免疫磁珠纯化小鼠精原干细胞的研究

doi:10.13523/j.cb.20140706

中国生物工程杂志

2014, Vol. 34

Issue (7): 38-43 DOI: 10.13523/j.cb.20140706

技术与方法

免疫磁珠纯化小鼠精原干细胞的研究

贺亚南¹, 陈晓丽¹, 任晓霞¹, 郝海生¹, 秦彤¹, 赵学明¹, 路永强², 王栋¹

1. 中国农业科学院北京畜牧兽医研究所农业部畜禽遗传资源与利用重点开放实验室北京 100193;
2. 北京畜牧兽医总站北京 10017

Research on Rurification of Mouse Spermatogonial Stem Cells Using Magnetic Microbeads

HE Ya-nan¹, CHEN Xiao-li¹, REN Xiao-xia¹, HAO Hai-sheng¹, QIN Tong¹, ZHAO Xue-ming¹, LU Yong-qiang², WANG Dong¹

1. The Key Larboratory for Farm Animal Genetic and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Animal Husbandry and Veterinary Station of Beijng, Beijng 100107, China

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摘要：

精原干细胞（spermatogonial stem cells，SSCs）富集纯化是利用SSCs进行基因修饰新方法等研究的前提基础。采用免疫磁珠分选法，使用干细胞抗体CD90.2进行小鼠SSCs的纯化富集，并采用流式细胞分析法和定量PCR验证了磁珠分选效率。流式细胞分析结果：免疫磁珠分选后SSCs纯度为50.11%。荧光定量PCR检测结果：磁珠分选后支持细胞特异表达基因 GATA4 显著下调（6倍）、SSCs表达基因 GFRα-1 上调（6.5倍）、生殖干细胞特异表达基因 OCT4 极显著上调（5.9倍），3个基因相对表达量的变化说明，免疫磁珠分选效率为6倍。流式细胞分析法所产生的偏差可能是受到了未解离磁珠及SSCs本身转基因荧光的影响。

关键词： 免疫磁珠分选; 流式细胞分析; 荧光定量PCR

Abstract:

Enrichment and purification of spermatogonial stem cells (SSCs) is a prerequisite for genetic modification methods by SSCs. Stem cells antibody CD90.2 was used to enrich and purify mice SSCs through magnetic activated cell sorting(MACS) method, flow cytometry analysis(FACS) and Real-Time Polymerase Chain Reaction (RT-PCR) were used to detect the sorting efficiency. The sorting result using FACS showed that the purity of SSCs was 50.11% after sorting. However, the data by RT-PCR suggested that the expression of GATA4, a special-expression gene of sertoli cells, was downregulated about 6 times, and the expression of GFRα-1 and OCT4, two special-expression genes of germ stem cells, was upregulated 6.5 and 5.9 times respectively after-sorting. The relative expression of those three genes indicated that the enrichment efficiency using MACS was 6 times. The deviation between FACS and RT-PCR may come from undissociated magnetic bead and genetically modified fluorescence of SSCs.

Key words: Magnetic activated cell sorting Flow cytometric analysis Real-Time Polymerase Chain Reaction

收稿日期: 2014-04-08 出版日期: 2014-07-25

ZTFLH:

Q819

基金资助:

国家“十二五”科技支撑计划（2011BAD19B02），中国农科院科技创新团队家畜胚胎工程与繁殖团队项目（cxgc-ias-06-2013）资助项目

通讯作者: 路永强, 王栋 E-mail: luyongqiang@163.com;dwangcn2002@vip.Sina.com.cn

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引用本文:

贺亚南, 陈晓丽, 任晓霞, 郝海生, 秦彤, 赵学明, 路永强, 王栋. 免疫磁珠纯化小鼠精原干细胞的研究[J]. 中国生物工程杂志, 2014, 34(7): 38-43.

HE Ya-nan, CHEN Xiao-li, REN Xiao-xia, HAO Hai-sheng, QIN Tong, ZHAO Xue-ming, LU Yong-qiang, WANG Dong. Research on Rurification of Mouse Spermatogonial Stem Cells Using Magnetic Microbeads. China Biotechnology, 2014, 34(7): 38-43.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140706 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I7/38

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