小鼠睾丸精子细胞的分离与鉴定  

Isolation and Identification of Spermatids from Mouse Testis

作　　者：朱培元[1] 黄宇烽[2] 徐建平[2] 

机构地区：[1]南京医科大学病原生物学和免疫学学系,江苏南京210029 [2]南京军区南京总医院生殖遗传研究室,江苏南京210002

出　　处：《中华男科学杂志》2002年第1期28-31,共4页National Journal of Andrology

基　　金：江苏省生殖医学重点实验室开放课题基金资助 (K990 62 )

摘　　要：目的 :建立一种简单、可有效分离小鼠睾丸精子细胞的方法。　方法 :用组合酶消化成年小鼠睾丸制备睾丸细胞悬液 ,然后经 6层非连续Percoll梯度 (15 %、2 2 %、30 %、4 0 %、5 0 %和 6 0 % )离心法分离 ,通过形态学和流式细胞术鉴定各个Percoll梯度中精子细胞的含量 ,并以伊红Y排斥试验测定细胞的存活率。　结果 :组合酶消化后获得的睾丸细胞悬液中 ,97%以上细胞仍然存活。经Percoll梯度离心 ,共形成 6条细胞带 ,其中 2 2 %梯度中主要为精子细胞 (平均 86 .7% ,P <0 .0 5 ) ,85 .5 %以上细胞存活 ;而 30 %梯度中细胞密度最高 (P <0 .0 5 ) ,含有各级未成熟生精细胞 ,存活细胞超过 92 %。　结论 :采用组合酶消化结合非连续Percoll梯度离心法 。Objectives: To develop a simple and effective method by which spermatids can be isolated from mouse testis. Methods: Combination of enzymatic digestion was used to prepare suspension of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient (15%,22%,30%,40%,50%,60%) centrifugation method was introduced to isolate spermatids from the cellular suspension. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright Giemsa staining) and flow cytometry analysis, and the viability of spermatogenic cells was assessed using Eosin Y exclusion test. Results: More than 97% of the testicular cells remained their viability after enzymatic digestion. After Percoll centrifuged, six fractions were formed. In each isolated fraction, the 22% fraction contained mostly spermatids(mean 86.7%) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability. Conclusions: A large of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined discontinuous Percoll gradient centrifugation method.

关 键 词：精子细胞 Percoll梯度离心 睾丸 分离 鉴定 组合酶消化 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]