山羊β防御素124促精子成熟机制的研究

Beta-defensin family in male reproductive tract exhibited broad spectrum antimicrobial properties in host defense. The majority of beta-defensins played crucial roles in non-immunological processes such as sperm maturation and capacitation. In our previous experiment, a novel epididymis-specific antimicrobial peptide, goat beta-defensin 124 (gDB 124) was identified in pseudostratified ciliated columnar epithelium and luminal fluid of epididymal caput by immunofluorescence technique, which expression was the highest in beta-defensin family of caput epididymis. Little is known about the functional mechanism in sperm maturation. The plasmid vector with knock-down and konck-in of gDB 124 gene combined by CRISPR/Cas9 system were used in construction of caput epididymal epithelium cell model and animal model. The roles of gDB 124 in sperm maturation were determined by flow cytometry, real time cellular analysis and laser scanning confocal microscopy. The interaction of gDB 124 and its cell surface ligand were verified by co-immunoprecipitation and surface plasmon resonance. Transcriptome of epididymal caput at different ages and gDB 124 knock-out and konck-in cell were analyzed by the high-throughput sequencing. The downstream signaling molecules were identified by bioinformatics and verified by signal pathway inhibitors used in caput epididymal epithelium cell. The molecular signaling pathway of gDB 124 was constructed by pathway assist software for uncovering the molecular mechanisms of gDB 124 protein. The results will enrich endocrine theory in physiology of animal reproduction, and lay a foundation for improvement of the reproductive rate and cure of male infertility of domestic animals.

雄性生殖器官中的β防御素家族是保护精子免受微生物侵袭的特殊免疫系统，同时对精子成熟和获能有重要作用。本项目组的前期研究发现山羊附睾头中表达有30种β防御素，其中β防御素124（gDB 124）的表达量最高，定位在附睾头假复层柱状上皮细胞和管腔液中，推测其具有促进精子成熟作用。但关于gDB124促精子成熟作用的机制尚不清楚。本项目拟用Crispr/cas9技术构建gDB 124基因敲除和过表达的细胞模型和动物模型，利用流式细胞术、实时无荧光标记细胞分析技术、激光扫描共聚焦显微技术、免疫共沉淀技术、表面等离子体共振技术、RNA-Seq等技术，研究gDB124蛋白如何促进精子成熟，并探寻与gDB124相互作用的膜配体，初步找出gDB124作用的分子信号通路，探明gDB124作用的分子机制。研究结果可丰富动物生殖生理中内分泌学的理论，为动物繁殖力的提高和繁殖障碍的治疗提供理论依据。

β防御素124（gBD124）是公山羊附睾头表达量最高的β防御素。gBD124是发挥免疫作用，还是促精子成熟作用呢？本研究利用RNAi技术、RNA-Seq技术、生物信息学技术、FISH技术、激光扫描共聚焦显微等技术，应用gBD124基因沉默和过表达细胞模型，研究gBD124在附睾头细胞发挥作用的信号通路。取得结果有：（1）成功构建gBD124基因沉默慢病毒载体和过表达慢病毒载体，转染山羊附睾头细胞经嘌呤霉素二次筛选分别建立gBD124基因沉默和过表达的附睾头细胞稳转系；（2）重组gBD124蛋白、鼠来源gBD124单克隆抗体和兔来源的多克隆抗体；Western blot结果显示gBD124蛋白可能存在3种形式：单体、二聚体和三聚体；（3）基于30个附睾样品RNA-Seq测序数据分析，应用生物信息学技术将30种β防御素分为4个基因簇分别定位到山羊8、13、23和27号染色体上，8号染色体上gBD131、134和30，与13号染色体上除gBD123-126和128在30种组织中均表达外，其余β防御素是在繁殖器官特异性表达；23号染色体上gBD110-114是在繁殖器官中高度表达，27号染色体gBD104-109tr2在繁殖器官特异性表达；（4）利用FISH技术、激光扫描共聚焦显微技术的结果显示gBD124在细胞核周围高度聚集，细胞核内和胞质内均有分布；再结合转录组差异显著基因富集在MAPK信号通路结果，发现gBD124基因沉默后显著降低了MAPK信号通路中总p38MAPK、AP1亚型c-JUN和c-FOS蛋白表达，以及p38MAPK和c-JUN蛋白磷酸化，上调了MAPK通路下游IL-1β及其受体IL-1R2、IL-8和趋化因子CCL6和CCL21基因表达，下调了CCL5和IL-1α基因表达（P<0.05）；gBD124基因过表达后显著增加总p38MAPK和总c-FOS蛋白表达，以及p38MAPK蛋白磷酸化，上调趋化因子CCL21和细胞因子受体IL-1R2基因的表达，下调趋化因子CCL5、CCL6和IL-8的基因表达（P<0.05）。⑤初步绘制了β防御素124免疫调节的信号通路图。说明gBD124在附睾头中可通过发挥免疫调节作用间接起到保护精子的功能，哪种构型的gBD124发挥作用？是否对精子成熟有影响等问题仍需深入研究，并找到其配体，探明其功能及其作用信号通路。