金丝桃苷纳米粒与骨髓间充质干细胞协同给药修复子宫内膜损伤

Hyperoside nanoparticles loaded with bone marrow mesenchymal stem cells synergistically repair endometrial injury

目的体外细胞实验评价金丝桃苷壳聚糖纳米粒(hyperoside/chitosan-nanoparticles,Hyp/CS-NPs,Hyp-NPs)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的影响及作用机制;体内动物实验探讨Hyp-NPs与BMSCs协同修复大鼠子宫内膜损伤的效果。方法采用流式实验鉴定BMSCs;采用离子交联法制备Hyp-NPs,激光粒度分析仪及透射电镜进行表征评价;采用划痕实验检测Hyp-NPs对BMSCs的迁移作用;以及构建NRF2慢病毒载体,检测Hyp-NPs对BMSCs的氧化损伤模型的作用机制;动物实验将Hyp-NPs与BMSCs协同移植入子宫内膜损伤大鼠模型宫腔中,利用HE、Masson、IHC、TUNEL及ELISA实验从子宫内膜的一般病理、纤维化、干细胞募集及炎症情况,评价其对大鼠子宫内膜损伤的修复作用及妊娠功能。结果成功分离培养BMSCs;成功制备Hyp-NPs;划痕实验表明Hyp-NPs可促进BMSCs迁移;通过成功构建BMSCs的慢病毒NRF2载体以及细胞氧化损伤模型,表明Hyp-NPs可通过激活NRF2调控BMSCs生物轴;动物实验表明Hyp-NPs与BMSCs协同给药可增加子宫内膜厚度及腺体数量,抗纤维化、抗凋亡及抗炎等促进干细胞归巢,恢复子宫内膜损伤大鼠的妊娠功能。结论Hyp-NPs与BMSCs协同给药可修复子宫内膜损伤。

Aim To evaluate the effect of hyperoside/chitosan-nanoparticles(Hyp-NPs)on bone marrow mesenchymal stem cells(BMSCs)in vitro cell experiments and the underlying mechanism,and to conduct in vivo animal experiments to investigate the synergistic effect of Hyp-NPs and BMSCs on repairing endometrial damage in rats.Methods BMSCs were identified by flow cytometry.Hyp-NPs were prepared by ion crosslinking method,characterized and evaluated by laser particle size analyzer and transmission electron microscopy.The effects of different concentrations of Hyp-NPs on the migration of BMSCs were evaluated by scratch assay and immunofluorescence.NRF2 lentivirus vector was constructed to explore the mechanism of Hyp-NPs on BMSCs.In animal experiments,Hyp-NPs loaded with BMSCs were co-transplanted into the uterine cavity of a rat model of endometrial injury.HE,Masson,IHC,TUNEL,and ELISA experiments were used to systematically evaluate the repair effect and pregnancy function of the composite formulation on rat endometrial injury from multiple aspects and angles,including general pathology,fibrosis,receptivity,cell proliferation,angiogenesis,stem cell recruitment,and inflammation of the endometrium.Results BMSCs were successfully isolated and cultured.Hyp-NPs with high stability and small particle size were successfully prepared.Scratch experiments indicated that Hyp-NPs could promote the migration of BMSCs.By successfully constructing a lentiviral NRF2 vector and oxidative damage model for BMSCs,immunofluorescence experiments showed that Hyp-NPs could regulate the biological axis of BMSCs by activating NRF2.Animal experiments showed that the synergistic administration of Hyp-NPs and BMSCs could increase endometrial thick-ness and glandular quantity,promote stem cell homing through anti-fibrotic,anti-apoptotic,and anti-inflammatory effects,and restore pregnancy function in rats with endometrial injury.Conclusion The synergistic administration of Hyp-NPs and BMSCs could repair endometrial injury.