人睾丸中各级生精细胞的鉴定

Identification of human testicular spermatogenic cells at different stages

目的:建立人睾丸生精细胞鉴定方法。方法:用机械法离散人睾丸细胞,涂片,用D iff-Qu ik法染色显示细胞形态。在Hoffm an光学镜头下根据细胞形态进行活细胞分类,选取各类细胞分别涂片,行17号染色体着丝粒探针化学显色原位杂交和c-k it抗原表达免疫细胞化学染色。结果:在配置Hoffm an镜头的倒置显微镜下人睾丸圆形细胞主要可分为4群:支持细胞(Sertoli)细胞,粗线期初级精母细胞,精原细胞,圆形精子细胞。原位杂交结果为:Sertoli细胞、粗线期初级精母细胞、精原细胞均显示2个着丝粒信号,圆形精子细胞及精子显示单个着丝粒信号。抗c-k it抗体免疫化学染色显示:粗线期初级精母细胞、精原细胞反应呈阳性,Sertoli细胞、圆形精子细胞、精子反应呈阴性。结论:人睾丸各类细胞在活体形态、染色形态、染色体倍性、抗原表达上均有各自显著的特征,其中据Hoffm an成像形态区分各级生精细胞是在活体状态下一种简便有效的鉴别人生精细胞的方法。

Objective： To establish the method to identify human testicular spermatogenic cells. Methods： Cells were dispersed by mechanic disintegration on testicular biopsy samples of obstructive azoospermic patients. Diff-Quik staining was applied to mixed cell smears. Live cells were observed under inverted microscope equipped with Hoffman modulation contrast optics, and classified according to their morphology. Chromogenic in situ hybridization （CISH） using chromosome 17 centromere probe and anti-c-kit immunocytochemistry staining were applied to classified cell smears. Results： Sertoli cell, primary pachytene spermatocyte （PPS）, spermatogonia, round spermatid were the main four round cell groups that can be classified under Hoffman optics. In CISH, Sertoli cell, PPS and spermatogonia displayed 2 centromere signals, while round spermatid and elongated spermatid/sperm displayed 1 centromere signal. In immunocytochemistry staining, PPS and spermatogonia displayed positive staining, while Sertoli cell, round spermatid and sperm displayed negative staining. Conclusion： Dispersed human testicular cells displayed different characteristics in live/staining morphology, ploidy analysis, cell surface membrane antigen expression. All of these methods can be chosen to identify human testicular spermatogenic cells at different stages, while cell morphology classifying under Hoffman optics is a simple and effective method for live cell identification.