山羊精原干细胞的分离纯化及鉴定

doi:10.11843/j.issn.0366-6964.2013.10.007

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (10): 1554-1560.doi: 10.11843/j.issn.0366-6964.2013.10.007

山羊精原干细胞的分离纯化及鉴定

孔群芳，罗奋华，萨初拉，包佳婧，刘代艳，吴应积^*

（内蒙古大学哺乳动物生殖生物学与生物技术教育部重点实验室, 呼和浩特 010021）

收稿日期:2013-04-03 出版日期:2013-10-23 发布日期:2013-10-23
通讯作者: 吴应积，教授，主要从事精原干细胞、精子发生过程及乳腺生物反应器研究，E-mail:wuyj1211@163.com
作者简介:孔群芳（1988-），女，内蒙古赤峰人，硕士生，主要从事生殖生物学与生殖技术研究，E-mail:kongqf1113@163.com
基金资助:
教育部创新团队计划（IRT0833）;高等学校博士学科点博导类基金（20101501110001）

Enrichment and Identification of Spermatogonial Stem Cells from Goat Testis

KONG Qun-fang, LUO Fen-hua, WU Sachula, BAO Jia-jing, LIU Dai-yan, WU Ying-ji^*

(The Key Laboratory for Mammalian Reproductive Biology and Biotechnology，Ministry of Education，Inner Mongolia University，Hohhot 010021，China)

Received:2013-04-03 Online:2013-10-23 Published:2013-10-23

摘要/Abstract

摘要：

旨在探究1种简便高效分离纯化山羊精原干细胞的方法。采用三步酶消化对4月龄的山羊睾丸进行处理，分离获得单细胞悬液。将细胞依次培养于明胶、大鼠鼠尾胶原和层粘连蛋白涂层的细胞培养皿中，根据各类细胞贴壁能力和贴壁速度不同进行精原干细胞的富集和纯化，并在各步纯化过程中用精原干细胞特异标记分子PLZF和CDH1进行免疫荧光标记染色鉴定，统计精原干细胞所占的比率，并对获得的细胞进行初步培养和鉴定。每克睾丸中分离出1.36×10⁷个细胞。纯化后每克睾丸组织获得8.7×10⁴个细胞，其中精原干细胞纯度达87.0%。将分离纯化后的细胞在体外培养可形成细胞簇，并表达精原干细胞标记分子CDH1和PLZF。结果表明，通过三步酶消化和差异贴壁方法已经建立起山羊精原干细胞的分离纯化技术。用此技术纯化的山羊精原干细胞(SSCs)能在体外培养增殖, 形成细胞簇。

Abstract:

The present study was aimed to establish a novel method that can efficiently isolate and purify spermatogonial stem cells (SSCs) from goat testis. Single cell suspension was prepared from testis of 4-month old goat by three-step enzymatic digestion. The SSCs were enriched from the suspended cells via differential attachment in gelatin, collagen, and laminin coated dishes respectively, according to the differential adherence capability in different kinds of cells. Immunofluorescent staining by using SSC molecular marker PLZF and CDH1 was performed for analysis of enrichment efficiency of the SSCs. The percentages of SSCs were calculated through PLZF- and CDH1- positive cells. The enriched cells were primarily cultured and carried out identification. 1.36×10⁷ suspended cells were harvest from per gram of goat testis. 8.7×10⁴ cells were obtained after purification, in which the purity of SSCs was 87.0%. The purified cells could be cultured to form clusters in vitro, and CDH1 and PLZF were identified to express in the cells by immunofluorescent staining. The results show that a technique for purification of goat SSCs has been established by three-step enzymatic digestion and differential attachment. Goat SSCs purified by this technique can be cultured in vitro for proliferation and forming clusters.

中图分类号:

S827
Q28

孔群芳，罗奋华，萨初拉，包佳婧，刘代艳，吴应积. 山羊精原干细胞的分离纯化及鉴定[J]. 畜牧兽医学报, 2013, 44(10): 1554-1560.

KONG Qun-fang, LUO Fen-hua, WU Sachula, BAO Jia-jing, LIU Dai-yan, WU Ying-ji. Enrichment and Identification of Spermatogonial Stem Cells from Goat Testis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(10): 1554-1560.

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山羊精原干细胞的分离纯化及鉴定

Enrichment and Identification of Spermatogonial Stem Cells from Goat Testis

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