不育病人精液中精子细胞的分离与鉴定

Isolation and Identification of Spermatids in Semen of Male Infertile Patients

目的 :建立一种可从不育病人精液中分离出较多、较纯精子细胞的新方法。 方法 :收集 15例含细胞成分的不育病人精液 ,利用一种改良的非连续Percoll梯度 (15 %、2 2 %、30 %、4 0 %、5 0 %和 6 0 % )离心法分离精子细胞。于 18℃ 2 0 0 0r/min离心 30min后 ,精液细胞被分成单个的细胞组分 ;然后通过细胞计数、形态学、流式细胞术 (FCM )和免疫细胞化学等方法进行分析 ,选择含精子细胞纯度最高的组分。 结果 :经Percoll梯度离心后获得6个细胞组分 ,形态学和FCM分析表明 ,2 2 %组分中主要为精子细胞 [(91.85± 5 .18) % ,P <0 .0 0 5 ],平均密度为(1.0 10± 0 .786 )× 10 5/ml;30 %组分中含包括精子细胞在内的各种未成熟生精细胞 ;而白细胞主要位于 6 0 %组分中。 结论 :利用这种改良的非连续Percoll梯度离心法 ,可从不育病人的精液中分离到较多。

Objectives: To develop a method by wich large and purified populations of spermatids can be isolated in semen of male infertile patients. Methods: A total of fifteen ejaculates containing cellular elements from infertile patients with various andrological pathologies were obtained after a 24 hour abstinence. A modified discontinuous Percoll gradient(15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was used to isolate the spermatids. After centrifugation at 2 000 r/min for 30 min at 18 ℃,the single Percoll fractions were separated and analyzed in order to select the one with the greateast purity of spermatid. The germinal cells in each isolated fraction were counted using a Macro sperm counting chamber, then the contents of spermatids were determined by morphology (Wright Giemsa staining method) and flow cytometry (FCM) analysis,while the contaminated leukocytes were assessed by anti CD45 immunocytochemistry. Results: After Percoll centrifuged, six single fractions were obtained. Morphology and FCM analysis showed that the 22% fraction contained mostly spermatids \[( 91.85 ± 5.18 )%, P <0.005\] and the mean density in this fraction was ( 1.010 ± 0.786 )×10 5 /ml. While in the 30% fraction, various immature spermatogenic cells including spermatids were present and leukocytes mostly presented in the 60% fraction. Conclusions: A lagre population of relatively purified spermatids can be isolated from the ejaculates of infertile patients by using this modified discontinuous Percoll gradient centrifugation method.