外泌体介导的miRNA传递在脂肪干细胞微球治疗阴茎勃起功能障碍中的功能研究

Due to the scarcity of evidence supporting cellular differentiation, most researchers hold the opinion that paracrine action is the principal therapeutic mechanism of stem cell therapy for erectile dysfunction (ED). However, the majority of paracrine factors in vivo have short half-life period which need to exist continuously in order to play an effect. Under the protection of the outer lipid membrane, small molecular genetic material carried by exosome can regulate the functions and phenotypes of its target cells in a more stable way. Therefore, applicant put forward the scientific hypothesis: intercellular communication mediated by exosome is the internal mechanism of stem cell therapy for ED. The aim of this project is to promote the therapeutic potential of adipose derived stem cell-based microtissues with its internal hypoxia and nutrient deficiencies, and focus on the role of exosome-shuttle miRNA on its therapeutic effect. Firstly, the role of exosomes on stem cell therapy for ED will be verified with the aid of in vitro experiments and ED model. Secondly, the differentially expressed miRNA will be identified, and their regulatory networks will be built. Based on the above, the key miRNA which may play an important role on ED repair can be identified. Finally, the role of selected miRNA in the ED model was validated using specific inhibition and overexpression techniques. Through this study, it is expected to reveal the molecular mechanism of stem cell therapy for ED on the level of post-transcriptional regulation of gene expression.

由于缺乏分化证据，普遍认为旁分泌作用是干细胞参与阴茎勃起功能障碍（ED）修复的主要方式。但是，多数旁分泌因子在体内半衰期短，需持续存在方能发挥效应。外泌体受外层脂质膜保护，可实现细胞间小分子遗传物质的稳定传输，并以此调控靶细胞表型和功能。由此，申请者提出“干细胞通过外泌体介导的信息传递参与ED修复”的科学假设。在前期研究基础上，该项目拟利用干细胞微球内部低氧、营养缺乏的“应激微环境”提高脂肪干细胞治疗潜能，并重点研究其外泌体携带的miRNA在ED修复中的功能。首先，借助体外实验和ED动物模型验证外泌体在ED干细胞疗法中的作用。然后，识别脂肪干细胞微球外泌体中的差异表达miRNA并构建其可能参与调控的功能网络，从中挑选出可能对ED修复起关键作用的miRNA。最后，通过特异性上调或抑制备选miRNA，进一步验证其在ED修复中的作用。通过本研究，有望从转录后调控水平揭示ED干细胞疗法的分子机制。

经海绵体注射脂肪干细胞（ADSCs）对神经损伤等原因造成阴茎勃起功能障碍（ED）具有良好的治疗效果。但是，阴茎海绵体是与血液循环直接相通的血窦结构，经海绵体注射单细胞悬液存在局部滞留率低、细胞进入外周循环造成肺栓塞等风险。该研究旨在利用悬滴培养法将ADSCs体外聚集为集团化的干细胞微球，通过调整干细胞微球自身体积大小，提高干细胞在阴茎海绵体的滞留率，并减少游离干细胞通过血液循环进入肺脏造成的相关风险。将不同粒径的聚丙乙烯微球（PSMs）混悬液经海绵体注射，通过检测下腔静脉和腹主动脉血液中的PSMs，据此选择合适粒径大小的ADSCs微球用于海绵体内注射。通过损伤双侧海绵体神经建立神经性ED动物模型，将ADSCs单细胞悬液和ADSCs微球经海绵体注射，对比评估各组阴茎勃起功能、组织病理变化及干细胞分化等。研究数据表明大鼠阴茎海绵体引流静脉可允许直径小于80~100μm的PSMs通过，含有1000个细胞的ADSCs微球直径约153.26±23.74μm，能够利用自身较大的体积滞留于阴茎海绵体内。ADSCs单细胞悬液经海绵体注射后，短时间就有大量细胞进入外周循环并滞留于肺脏微循环内。而且，随着经海绵体注射ADSCs总细胞数量增多，肺栓塞及实验动物死亡数量也逐渐增多。当总细胞数量不高于1×10^6时，尽管能在肺脏微循环内观察到大量栓塞，实验动物死亡率较低。在相同细胞数量条件下，经海绵体注射ADSCs微球对阴茎勃起功能及nNOS+神经纤维的修复作用更明显，且无明显肺栓塞及实验动物死亡现象发生。经海绵体注射ADSCs单细胞悬液或微球后，未观察到ADSCs向其他功能细胞分化的证据。该研究表明，经海绵体注射ADSCs单细胞悬液或微球，对神经性ED动物模型的阴茎勃起功能及组织病理变化均有改善作用，但其作用机制可能与干细胞分化无关。由于ADSCs微球在阴茎阴茎海绵体具有较高的滞留率，在总细胞数量相同时比ADSCs单细胞悬液疗效更好，并有效减少肺栓塞发生。