不成熟卵泡体外培养在肿瘤患者生育力保存中的应用研究

The cryopreservation of ovarian tissue can provide fertility preservation for female tumor patients , but in patients with malignant tumor of ovary, it is not appropriate to retransplant frozen ovaries.It is needed to develop new technology of follicle in-vitro culture. Our team has developed in vitro maturation medium in the early stage. The main bottleneck is the low efficiency of follicle activation, which affects the success rate of in vitro culture . The PTEN-PI3K-AKt is known to play an important role in follicle activation, and GDF9 and BMP 15 promote follicle growth and development. Therefore, we propose the following idea: combining physical stimulation with PTEN inhibitor and Akt agonist, adding GDF9 and BMP 15 can improve the activation efficiency and the success rate of follicle culture in vitro. We are planning to combine physical stimulation with PTEN inhibitor, Akt agonist, GDF9 and BMP 15 to activate primary follicles, followed by in vitro maturation culture in sequential double pool Co-culture system. We aim to establish a complete follicle culture system in vitro and confirm whether combined with physical and biochemical factors can improve follicle activation efficiency and success rate of follicle in vitro culture . The completion of this project will fill the gap of fertility preservation technology in patients with ovarian malignancy and provide the basis for elucidating follicle development mechanism.

卵巢组织冷冻可为生育期女性肿瘤患者提供生育力保存，但卵巢有恶性肿瘤的患者，不宜回移冻存的卵巢，需要研发卵泡体外培养的新技术。本团队在前期已自主研制体外成熟液配方，目前存在的主要瓶颈是卵泡激活的效率低，影响卵泡体外培养的成功率。已知PTEN-PI3K-AKt通路在卵泡激活中起重要作用，GDF9、BMP15对卵泡的生长发育起促进作用；据此，我们提出以下设想：结合物理刺激及PTEN抑制剂、Akt激动剂，添加GDF9、BMP15，是否可提高激活效率和卵泡体外培养的成功率？本项目拟结合物理刺激和PTEN抑制剂、Akt激动剂、GDF9、BMP15激活始基卵泡，采用自主研制体外成熟液及序贯双池共培养系统培养至体外成熟。旨在建立完整的卵泡体外培养系统，并证实结合物理及生化因子方法是否可提高卵泡激活效率和卵泡体外培养的成功率。本项目的完成将填补卵巢恶性肿瘤患者生育力保存技术空白，为阐明卵泡发育机制提供依据。

卵巢组织冷冻可为生育期女性肿瘤患者提供生育力保存，但卵巢有恶性肿瘤的患者，不宜回移冻存的卵巢，需要研发卵泡体外培养的新技术。目前存在的主要瓶颈是卵泡激活的效率低，影响卵泡体外培养的成功率。本研究任务是结合物理刺激和优化因子激活始基卵泡，行卵泡体外培养，再采用自主研制体外成熟液及序贯双池共培养系统培养至体外成熟，旨在建立完整的卵泡体外培养系统并评估其成功率，为卵泡体外培养的临床应用奠定基础，以期填补卵巢恶性肿瘤患者生育力保存技术空白，为阐明卵泡发育机制提供依据。本研究的主要研究任务已经按计划完成。首先成功建立小鼠窦前卵泡海藻酸钠3D体外培养体系，并在此基础上探究PTEN抑制剂对体外培养小鼠颗粒细胞增殖与雌激素合成功能的作用；进而筛选可促进卵泡体外生长的因子，证实神经营养因子4（NT4）可促进小鼠卵泡体外生长及成熟，可能通过下调PTEN通路、影响卵母细胞成熟相关基因的表达来实现；最终结合优化因子及自主研制的体外成熟系统，成功构建国内首个可获得成熟卵母细胞的人卵泡体外培养体系，并检验NT4对人卵泡体外生长的促进作用，部分成果已申请国家发明专利，发表在Reproductive Biology and Endocrinology等期刊。本研究的主要研究目标目前已经实现，建立了高效、完整的人卵泡激活及卵泡体外培养体系，并评估其有效性。发表SCI论文2篇，核心期刊3篇，申请2项国家发明专利，培养硕士研究生2名，博士研究生5名。