Beta-防御素3对猪卵泡发育的调节及其机制

Beta defensins mainly involved in anti-infection and immunomodulation, its funtion in female fertility has never been reported. The previous studies showed that beta defensin 3 expression level in mural granulosa cells varied during swine follicular development, moreover, BD3 was proved to induce mural granulosa cell proliferation and migration. However, neither regulatory mechanisms underlying BD3 expression nor its function in swine follicle is clear. This project intends to use swine follicular granulosa cells as cell model to study the regulatory mechanism underlying the BD3-induction in mammalian follicular development, using western-blot and PathDetect in vivo signal transduction pathway trans-reporting system to identify the pathways involved in the regulatory effect of BD3 on granulosa cell, furthermore, using siRNA to delete the key kinase in the signaling pathway and identify its effect on the BD3 funtion. Meanwhile, to study the regulatory effect of follicle stimulating hormone on BD3 mRNA and protein expression, the pathways involved in its regulation and its regulatory mechanism. The project result will not only reveal the regulatory effect of BD3 on swine follicular development for the first time, elucidate its regulatory mechanism, but also provide basis for a novel application of defensins in mammalian fertility.

Beta-防御素（BDs）的功能主要为抗感染和免疫调节，其在雌性动物生殖发育中的作用未曾报道过。实验室前期试验结果显示在猪卵泡发育不同阶段BD3在壁颗粒细胞中表达量不同，且BD3对猪颗粒细胞的增殖和迁移有促进作用，但其在猪卵泡中的表达调控和功能调节机制并不清楚。本项目拟在前期基础上以猪卵泡颗粒细胞为细胞模型，研究BD3在哺乳动物卵泡发育中的调控机制，采用Western-blot和体外PathDetect信号传导通路trans-报告系统检测BD3调节颗粒细胞功能的信号通路，并检测用siRNA缺失待检信号通路中关键激酶后对BD3功能调节作用的影响。同时，研究促卵泡素对BD3在猪颗粒细胞中mRNA和蛋白表达水平的调控作用，调控作用的信号通路和表达调控机制。研究结果不仅首次揭示BD3在猪卵泡发育中的调控作用，并从理论上阐明BD3的作用机制，还为防御素在哺乳动物生殖发育领域中的创新型应用提供基础。

抗菌肽的功能主要为抗感染和免疫调节，其在雌性哺乳动物生殖发育中的作用未曾报道过。卵泡中主要的体细胞是壁颗粒细胞，在卵泡生长发育过程中发挥着重要生物学功能。卵泡壁颗粒细胞的增殖、迁移、激素合成和凋亡受到多种机制调控，与卵泡的发育紧密相关。本项目以猪卵泡壁颗粒细胞为细胞模型，通过研究防御素中β-防御素3(BD3)和Cathelicidins类中Protegrin 1(PG1)对壁颗粒细胞功能的调节及其作用机制来阐明抗菌肽在卵泡发育中的作用。通过细胞计数和CCK-8试验证实BD3对猪壁颗粒细胞的增殖有显著促进作用，且RT-qPCR试验证实BD3对壁颗粒细胞增殖相关因子CCNB1, CDK1和PCNA mRNA表达水平有显著促进作用。运用transwell试验证实BD3能显著促进壁颗粒细胞的迁移。并进一步采用Western-blot、体外PathDetect信号传导通路trans-报告系统检测证实BD3激活MAPK-ERK1/2信号通路，使用ERK1/2磷酸化特异性抑制剂后显著抑制BD3诱导的ERK1/2磷酸化和其对壁颗粒细胞的增殖和迁移的促进作用，从而证实BD3通过MAPK-ERK1/2信号通路调节猪源卵泡壁颗粒细胞的增殖和迁移功能。同时，也运用上述方法证实了在另一类抗菌肽Cathelicidins中，PG1能显著促进猪壁颗粒细胞的增殖能力，并上调增殖相关因子CCND1和CCND2 的mRNA表达水平，采用流式细胞仪分析显示PG1能显著促进壁颗粒细胞的抗凋亡能力，并且证实PG1通过EGFR-ERK1/2信号通路促进猪卵泡壁颗粒细胞的增殖及抗凋亡能力。本项目研究结果不仅首次揭示抗菌肽在猪卵泡发育中的调控作用，并从理论上阐明抗菌肽的作用机制，还为抗菌肽在哺乳动物生殖发育领域中的创新型应用提供基础。