靶向纳米壳寡糖通过改善卵巢生殖干细胞的功能在延缓卵巢衰老中的作用

Regulating the proliferation and differentiation of ovarian germline stem cells (OGSCs) is crucial for increasing primordial follicle pool, seeking new preventive and curative ways in female infertility, and preventing ovarian aging. Thus, the search for relevant natural drug definitely has significance for the prevention and treatment of physiological and pathological recession of ovary. Our recent studies have revealed multiple signaling pathways in the regulation of mammalian OGSCs function. In addition, we found that Chito oligosaccharide, a widely existing nature chemical which has little toxic side effects and may improve immunity, could regulate OGSCs proliferation effectively. Based on these preliminary results, we hypothesize that Chito oligosaccharide possesses the ability to delay ovarian aging. By synthesizing Chito oligosaccharide Liposome nanoparticles of Follicle Stimulating Hormone（FSH-Lipo-CS）, in the in vitro studies we will investigate the binding location between OGSCs and the roles and mechanism of FSH-Lipo-CS on proliferation and differentiation and aging for both human and mouse OGSCs. Furthermore, by utilizing the transgenic mice carrying Pou-5fl-EGFP and human ovarian tissue specimens, in the in vivo studies we will analyze the ovarian salvation effects of FSH-Lipo-CS on the female infertility and aging mouse models together with the clinical ovarian function damage caused by X-ray. The efficiency and pharmacokinetics differences among different administration methods such as ovary micro injection, intravenous injection and intramuscular injection will also be compared. The findings of this study will add new tactics and candidate molecule on prevention and treatment of infertility patients and aging women.

调控卵巢生殖干细胞(OGSCs)的增殖分化是增加原始卵泡池、防治女性不孕症和卵巢衰老的重要手段，而寻找相关天然药物对于防治卵巢衰老具有重要意义。申请人近期研究表明，多种信号通路参与哺乳动物OGSCs功能的调控，且在自然界中广泛存在、无毒副作用并能提高免疫力的壳寡糖能调控OGSCs的增殖。因此我们假设壳寡糖能有效地应用于延缓卵巢衰老。本研究通过合成能长效靶向卵巢的壳寡糖卵泡刺激素脂质体纳米，离体研究其与人和小鼠OGSCs的结合位点及对OGSCs增殖分化和衰老的作用及机理；利用携带 Pou-5fl-EGFP 转基因小鼠和人卵巢组织，观察其对生理性卵巢功能衰退及模拟化疗和放疗造成的病理性卵巢功能衰退的预防和拯救作用；并比较经卵巢内微注射、静脉注射和肌肉注射等多途径给药对上述卵巢衰老模型的预防和拯救作用及药代动力学差异。本课题旨在为不孕症的治疗、重塑和保护卵巢功能提供新的技术方法和药物候选分子。

通过调控卵巢生殖干细胞(OGSCs)的增殖分化，是增加原始卵泡池防治女性不孕症和卵巢早衰及衰老的关键手段，故寻找促进 OGSCs 增殖和分化的天然药物对防治卵巢生理性和病理性的衰退具有重要的意义。本课题以小鼠和人卵巢组织为实验对象，主要包含以下研究内容：①采用微乳液法合成了FSH-Lipo-COS，并且用透射电镜、激光共聚焦、核磁共振氢谱和高压液相等对其粒径、聚合度等进行了鉴定；②在体观察了FSH-Lipo-COS经不同给药途径在体内的药代动力学；③改良了OGSCs分离和培养方法；④在体观察COS对不孕模型和衰老模型小鼠卵巢功能的拯救和和预防作用；⑤离体观察了COS与OGSCs的结合位点，在OGSCs增殖分化中的作用和作用机理。研究结果发现：①选择FSH 33-53结合片段作为卵巢组织药物递送系统的靶向头基，采用微乳液法合成的纳米粒子壳寡糖卵泡刺激素脂质体(FSH-Lipo-COS)其粒径工艺控制在100-200nm左右，既可避免血液的首过清除效应又可避免被网状内皮系统摄取，且可见其表面明显附着COS颗粒，提示其有良好的载药效果；②分光光度计测得通过腹腔注射FITC-FSH-Lipo-COS到达卵巢的效果最佳，激光共聚焦结果显示其能选择性地富集到卵巢上皮；③改良两步酶法收集的OGSCs细胞数和纯度最高，以巨噬细胞作为滋养层后细胞增殖曲线最好；④在体观察到COS通过维持免疫器官和细胞的功能、平衡细胞因子的分泌改善化疗引起的卵巢功能损伤；⑤离体观察到COS能进入OGSCs，刺激OGSCs增殖并抑制其衰老，同时伴有IL-2、TNF-ɑ的增高和IL-10、TGF-β的降低。以上结果提示，COS可直接或通过调节免疫系统，改善卵巢微环境，促进OGSCs的增殖；合成FSH-Lipo-COS有望为不孕症的治疗、重塑和保护卵巢功能提供新的药物作用靶点。