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

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.