小熊猫精原干细胞的分选与培养
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1.四川省濒危野生动物保护生物学重点实验室;2.①成都大熊猫繁育研究基地,②四川省濒危野生动物保护生物学重点实验室,③四川省大熊猫科学研究院;3.①成都大熊猫繁育研究基地,②四川省濒危野生动物保护生物学重点实验室;4.成都大熊猫繁育研究基地

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四川杰出青年科技人才项目(No. 2020JDJQ0074),成都大熊猫繁育研究基金会项目(No. CPF2017-15),成都大熊猫繁育研究基地自立课题(No. 2020CPB-B02)


Purification and Culture of Spermatogonial Stem Cells from Red Panda (Ailurus fulgens)
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①Chengdu Research Base of Giant Panda Breeding,② Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife,③Sichuan Academy of Giant Panda

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    摘要:

    野生动物的保护手段主要包括就地保护、易地保护与离体保护。精原干细胞(SSCs)是雄性动物维持生殖能力的根本,既能通过自我更新产生新细胞,也能通过分化产生精子,在小熊猫(Ailurus fulgens)离体保护方面具有广阔的应用前景。动物睾丸中精原干细胞数量极少,分离纯化与体外培养对于其研究和应用至关重要。本研究选择整合素α6(ITGA6)蛋白作为精原干细胞分子标记,采用免疫磁珠分选(MACS)技术富集了3月龄小熊猫睾丸中的ITGA6阳性细胞。流式细胞术检测发现分选后ITGA6阳性细胞纯度可达74.27% ± 8.73%,显著高于分选前(32.60% ± 3.06%)。将分选后的细胞接种到层粘连蛋白包被的细胞培养板中,用含胶质细胞源性神经营养因子(GDNF)、表皮细胞生长因子(EGF)与成纤维细胞生长因子(bFGF)的培养基进行体外培养。培养10 d后,在显微镜下可观察到典型的精原干细胞集落,结合逆转录PCR(RT-PCR)和细胞免疫荧光染色发现这些细胞集落特异性表达精原干细胞分子标记蛋白ITGA6、早幼粒细胞白血病锌指蛋白(PLZF)和胸腺细胞分化抗原1(THY1),同时也表达生殖细胞标记蛋白VASA和DAZL。本研究结果证实,ITGA6可作为小熊猫精原干细胞的分子标记用于细胞分选富集,同时初步建立的培养体系也为小熊猫精子发生机制与应用研究提供材料。

    Abstract:

    In situ, ex situ, and in vitro conservation are the principal means of wild animal protection at present. Spermatogonial stem cells (SSCs) not only continuously produce new cells through self-renewal, but also produce sperm through cell differentiation, and thus they have broad application potential for in vitro conservation of Red Panda (Ailurus fulgens). However, SSCs in testes are rare, and their purification and culture are very important for scientific research and applications. [Objectives] To explore the feasibility of SSCs enrichment and culture in Red Panda. [Methods] Testes were collected post mortem from a 3-month-old Red Panda and were used to generate cell suspensions following two-step enzyme (1 g/L collagenase Type IV and 0.25% trypsin-EDTA) digestion. ITGA6 was used as a molecular marker of SSCs, and the ITGA6- positive cells were enriched by magnetic-activated cell sorting (MACS). ITGA6-positive cells were seeded into the cell culture plate coated with laminin and cultured with the medium containing GDNF (20 μg/L), EGF (10 μg/L) and bFGF (10 μg/L). RT-PCR and immunofluorescence staining were used for the cell colonies identification. [Results] The purity of ITGA6-positive cell after sorting was 74.27% ± 8.73%, which was significantly higher than that before sorting (32.60% ± 3.06%) (Fig. 1). After 10 days of culture, SSC colonies were observed under the microscope (Fig. 2). The results of RT-PCR and immunofluorescence staining showed that ITGA6, PLZF, THY1 (SSC markers), VASA, and DAZL (germ cell markers) were specifically expressed in MGSC colonies (Fig. 3 and 4). [Conclusion] The overall results demonstrated the feasibility of ITGA6 as a molecular marker of Red Panda SSCs for cell purification and cultivation.

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封同英,刘玉良,陈依娇,史可瑜,蔡志刚,王东辉,张名岳,李博琦,侯蓉,安俊辉.2022.小熊猫精原干细胞的分选与培养.动物学杂志,57(6):897-904.

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  • 收稿日期:2022-02-23
  • 最后修改日期:2022-08-30
  • 录用日期:2022-08-26
  • 在线发布日期: 2022-12-15
  • 出版日期: 2022-12-15