CD117(c-kit)在人类精液生精细胞中表达研究

Research on expression of CD117 on germ cells in human semen

目的从精液中分离和鉴定各类生精细胞,探讨用c-kit从精液中分离生精细胞的可能性。方法对精液中圆细胞超过3×106的标本进行联苯胺兰染色,排除白细胞,再用改良非连续percoll梯度离心,对生精细胞进行分离纯化。结合细胞形态学、荧光原位杂交,鉴别各类生精细胞的组成,运用抗人CD117单克隆抗体对纯化的细胞进行免疫细胞化学检测。结果经Percoll分离,对分离的细胞用X/Y混合探针进行荧光原位杂交,可对除精原细胞和初级精母细胞外的各级生精细胞进行准确的分类,结合形态学分析,可确定分离细胞中生精相关细胞的组成比例。在分离纯化的圆细胞中,无核胞浆小体占整个细胞比例为28.5%,细胞免疫化学研究表明,有c-kit表达的细胞占整个总细胞的比例为66.3%,且精子也表达c-kit。结论c-kit在人类精液中各级生精细胞及精子上均表达,为从人类精液中分离各类生精细胞用于临床及科研提供了可能。

To isolate and identify all sorts of germ cells, and probe into the possibility of separation of germ cells from sperm through CD117(c-kit). The round cells of human sperm over 3×106 were ortho-toluidine stained to exclude leukocytes, a modified discontinuous Percoll centrifugation used to separate and purify the seminiferous cells. Combining the cell morphology, fluorescence in situ hybridization to distinguish the constitution of seminiferous cells. Anti-human CD117 mono-antibody was used to examine the purification of collected cells. After Percoll centrifugation, employing fluorescence in situ hybridization on the purified cells with X/Y mixed probe, to classify all kinds of seminiferous cells except for spermatogonia and primary spermatocyte exactly. From the analysis of morphology, the constitution ratio of seminiferous related cells can be identified. Among the separated and purified round cells, the cytoplasmic mass consists 28.5%, c-kit expressing cells composes 66.3%, and sperms express c-kit either. [Conclusion] c-kit expresses on all stages of seminiferous cells and sperm in human semen, and provides the possibility of applying the separation of seminiferous cells to the clinical and scientific research.