾丸类器官培养尚处于探索阶段。本研究无菌采取莎能奶山羊(Capra hircus)睾丸组织,用混合酶消化法分离培养睾丸细胞。用含低熔点琼脂糖的混合培养基在三维环境中培养睾丸细胞。采用光学显微镜和免疫荧光检测技术对形成的类器官进行记录和评价,比较不同来源的睾丸细胞、不同数量的睾丸细胞和不同浓度的低熔点琼脂糖对睾丸类器官的培养效果。进一步通过脂多糖(lipopolysaccharide, LPS)感染验证睾丸类器官的模型功能。结果表明1%低熔点琼脂糖、含量为2×10^(5)~3×10^(5)的细胞更有利于山羊睾丸类器官的形成。形成的类器官具有生精小管样结构,并且精原干细胞和支持细胞均匀分布其中。LPS感染的睾丸类器官较正常的类器官小,并有细胞碎片,同时伴有炎性因子的显著增加,表明该类器官能够响应外界刺激,一定程度上模拟真实器官的免疫反应,可作为体外研究睾丸疾病的模型。本研究不仅推动了睾丸类器官培养技术的发展,而且为生殖生物学、疾病模型建立、药物筛选、再生医学以及个性化医疗等多个领域提供了重要的科学依据和实验平台。Organoid is a tissue substitute that is structurally and functionally similar to the origin organ.At present,many organoid culture systems have been established,however testicular organoid culture is still in the exploratory stage.In this study,testicular tissues of Saanen dairy goats(Capra hircus)were taken,and testicular cells were isolated and cultured by mixed enzyme digestion.Testicular cells were cultured in three dimensions using a mixed medium containing low melting point agarose.The formed organoids were evaluated using light microscope and immunofluorescence detection techniques,and the culture effects of different sources of testicular cells,different numbers of testicular cells and different concentrations of low melting point agarose on testicular organoids were compared.Finally,the model function of the testicular organoid was verified by lipopolysaccharide(LPS)infection.The result showed that cells ranging from 2×10^(5) to 3×10^(5),in 1%low-melting-point agarose were more favorable for the formation of goat testicular organs in goats.The organoids formed possess seminiferous tubule-like structures,with spermatogonial stem cells and Sertoli cells evenly distributed within.Testicular organoid infected with LPS were smaller than normal organoids,exhibited cellular debris,and were accompanied by a significant increase in inflammatory factors,indicating that these organoids could respond to external stimuli and simulate the immune response of organs to a certain extent,making them a suitable model for the in vitro study of testicular diseases.This study not only promotes the development of testicular organoid culture technology,but also provides an important scientific basis and experimental platform for a variety of fields,including reproductive biology,disease modeling,drug screening,regenerative medicine,and personalized medicine.

关 键 词：睾丸类器官 低熔点琼脂糖胶 山羊 

分 类 号：S827[农业科学—畜牧学] Q813.1[农业科学—畜牧兽医] Q5-33[生物学—生物工程]