利用流式分选技术富集小鼠单倍体精子细胞

Enrichment of haploid spermatids in mice by flow sorting

目的:建立并完善流式分选单倍体精子细胞的方法。方法:制备小鼠睾丸单细胞悬液;活细胞DNA染料Hoechst 33342孵育细胞;利用流式细胞术分选富集单倍体圆形精子细胞或长型精子细胞;在分选富集得到的精子细胞中,通过RT-PCR方法检测多种组蛋白乙酰化修饰酶基因的表达情况;检测组蛋白乙酰基转移酶抑制剂姜黄素对上述基因表达的影响。结果:用Hoechst 33342染色结合流式分选方法可以高效地富集高纯度的精子细胞,还可以进一步纯化圆形精子细胞与长型精子细胞。RT-PCR结果表明,与睾丸整体组织相比,精子细胞中组蛋白乙酰化修饰酶基因的表达具有特异性,并受姜黄素处理的影响。结论:该流式分选方法提高了富集单倍体精子细胞的效率和纯度,为阐明精子形成的机制提供了简便易学、适于推广的技术平台。

Objective： To establish an effective method for haploid spermatid enrichment by Hoechst 33342 staining and flow sorting. Methods ： Mouse testicular monoplast suspension was prepared by two-step enzyme digestion, and the cells were incubated in the medium containing Hoechst 33342 and Verapamil. Haploid spermatids were separated and enriched according to their DNA content by flow sorting. The gene expressions in the spermatids of several histone-modified enzymes, including the histone acetylases （HAT） and histone deacetylases （ HDAC）, were examined by RT-PCR and compared with that in the HAT-inhibitor curcumin-treated counterparts. Results ： We successfully enriched the haploid spermatids with high purity and further purified the round and elongated spermatids. RT-PCR results indicated the specificity of the expression of the HAT gene in the spermatids, and that it was influenced by curcu- min. Conclusion ： Flow sorting can efficiently improve the purity of haploid spermatid enrichment, which helps a lot to elucidate the mechanisms of spermiogenesis.