流式细胞仪富集大鼠A1～A4型精原干细胞

目的:探讨A型精原干细胞的A1~A4亚型细胞的分离纯化方法。方法:先使用不连续密度梯度法分离A型精原细胞,再经流式细胞分选技术分选有干细胞因子受体(c-kit)表达的亚型细胞。并采用免疫组化检测c-kit在睾丸中的表达,电镜下观察分选后细胞超微结构。结果:经密度梯度分离后的精原细胞内含c-kit阳性细胞比例为(18.65±1.69)%,未经密度梯度分离的睾丸细胞内含c-kit阳性细胞比例为(3.16±0.84)%,两者比较差异有显著性(P<0.01);c-kit阳性细胞经流式细胞术分选,阳性细胞回收率(65.90±1.24)%,活性(85.60±1.14)%。结论:以c-kit为标记物,经不连续密度梯度分离初步纯化后,使用流式细胞分选技术能有效分离A型精原干细胞亚型。

Enrichment of Type A1-A4 Spermatogonia by Flow Cytometry

OBJECTIVE: To explore a method to isolate and purify the subtype of type A spermatogonial stem cells (SSCs). METHODS: We isolated spermatogonia by discontinuous density gradient centrifugation, sorted c-kit-expressed cells with the fluorescence-activated cell sorter (FACS), observed their ultrastructure by electron microscope, and performed immunohistochemistry to determine the expression of c-kit in the testis. RESULTS: The c-kit positive cells constituted (18.65 +/- 1.69) % of the testis cells that were isolated by density gradient centrifugation, but made up only (3.16 +/- 0.84) % of those that were not (P < 0.01). The rates of recovery and viability of the c-kit positive cells sorted by FACS were (65.90 +/- 1.24)% and (85.60 +/- 1.14)%, respectively. CONCLUSION: With c-kit as the marker, FACS can effectively isolate and purify the subtype of SSCs after preliminarily purified by discontinuous density gradient centrifugation.