南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (09): 1059-.doi: 10.12122/j.issn.1673-4254.2019.09.09

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CEP55 可能是治疗成熟阻滞型非梗阻无精子症的分子靶标

朱永通，刘君婷，张为青，吴嘉敏，李文锋，李惠溪，褚庆军，罗琛   

  • 出版日期:2019-09-20 发布日期:2019-09-20

CEP55 may be a potential therapeutic target for non-obstructive azoospermia with maturation arrest

  • Online:2019-09-20 Published:2019-09-20

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摘要： 目的应用siRNA技术沉默CEP55基因表达对小鼠精原细胞增殖的影响。方法选取2017年1月1日~12月31日在南方 医科大学南方医院妇产科生殖中心就诊的无精子症男性不育症患者，根据其睾丸病理切片诊断分为成熟阻滞组和生精正常组 各3 名。应用核素标记相对和绝对定量（iTRAQ）技术，发现两组患者的睾丸组织表达差异蛋白质CEP55 蛋白。设计并合成 CEP55 基因特异性的siRNA 序列，转染小鼠精原细胞，分为空白对照组、阴性对照组及siRNA 转染组，应用Western blot 和 qPCR检测在siRNA 对CEP55的影响，CCK8实验观察siRNA抑制CEP55后对小鼠精原细胞增殖的影响。结果通过iTRAQ 核素标记结合LC/MS/MS分析，共鉴定出差异蛋白共两百多个，CEP55在基因表达水平差异最显著。Western blot结果显示， siRNA转染组CEP55蛋白的相对表达水平明显低于空白对照组和阴性对照组（P<0.05）。qPCR结果显示，siRNA转染组CEP55 的mRNA表达量低于空白对照组和阴性对照组（P<0.05）。CCK8实验发现siRNA干扰CEP55表达后，小鼠精原细胞生长明显 受到抑制（P<0.05）。结论CEP55可能在精子发生过程中起到关键作用，CEP55可能成为治疗成熟阻滞型非梗阻无精子症的分 子靶标。

Abstract: Objective To explore the effect of small interfering RNA (siRNA)-mediated CEP55 gene silencing on the proliferation of mouse spermatogonia. Methods Six patients with azoospermia diagnosed to have maturation arrest (3 cases) or normal spermatogenesis (3 cases) based on testicular biopsy between January 1 and December 31, 2017 in our center were examined for differential proteins in the testicular tissue using isobaric tags for relative and absolute quantitation (iTRAQ), and CEP55 was found to differentially expressed between the two groups of patients. We constructed a CEP55 siRNA for transfection in mouse spermatogonia and examined the inhibitory effects on CEP55 expressions using Western blotting and qPCR. The effect of CEP55 gene silencing on the proliferation of mouse spermatogonia was evaluated with CCK8 assay. Results In the testicular tissues from the 6 patients with azoospermia, iTRAQ combined with LC/MS/MS analysis identified over two hundred differentially expressed proteins, among which CEP55 showed the most significant differential expression between the patients with maturation arrest and those with normal spermatogenesis. The cell transfection experiment showed that compared with the cells transfected with the vehicle or the negative control sequence, the mouse spermatogonia transfected with CEP55 siRNA showed significantly lowered expressions of CEP55 mRNA and protein (P<0.05) and significantly decreased proliferation rate as shown by CCK8 assay (P<0.05). Conclusion CEP55 may play a key role in spermatogenesis and may serve as a potential therapeutic target for non-obstructive azoospermia with maturation arrest.