精原干细胞体外培养体系的建立

doi:10.3969/j.issn.1004-1524.2021.12.05

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (12): 2254-2263.DOI: 10.3969/j.issn.1004-1524.2021.12.05

精原干细胞体外培养体系的建立

张茂¹(), 赵鑫², 蔡更元², 杨化强²^,^*()

1.韶关学院英东生物与农业学院,广东韶关 512005
2.华南农业大学动物科学学院国家生猪种业工程技术研究中心,广东广州 510642

收稿日期:2020-09-01 出版日期:2021-12-25 发布日期:2022-01-10
通讯作者: 杨化强
作者简介:* 杨化强,E-mail: yangh@scau.edu.cn
张茂(1985—),男,云南大理人,博士,讲师,研究方向为猪遗传育种。E-mail: 281943141@qq.com
基金资助:
国家自然科学基金(31772555);国家转基因生物新品种培育重大专项(2016ZX08006002)

Establishment of in vitro culture system of pig spermatogonial stem cells

ZHANG Mao¹(), ZHAO Xin², CAI Gengyuan², YANG Huaqiang²^,^*()

1. Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China
2. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received:2020-09-01 Online:2021-12-25 Published:2022-01-10
Contact: YANG Huaqiang

摘要/Abstract

摘要：

精原干细胞(spermatogonial stem cells,SSCs)是雄性哺乳动物精子发生及具有生育能力的保障。精原干细胞的体外培养不仅为精子发生的研究提供材料,还有助于开发新的家畜保种方法和动物遗传修饰。为了探索猪精原干细胞体外培养体系的建立方法,本研究采用胶原酶Ⅳ-胰酶两步酶法对3~7日龄大白仔猪睾丸进行消化得到单细胞悬液,利用不同时间程序的差速贴壁对精原干细胞进行纯化,选择大白仔猪睾丸支持细胞作为饲养层,添加不同细胞因子研究精原干细胞的增殖情况以期得到最佳的培养体系。结果显示,通过差速贴壁得到的UCHL-1阳性生殖细胞比例最高为18.59%±0.94%;不同细胞因子组合添加试验发现精原干细胞添加20 ng·mL^-1 GDNF、10 ng·mL^-1 IGF和20 ng·mL^-1 bFGF的增殖效果最佳;以支持细胞作为饲养层、在DMEM/F12中添加1%FBS以及上述细胞因子组合对精原干细胞进行培养15 d后可见大量的精原干细胞集落,通过免疫荧光、AKP染色、荧光定量PCR等试验证明精原干细胞进行了大量增殖。本研究初步建立了猪精原干细胞的体外培养体系,可通过体外培养大量增殖精原干细胞,为后续精原干细胞的研究奠定基础。

关键词: 猪, 精原干细胞, 细胞因子, 体外培养

Abstract:

Spermatogonial stem cells (SSCs) are the guarantee of spermatogenesis and fertility in male mammals. In vitro culture of spermatogonial stem cells not only provides materials for the study of spermatogenesis, but also helps to develop new methods of livestock conservation and animal genetic modification. In order to find out the better culture system of porcine spermatogonial stem cells in vitro, two step enzymatic method of collagenase Ⅳ-trypsin was used to digest the testis of 3-7 day Yorkshire piglets to obtain single cell suspension. And the spermatogonial stem cells were purified using differential adherent with different time programs. In addition, different cytokines were added to study the best culture system for the proliferation of spermatogonial stem cells. The results showed that the highest percentage of UCHL-1 positive germ cells was 18.59%±0.94%, and the result of cytokine addition showed that spermatogonial stem cells added with 20 ng·mL ^-1 GDNF, 10 ng·mL^-1 IGF and 20 ng·mL^-1 bFGF had the best effect of proliferation. Spermatogonial stem cells were cultured in DMEM/F12 with 1% FBS and the cytokines described above, taking Sertoli cells as feeder laye. After 15 days, a large number of spermatogonial stem cell colonies were observed. The proliferation of spermatogonial stem cells was confirmed by immunofluorescence, AKP staining and fluorescent quantitative PCR detcetion. In brief, we initially established the culture system of porcine spermatogonial stem cells in vitro, through which a large number of spermatogonial stem cells can be cultured in vitro, laying the foundation for subsequent research on spermatogonial stem cells.

