小鼠精原干细胞体外培养体系建立及功能鉴定

doi:10.11843/j.issn.0366-6964.2021.02.013

摘要/Abstract

摘要： 旨在通过探索ICR品系小鼠精原干细胞（spermatogonial stem cells，SSCs）的体外稳定培养，为别的品系小鼠和大动物的SSCs体外培养提供参考。本研究采集6～8日龄ICR乳鼠的睾丸，用两步酶消化法（胶原酶和胰蛋白酶）和差速贴壁法来纯化SSC细胞。用不同的饲养层（小鼠胎儿成纤维细胞（mouse embryonic fibroblasts，mEF）作为饲养层和层黏连蛋白与多聚赖氨酸联合铺板），不同的培养基（StemPro-34培养基和DMEM培养基），添加不同的生长因子（GDNF、LIF、EGF、bFGF、IGF1）来观察体外SSCs的生长。将传代至第6代的SSCs用免疫染色法和分子检测法来评估细胞增殖。最后将稳定增殖的SSCs移植到受体睾丸中进行功能鉴定。结果显示，通过两步酶消化法和差速贴壁法获得纯度约79%以上的SSCs细胞；mEF作为饲养层，StemPro-34作为培养基，组合添加GDNF、LIF、EGF、bFGF、IGF1生长因子能促进SSCs的体外长期培养；SSCs克隆集落用碱性磷酸酶（alkaline phosphatase，AKP）染色、PLZF和UCHL1蛋白免疫荧光检测均显示为阳性，用反转录聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）和荧光实时定量PCR （quantitative real-time PCR，qRT-PCR）检测显示，克隆集落高表达多能基因和自我更新基因，说明体外培养的SSCs实现了稳定增殖；SSCs （带有报告基因EGFP）移植到受体睾丸后，受体附睾中精子头部发出绿光，证明精子为供体SSCs来源。研究表明，本试验中的培养体系适用于ICR品系小鼠的体外培养，且稳定培养后的SSCs具有正常生物学功能。本研究为别的品系小鼠和大动物SSCs的体外培养提供了参考和借鉴。

关键词: SSCs, 体外培养, 细胞增殖, 功能鉴定

Abstract: The objective of the present study was to provide a reference for in vitro culture of spermatogonial stem cells (SSCs) for other strain mice and megafauna by exploring the SSCs stable culture from ICR strain mice. Testes were harvested from 6-8 days old postpartum ICR male pups and digested using a two-step enzymatic (collagenase and trypsin) digestion protocol, then SSCs were purified by differential adherent method. The growth of SSCs in vitro culture was detected by using different feeder layers (mouse embryonic fibroblasts as feeder layer or laminin combined with polylysine), different media (StemPro-34 medium or DMEM medium) and different growth factors(recombinant rat glial-cell-line-derived neurotrophic factor (GDNF), recombinant mouse leukemia inhibitory factor (LIF), epidermal growth factor (EGF), recombinant human basic fibroblast growth factor (bFGF), insulin-like growth factors-1 (IGF1)). Proliferation of SSCs in the F6 was evaluated by immunostaining and molecular detection. Finally, stable proliferative SSCs in vitro were transplanted into recipient testes for functional identification. The results showed that SSC cells with purity higher than 79% could be achieved by two-step enzymatic digestion and differential adherent method; Using mouse embryonic fibroblasts as feeder layer, StemPro-34 as base medium, and adding GDNF, LIF, EGF, bFGF, IGF1 composite growth factors, the proli-feration and long-term culture of SSCs in vitro were observed. SSC colonies showed positive signals by alkaline phosphatase (AKP) immunostaining, and promyelocyte leukemia zinc-finger factor (PLZF) and ubiquitin C-terminal hydrolase L1 (UCHL1) immunofluorescence detection. Moreover, the high expression of pluripotent and self-renewal genes in colonies indicated the stable proliferation of SSCs by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) testing. After transplanting SSCs (with the enhanced green fluorescent protein (EGFP) reporter gene) into recipient testes, the heads of sperm in the epididymides emitted green light, indicating the sperm originated from the donor-SSCs. The results illustrated that the culture system obtained in this study was suitable for SSCs in vitro culture of ICR strain mice, and cells cultured in vitro had normal biological function. This study provided a reference for in vitro culture of spermatogonial stem cells of other strain mice and megafauna.

Key words: SSCs, culture in vitro, cell proliferation, functional identification

中图分类号:

S814.1

张仙玉, 赵鑫, 李国玲, 邢萍萍, 李紫聪, 杨化强, 吴珍芳, 陈斌. 小鼠精原干细胞体外培养体系建立及功能鉴定[J]. 畜牧兽医学报, 2021, 52(2): 408-419.

ZHANG Xianyu, ZHAO Xin, LI Guoling, XING Pingping, LI Zicong, YANG Huaqiang, WU Zhenfang, CHEN Bin. Establishment of the Culture System in Vitro and Functional Identification of Mouse Spermatogonial Stem Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 408-419.

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