CTF1修饰的ADSC外泌体促进子宫内膜微血管新生的作用及机制研究

Endometrial receptivity decline due to abnormal endometrial angiogenesis is an important cause for repeated embryo implantation failure (RIF). Studies in recent years find that adipose-derived stem cell(ADSC) exosome has the effect of facilitating angiogenesis but with difference existing in actual curative effect, which might be related to action efficiency of exosome and microenvironment in lesion region. It’s found in our preliminary experiment that exosome has weak ability of facilitating endometrial microvascular endothelial cell proliferation. Recent studies find that gene modification of stem cells can enhance relevant functions of its exosome. Cardiotrophin (CTF)1 can not only significantly promote angiogenesis but also can improve efficiency of MSC implantation into target organ and increase MSC survival rate. It’s assumed that: using CTF1 to modify ADSC and elevate CTF1 level in exosomes can facilitate endometrial angiogenesis and improve endometrial receptivity more significantly. This project plans to use nuclear transfection to make CTF1 over-expression in ADSC so as to study influence of CTF1 transfer by its exosome (C-ADSC-Exo)on angiogenesis and endometrial receptivity. With relevant receptor antagonists of CTF1 combined, change of signal path will be discussed and mechanism of C-ADSC-Exo facilitating endometrial angiogenesis so as to provide a new idea for clinical improvement of endometrial receptivity and RIF treatment.

子宫内膜血管生成异常引起内膜容受性下降是胚胎反复移植失败（RIF）的重要原因之一。近年来研究发现脂肪干细胞(ADSC)外泌体具有促进血管新生的作用，但实际疗效存在差异，可能与外泌体植入效率及病变区域的微环境有关。我们预实验发现其促进子宫内膜微血管内皮细胞增殖能力较弱。最近发现利用基因修饰干细胞可增强其外泌体的相关功能。心肌营养素（CTF）1不仅能显著促进血管新生，亦能增加MSC植入靶器官的效率，提高MSC存活率。我们假设：利用CTF1修饰ADSC，提高外泌体中CTF1水平，能促进子宫内膜微血管新生、改善内膜容受性。本项目拟利用核转染使ADSC过表达CTF1，研究其外泌体（C-ADSC-Exo）传递CTF1等对微血管新生以及子宫内膜容受性的影响，结合CTF1相关受体拮抗剂探讨信号通路变化，阐明C-ADSC-Exo促进子宫内膜微血管新生的机制，为临床上改善内膜容受性、治疗RIF提供新思路。

间充质干细胞来源的外泌体作为细胞治疗的关键效应介质和有效替代工具在子宫内膜再生领域受到了越来越多的关注。CTF1可促进血管内皮细胞增殖、迁移，以及血管形成，并可诱导胚胎成功植入子宫内膜，通过使MSC过表达CTF1后提取CTF1修饰的MSC外泌体可能会更好的促进子宫内膜血管新生以及内膜修复。首先，我们验证了CTF1-MSC-exo对血管内皮细胞增殖、迁移、及成管功能的影响。利用慢病毒转染技术，建立一套稳定的使MSC 过表达CTF1的体系，明确转染CTF1的效率及CTF1表达持续时间；通过Brdu实验发现CTF1-MSC-exo的促进血管内皮细胞增殖能力明显更强；利用Transwell实验证实CTF1-MSC-exo可以促进血管内皮细胞的迁移能力，成管实验发现CTF1-MSC-exo促进血管内皮细胞成管能力明显增强；其次，我们探索了CTF1-MSC-exo对薄型子宫内膜的血管新生及修复作用。我们通过免疫荧光和免疫组化也证实了CTF1-MSC-exo有着显著更多的新生血管。Masson染色显示CTF1-MSC-exo治疗组胶原纤维面积明显减少，纤维化程度明显减少，除此我们还发现CTF1-MSC-exo治疗组薄型子宫内膜大鼠妊娠率明显提高。最后，我们揭示了CTF1-MSC-exo促进子宫内膜血管新生及修复的分子机制。不论在体外还是体内实验中，CTF1-MSC中及CTF1-MSC-exo中mTOR/JAK/PI3K/STAT3/AKT信号通路的磷酸化水平均明显上调。我们的研究结果阐明了CTF1修饰的干细胞外泌体促进薄型子宫内膜血管新生及修复的作用及其分子机制，该研究结果有希望应用于薄型子宫内膜的治疗。