白介素17及其受体对精子线粒体免疫损伤的分子机制研究

Orchitis is amongst the leading causes of male infterility. Our previous studies showed that overexpression of Th17 cells and IL-17 were found in human testis with chronic inflammation, and Th17 cells and IL-17 were involved in the destruction of blood-testis barrier and loss of germinal epithelium under chronic inflammatory conditions. Furthermore, IL-17R, the specific receptor for IL-17, was found to localize to the midpiece of sperm. Upregulated concentration of IL-17 can interrupt mitochondria membrane potential and sperm motility and increase the precetage of spermatozoa apoptosis and expression of p38 protein. The results indicate that IL-17/-17R injure sperm mitochondria possibly through activating p38 pathway. The outline of the present study is 1) to investigate the expression of IL-17/IL-17R in ejaculates and subcellular location of IL-17R on sperm from patients with chronic orchitis using flow cytometry and immunoelectron microscopy; 2) to build IL-17 and sperm co-culture system in vitro and investigate IL-17/IL-17R immune injury of sperm mitochondria by flow cytometry, PCR and immunoelectron microscopy; 3) to examine the effect of IL-17/IL-17R on the expression and phosphorylation of p38MAPK pathway, and investigate the impact of p38MAPK pathway changes on sperm mitochondria. The purpose of our project is to elucidate the molecular mechanism of IL-17/IL-17R immune injury of sperm mitochondria, and it will provide a new targeted approach and theoretical basis for the diagonsis and treatment of male infertility.

睾丸炎是导致男性不育的重要原因。本课题组前期研究显示，Th17细胞及其细胞因子IL-17在睾丸炎中破坏血睾屏障、损伤生精上皮以致男性不育。IL-17的受体IL-17R在人精子定位于颈部，高浓度IL-17体外培养可致精子线粒体膜电位升高、活力下降、凋亡增多和p38表达上调；据此推测IL-17/IL-17R可能通过激活p38信号途径损伤精子线粒体。本课题拟：1）采用流式细胞术和免疫电镜等，检测IL-17/IL-17R在慢性睾丸炎患者精液中表达和IL-17R亚细胞定位；2）通过流式细胞术、PCR和电镜等研究IL-17/IL-17R对精子线粒体损伤的影响；3）采用免疫细胞化学染色和蛋白免疫印迹技术等研究IL-17对p38蛋白家族表达和磷酸化的变化，及其变化对精子线粒体损伤的影响。本研究力图阐明IL-17/IL-17R对精子线粒体免疫损伤的分子机制，为男性不育诊治提供新的靶点和理论依据。

慢性生殖道炎症如睾丸炎、附睾炎是导致男性不育的重要原因之一，资料显示15%以上男性不育患者伴有慢性生殖道炎症。然而由于慢性生殖道炎症患者临床表现常以少、弱精子症为主，而缺乏相对特异性的精液和血清标志物，因此慢性生殖道炎症所致男性不育又是临床上容易被忽视的重要因素。本课题主要分为三部分研究内容：第一部分：检测Th17细胞和IL-17/IL-17R在慢性睾丸炎患者和正常健康男性精液中表达差异，和IL-17R在精子的亚细胞定位；第二部分： IL-17/IL-17R对精子活力、线粒体功能、DNA损伤和凋亡的影响；第三部分：IL-17/IL-17R激活p38MAPK信号途径对线粒体免疫损伤的分子机制。根据我们的实验结果发现，IL-17/IL-17R主要通过p38MAPK信号途径损害精子线粒体，从而导致精子活力下降以及凋亡增加；使用IL-17受体拮抗剂或p38MAPK蛋白抑制剂，可以减轻炎症反应、改善精子活力和数量。本研究阐明了IL-17/IL-17R对精子线粒体免疫损伤的分子机制和作用靶点，为诊治慢性生殖道炎症所致男性不育提供一个新的靶向途径。