雌激素作用下BMSCs结合改性PRP水凝胶支架的设计及其在子宫内膜损伤修复障碍中的应用

The current advances in assisted reproductive technology have made it possible to overcome many causes of female infertility. However, pregnancy is still notably difficult for patients with severe endometrial injury, which is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium. Regeneration of the endometrial cells is essential for patients with endometrial damage. However, severe damage to the basal layer may not be amenably repairable due to loss of the basal endometrial progenitor stem cells. Using Bone marrow-derived mesenchymal stem cells (BMSCs) to repair endometrium has become promising in the clinical research. BMSCs can differentiate into endometrial epithelium and accelerate the regeneration of injured endometrium. A major challenge of using stem cell, however, is the low percentage of cell retention after the transplantation to the wounded site. While in our previous study, platelet-rich plasma (PRP) as scaffolds for BMSCs has been proved to be able to greatly enhance BMSCs’ capability of proliferation and adherence, but we also found that the release of growth factors is temporal and PRP would degrade completely in just few days. Therefore, to address this problem, we plan to use PRP-Pluronic F127-pectin hydrogel scaffolds instead of PRP alone in this study because they are proteiform, degrade slowly and can have a controlled release of growth factors. In addition, considering that 17β-estradiol (E2) can effectively promote endometrial regeneration, we developed E2-PLGA nanoparticles (NPs) which keep E2 sustainedly releasing into the culture to induce the differentiation of BMSCs and the restoration and regeneration of injured endometrium. Furthermore, we intend to investigate the mechanism of BMSCs differentiation promoted by E2 via exosomes derived from endometrial stromal cell (ESCs). Our goal is to provide a novel and multidisciplinary approach to restore the endometrial function.

子宫内膜损伤修复障碍是不孕症治疗的瓶颈。损伤累及内膜基底层会造成内膜上皮再生障碍，无法自身修复。应用骨髓间充质干细胞(BMSCs)修复子宫内膜是临床研究的方向，但存在干细胞无法集聚于损伤部位，影响了干细胞增殖和分化的能力。我们前期用富血小板血浆(PRP)作为支架以利于BMSCs的生长，但其较快的降解速度使BMSCs的粘附和分化能力受限。课题组研制的Pluronic F127-果胶水凝胶降解缓慢，能延长生长因子释放，且能充分填充宫腔并原位凝固，将BMSCs局限于损伤部位。本项目中，我们将设计PRP-Pluronic F127-果胶水凝胶支架，对PRP改性，弥补单纯应用PRP的缺点；观察在包裹了雌激素（E2）的缓释系统持续稳定释放E2作用下，复合了PRP-Pluronic F127-果胶水凝胶支架的BMSCs治疗内膜损伤的效果。我们还将探索E2通过内膜间质细胞释放的外泌体促干细胞分化的机制。

子宫内膜损伤修复障碍是不孕症治疗的瓶颈。损伤累及内膜基底层会造成内膜上皮再生障碍，无法自身修复。应用骨髓间充质干细胞（BMSCs）修复子宫内膜是临床研究的方向，但存在干细胞无法集聚于损伤部位，影响了干细胞增殖和分化能力。为了克服这一难题，我们成功研制了：①果胶-Pluronic® F-127水凝胶支架。该支架不仅可为BMSCs提供得以附着的稳定的三维结构，还易于成型，可通过注射，到达子宫内膜损伤部位，并原位凝固，从而有效修复受损的子宫内膜；②制备了包封雌二醇（E2）的PLGA-E2纳米缓释载体微粒。该纳米微粒可使E2在7天内呈持续、稳定地释放；③我们发现，在PLGA-E2纳米缓释载体微粒的作用下，BMSCs联合果胶-Pluronic®F-127水凝胶支架可有效地促进BMSCs的分化和子宫内膜细胞的增殖和再生，恢复内膜功能，使其基本达到正常水平。.在此基础上，我们还评价了富血小板血浆（PRP）联合BMSCs治疗大鼠子宫内膜损伤的效果。证明了在PRP的参与下，BMSCs会进一步增强内膜血管的生成和平滑肌的增生；而PRP通过促进BMSCs合成和分泌IGF-1和IL-10，从而提高了BMSCs的分化能力，促进了大鼠受损子宫的修复。此后，我们进一步探讨了PRP诱导BMSCs中IL-10表达和分泌的分子机制，证明PRP通过p50亚基激活NFκB途径，促进IL-10及其下游IGF-1的表达。至此顺利完成了研究任务。此外，我们还利用高地水芹、三水合氯化金制备了具有抗菌活性的纳米金，增加了感染预防的益处，超额完成预设的研究目标。上述研究成果将为临床治疗严重的子宫内膜损伤修复障碍提供新的方法和理论依据。