彭波半细毛羊冷冻保存睾丸组织支持细胞分离、纯化及鉴定

doi:10.16431/j.cnki.1671-7236.2025.01.022

摘要/Abstract

摘要： 【目的】睾丸支持细胞(Sertoli cells,SCs)位于生精小管内上皮,为不同分化阶段雄性生殖细胞发育提供物理构架和能量物质支持,对精子发生过程产生至关重要的作用。试验旨在研究新鲜绵羊睾丸组织样品获取不便的情况下,从冷冻保存的彭波半细毛羊睾丸组织分离支持细胞的技术方法。【方法】将冷冻保存液预处理后冻存的彭波半细毛羊睾丸组织进行复苏,采用苏木精-伊红(HE)染色检测复苏后睾丸组织形态,利用组合酶消化法、差速贴壁法进行睾丸组织支持细胞分离纯化培养,观察细胞生长规律,绘制生长曲线,采用RT-PCR和免疫荧光染色(IF)技术进行睾丸支持细胞特异性基因鉴定。【结果】冻存复苏睾丸组织曲细精索及睾丸间质保存完好。组合酶消化后能获得满足试验需要的生精上皮细胞混悬液,分离培养2~4 h后支持细胞贴壁,形态呈梭形或不规则多边形,培养3~4 d后汇合,其中培养1~2 d生长速度缓慢,培养3~6 d进入对数期,增殖速度加快,培养7~8 d后增殖速度下降。RT-PCR检测显示,GNDF、WT1、ABP、SOX9基因均在彭波半细毛羊睾丸支持细胞中特异性表达;免疫荧光染色显示,特异性抗体GATA4、Vimentin免疫阳性。【结论】经冻存液预处理的冷冻保存睾丸组织复苏后,可分离出满足试验条件的原代支持细胞,应用于科学试验研究。

关键词: 彭波半细毛羊; 冷冻保存; 睾丸支持细胞; 分离鉴定; 细胞培养

Abstract: 【Objective】 Testicular Sertoli cells (SCs) are located in the epithelium of seminiferous tubules,which provide physical framework and energy material support for the development of male germ cells at different stages of differentiation,and are very important for spermatogenesis.The purpose of this experiment was to study the technical method of separating SCs from frozen testicular tissue in Pengbo Semi-fine wool sheep under the condition that it was inconvenient to obtain fresh testicular tissue samples in sheep. 【Method】 The cryopreserved testicular tissue of Pengbo Semi-fine wool sheep was resuscitated by cryopreservation solution.The morphology of the recovered testicular tissue was detected by hematoxylin-eosin (HE) staining.The testicular SCs were isolated,purified and cultured by combined enzyme digestion and differential adherence method,the growth pattern was observed,and the growth curve was drawn.The specific genes in testicular SCs were identified by RT-PCR and immunofluorescence staining (IF). 【Result】 The spermatic cord and leydig of frozen-thawed testis were well preserved.Spermatogenic epithelial cell suspension meeting the experimental requirements could be obtained after digestion with combined enzymes.After 2-4 h of separation and culture,the cells adhered to the wall,and the shape was spindle or irregular polygon.After 3-4 d of culture,they converged,and the growth rate was slow after 1-2 d of culture.The proliferation rate was accelerated after 3-4 d of culture,and,the proliferation rate was reduced after 7-8 d of culture.RT-PCR detection showed that GNDF,WT1,ABP and SOX9 genes were specifically expressed in testicular SCs of Pengbo Semi-fine wool sheep,and IF results showed that the specific antibodies GATA4 and Vimentin were immune positive. 【Conclusion】 The primary SCs that met the experimental conditions could be isolated and applied to scientific experimental research after the cryopreservation of testicular tissue pretreated with antifreeze was revived.

Key words: Pengbo Semi-fine wool sheep; cryopreservation; testicular Sertoli cells; separation and identification; cell culture

中图分类号:

S814.8

刘海霞, 王健, 平措班旦, 朱爱文, 德庆卓嘎, 王军, 格桑加措. 彭波半细毛羊冷冻保存睾丸组织支持细胞分离、纯化及鉴定[J]. 中国畜牧兽医, 2025, 52(1): 249-257.

LIU Haixia, WANG Jian, PINCUO Bandan, ZHU Aiwen, DEQING Zhuoga, WANG Jun, GESANG Jiacuo. Isolation,Purification and Identification of Sertoli Cell in Cryopreserved Testis of Pengbo Semi-fine Wool Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 249-257.

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