免疫磁珠纯化小鼠精原干细胞的研究  

Research on Rurification of Mouse Spermatogonial Stem Cells Using Magnetic Microbeads

作　　者：贺亚南 陈晓丽[1] 任晓霞[1] 郝海生[1] 秦彤[1] 赵学明[1] 路永强 王栋[1] 

机构地区：[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

出　　处：《中国生物工程杂志》2014年第7期38-43,共6页China Biotechnology

基　　金：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

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。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-1and OCT4,two special-expression genes of germ stem cells,was upregulated 6. 5 and 5. 9 times respectively aftersorting. The relative expression of those three genes indicated that the enrichment efficiency using MACS was 6times. The deviation between FACS and RT-PCR may come from undissociated magnetic bead and genetically modified fluorescence of SSCs.

关 键 词：免疫磁珠分选 流式细胞分析 荧光定量PCR 

分 类 号：Q819[生物学—生物工程]