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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 ,可从小鼠睾丸中分离到较多较纯的存活精子细胞
Objective: To establish a simple and effective method for the isolation of mouse testicular sperm cells. METHODS: Adult mouse testes were digested with combinatorial enzymes to prepare testicular cell suspensions and were then separated by six layers of non-continuous Percoll gradient centrifugation (15%, 22%, 30%, 40%, 50% and 60%) , The content of sperm cells in each Percoll gradient was identified by morphology and flow cytometry, and the viability of the cells was determined by the Eosin Y exclusion assay. RESULTS: More than 97% of the cells in testicular cell suspensions obtained after digestion with combined enzymes still survived. After Percoll gradient centrifugation, a total of 6 cell bands were formed, of which 22% were mainly sperm cells (average 86.7%, P <0.05), and 85.5% The highest cell density (P <0.05), containing all levels of immature spermatogenic cells, survival of cells over 92%. Conclusion: More pure and more viable sperm cells can be isolated from mouse testis by combinatorial enzyme digestion combined with discontinuous Percoll gradient centrifugation