Key words: pig, spermatogonial stem cells, cytokines, in vitro culture

中图分类号:

S828

张茂, 赵鑫, 蔡更元, 杨化强. 精原干细胞体外培养体系的建立[J]. 浙江农业学报, 2021, 33(12): 2254-2263.

ZHANG Mao, ZHAO Xin, CAI Gengyuan, YANG Huaqiang. Establishment of in vitro culture system of pig spermatogonial stem cells[J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2254-2263.

图/表 5

表1 引物设计

Table 1 Primers design

基因 Gene	引物 Primer (5'→3')	产物 Product/bp	Tm/ ℃
GAPDH	F:AGGGCTGCTTTTAACTCTGGCAA	180	56
	R:GATGGTGATGGCCTTTCCATTG
UCHL-1	F:TCCGGAAGACAGAGCAAAATGC	150	56
	R:AGTTCATAGAGGTGGCCATCCA
Thy-1	F:GTGCTCTTGGGCACTGTGGG	178	57
	R:TCTTGCTGGAGATGCTGGGC
Gfra-1	F:GAACGGAGGCGGCAGACCAT	242	57
	R:AAGCCCAGAGTAGGCGAGGAG
C-kit	F:GATGCCTTCAAGGATTTGGA	181	53
	R:ATGGAATCTGAGGCCTTCCT
Oct4	F:CGCGAAGCTGGACAAGGAGA	151	56
	R:CAAAGTGAGCCCCACATCGG
Nanog	F:AACCAAACCTGGAACAGCCAGAC	152	56
	R:GTTTCCAAGACGGCCTCCAAAT
Dazl	F:ACAGTGGCCTGCTGGGGAAC	153	56
	R:TGTGGGCCATTTCCAGAGGA

图1 纯化后精原干细胞的检测 A,免疫荧光检测(200×),a1-a3,方法A纯化后免疫荧光;b1-b3,方法B纯化后免疫荧光;c1-c3,方法C纯化后免疫荧光。B,AKP 染色,红色箭头为AKP染色阳性细胞(400×)。C,定量PCR分析,**表示差异极显著。

Fig. 1 Detection of purified SSCs A, Immunofluorescence detection (200×), a1-a3, Immunofluorescence after purification by method A; b1-b3, Immunofluorescence after purification by method B; c1-c3, Immunofluorescence after purification by method C. B, AKP staining, the red arrow indicates AKP staining positive cells (400×). C, Quantitative PCR analysis, ** represented the difference was significant at P<0.01.

图2 不同浓度丝裂霉素C对支持细胞的影响(上)和不同细胞因子组合添加对精原干细胞增殖的影响(下) 柱上没有相同小写字母的表示差异显著(P<0.05)。

Fig. 2 Effect of different concentrations of mitomycin C on Sertoli cells (up) and effect of different cytokine combinations on proliferation of spermatogonial stem cells (down) Bars marked without the same letters indicated that the difference was significant at P<0.05.

图3 SSCs的体外培养 A,接种的精原干细胞(200×);B,培养6 d的精原干细胞(200×);C,培养15 d的精原干细胞(100×);D,培养15 d的精原干细胞集落(200×)。

Fig. 3 In vitro culture of SSCs A, Inoculated spermatogonial stem cells (200×); B, Spermatogonial stem cells cultured for 6 days(200×); C, Spermatogonial stem cells cultured for 15 days(100×); D,Spermatogonial stem cell colonies cultured for 15 days(200×).

图4 增殖精原干细胞的检测 A,UCHL-1免疫荧光(200×),a1,常光,a2,UCHL-1染色。B,AKP染色(200×)。C,定量PCR检测,**表示差异极显著。D,RT-PCR检测,M,DL2000;泳道1~7分别为GAPDH、UCHL-1、Gfra-1、C-kit、Thy-1、Oct-4、Nanog基因。

Fig. 4 Detection of proliferating SSCs A, Immunofluorescence of UCHL-1 (200×), a1, white light, a2, UCHL-1 antibody staining. B, AKP staining (200×). C, Quantitative PCR detection, ** meaned the difference is extremely significant. D, RT-PCR detection, M, DL2000; lanes 1-7 are GAPDH, UCHL-1, Gfra-1, C-kit, Thy-1, Oct-4, Nanog genes, respectively.

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精原干细胞体外培养体系的建立

Establishment of in vitro culture system of pig spermatogonial stem cells

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