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目的:探寻小鼠睾丸支持细胞不同发育阶段和功能状态下的特异性标志蛋白。方法:通过对小鼠睾丸组织进行免疫组织化学染色和实时定量(real-time)RT-PCR,观察受精后16 d胚胎(E16)到出生后6周的雄性小鼠睾丸组织蛋白和m RNA表达。结果:雄激素受体(AR)在出生后1周、2周、6周小鼠的睾丸支持细胞、外周小管肌样细胞、间质细胞细胞核内都有表达,且表达量逐渐增强,但在E16不表达;Wilms肿瘤基因(WT1)在E16、出生后1周、2周、6周的睾丸支持细胞细胞核内表达,但不表达于其它细胞;苗勒氏管抑制素(AMH)在E16、出生后2周的睾丸支持细胞胞质中表达,而在出生后6周不表达;β-链蛋白(β-catenin)和胞质紧密粘连蛋白-1(Zo-1)是血睾屏障特异性蛋白,出生后2周时它们分布于整个曲细精管,而出生后4周时规则地分布于靠近外壁的一圈,此时它们的血睾屏障已经完全建立。Real-time PCR分析睾丸组织m RNA表达,佐证了这些结果。结论:WT1可以作为支持细胞的特异性标志,而AMH则标记不成熟的睾丸支持细胞;AR单独不适合作为睾丸支持细胞的标志物,但与其他标志物结合,可能可区分睾丸支持细胞的成熟与否。而通过β-catenin和Zo-1染色,可以判断血睾屏障是否完全形成,为支持细胞是否成熟提供判断标记。
Objective: To explore the specific marker proteins in mouse testis supporting cells at different developmental stages and functional states. Methods: The testis tissue protein and m RNA expression in male mice after 16 days of fertilization (E16) and 6 weeks after birth were observed by immunohistochemistry and real-time RT-PCR on mouse testis tissue . RESULTS: The androgen receptor (AR) was expressed in the testicular sertoli cells, peripheral tubule myoid cells and stromal cells in the nuclei of mice at 1 week, 2 weeks and 6 weeks after birth, and the expression was gradually increased The expression of Wilms tumor gene (WT1) in E16, 1 week, 2 weeks and 6 weeks after birth was not observed in the nuclei of testicular sertoli cells; Sertoli cells were expressed in the cytoplasm of testicular support cells 2 weeks after birth, but not expressed 6 weeks after birth; β-catenin and Zo-1 were blood-testis-barrier-specific Proteins, which are distributed throughout the seminiferous tubules 2 weeks after birth, regularly distribute around the outer wall 4 weeks after birth, when their blood-testis barrier is fully established. Real-time PCR analysis of testis tissue m RNA expression, corroborated these results. CONCLUSIONS: WT1 can serve as a specific marker for supporting cells, while AMH can label immature testicular support cells. AR alone is not suitable as a marker for testicular support cells, but binding to other markers may make it possible to distinguish mature testes from supporting cells Or not. By beta-catenin and Zo-1 staining, we can determine whether the blood-testis barrier is fully formed and provide a marker for supporting the maturation of cells.