间充质干细胞在卵巢组织移植中对卵泡存活和生长发育的作用及相关机制研究

Ovarian tissue cryopreservation and transplantation has been gradually acknowledged as a novel technique in fertility preservation especially for cancer patients. However, the main obstacle of the technique is massive follicle loss and follicle developmental block as a result of ischemia in the transplantation. Therefore, reconstruction of blood perfusion as soon as possible would be of great importance to enhance the survival and functional recovery of the ovarian grafts. In our previous studies, we found that ① mesenchymal stem cell (MSC) was capable of secreting various angiogenic factors ② and furthermore it could significantly enhance the migration capability of endothelial cell and vascular formation. Due to the previous findings, it may be hypothesized whether MSC could be applied to enhance the vascular formation and blood perfusion through secretion of certain key factors in order to preserve the fertility in the ovarian transplantation. Based on the above hypothesis, the study will make use of human ovarian tissues, construct the animal models of co-transplantation of MSC and ovarian tissues and combine the technique of follicle separation from the transplanted grafts and 3-D in vitro culturing model, to examine the effects of MSC on vascular formation, blood perfusion, follicle survival and subsequent development respectively. Then, the study will take advantage of cytokine array to screen the differential factors in the grafted tissues among different groups, and carry out functional verification to analyze the corresponding mechanisms. The completion of the study would supply the theoretical foundation of the clinical utilization of MSC in ovarian tissue transplantation and fertility preservation.

卵巢组织冷冻和移植正成为肿瘤患者生育力保存新的方向。然而移植后缺血缺氧损伤所致的大量卵泡丢失、卵泡活化和发育障碍是制约其临床应用的核心问题。尽早建立血流灌注是移植物存活和功能恢复的关键环节。前期研究申请者发现①间充质干细胞MSC可分泌多种促血管形成的关键因子②体外趋化和成管实验证实MSC可显著提高血管内皮细胞的迁移能力并促进管腔形成。上述现象提示：在卵巢移植中MSC可能分泌某些关键因子，介导血管新生并提高血流灌注，促进卵泡存活和生长发育，实现生育力的保存和恢复。本项目拟以人的卵巢组织为研究对象，建立MSC与卵巢组织皮下共移植动物模型，结合移植后卵泡分离和体外三维培养技术，从体内外两个层次分别验证MSC对血管新生、血流灌注、卵泡存活和生长发育的作用。继而利用细胞因子组织芯片，筛选移植物中差异表达的关键因子并进行效应验证，逐步阐明其分子机制，为MSC在卵巢移植和生育力保存中的应用提供理论基础。

本研究按计划完成了相关实验方法和平台的建立、稳定和优化，构建了人冷冻卵巢皮质组织裸鼠移植的动物模型，完成了MSC对血管形成、血流灌注、卵泡存活和生长发育等方面的效应研究。在此基础上，本项目为研究MSC共移植可提高移植后卵巢组织活力，利用人卵巢组织异种移植小鼠模型研究，在卵巢组织移植体系中加入间充质干细胞（MSC）共移植，发现MSC可促进卵巢组织移植后血管再生与卵泡存活。然后，利用蛋白芯片技术筛选了人卵巢组织与MSC共移植后表达显著上调的促血管生成相关因子，发现卵巢组织与MSC共移植组和卵巢组织单独移植组相比，Angiogenin（ANG）等细胞因子的表达水平显著上调。在核酸水平和蛋白水平敲减或阻断MSC相关因子ANG后的共移植实验发现，卵巢移植物的血管密度显著下降，卵泡丢失数量显著上升，卵泡凋亡比例显著上升，从而证实了在卵巢移植体系微环境中，ANG因子在MSC促进人卵巢组织移植物血管再生与防止人类卵泡丢失中起重要作用。本研究探索了MSC促进卵巢移植物中血管再生与卵泡存活的分泌机制，在局部微环境角度揭示了调控卵巢移植物中卵泡发育的外在分子机制，为攻克人卵巢组织移植技术难关，改善卵泡体外发育潜能与卵母细胞成熟的技术体系提供了有力支撑。

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