尿源性干细胞通过外泌体/miRNAs调控海绵窦内皮功能治疗糖尿病性勃起功能障碍大鼠的作用机制研究

The core pathologic mechanism of diabetic erectile dysfunction (DED) is endothelial dysfunction. However, current treatment for DED is insufficient. Our previous studies demonstrated that urine derived stem cells (USCs) could significantly improve the penile cavernous endothelial function and erectile function of DED rats, but the pathway and key regulation point of USCs repair the endothelial dysfunction remain unclear. Some other studies showed that paracrine mechanism mediated by the exosomes enrich miRNAs which were secreted by stem cells was an important mechanism for the stem cell therapy. Our pilot study showed the USCs could secrete exosomes and these exosomes enrich miRNAs. The abnormal miRNAs in the penis of DED rats would be partly corrected by USCs transplantation. Therefore, we hypothesis that USCs can restore the impaired endothelial function and improve the erectile dysfunction of DED rats by the exosomes enrich miRNAs. We are planning to analyze the cross-talk mechanisms of different signaling pathway mediated by the miRNAs regulation network by gene microarray, RPPA analysis and bioinformatics prediction et al. This project is aim to obtain the direct evidence that USCs can restore the endothelial dysfunction and erectile dysfunction by secreting exosomes enrich miRNAs. It will contribute to a better understanding of the mechanisms and key regulation node of USCs in the treatment of erectile dysfunction, which may also provide a theoretical basis for promoting the stem cell therapy in the treatment of DED.

糖尿病性勃起功能障碍(DED)的核心发病机制为阴茎海绵窦内皮功能障碍，现有疗法效果不佳。我们己证明尿源干细胞(USC)可改善DED大鼠阴茎海绵窦内皮及勃起功能，但其作用机制及调控节点尚不清楚。有研究发现由干细胞外泌体中miRNAs介导的旁分泌可能是干细胞治疗的主要作用机理。预实验发现USC在体外可分泌含miRNAs的外泌体，将USC移植入DED大鼠阴茎内可恢复部分miRNAs的表达。据此我们提出“USC通过外泌体中的miRNAs调控修复DED大鼠阴茎海绵窦内皮功能而改善其勃起功能”的假设。本项目结合基因芯片与蛋白芯片、分子生物学等方法探讨USC所分泌外泌体中的miRNAs介导不同信号通路之间交叉串话的机理，旨在获得USC通过外泌体调控修复DED大鼠内皮功能及勃起功能的直接证据，将助于更深刻地理解USC治疗DED的作用机制与调控节点，为推动干细胞疗法成为勃起功能障碍治疗的新手段提供理论依据。

糖尿病性勃起功能障碍(DED)的核心发病机制为阴茎海绵窦内皮功能障碍，现有疗法效果不佳。我们己证明尿源干细胞(USC)可改善DED大鼠阴茎海绵窦内皮及勃起功能，但其作用机制及调控节点尚不清楚。有研究发现由干细胞外泌体中miRNAs介导的旁分泌可能是干细胞治疗的主要作用机理。预实验发现USC在体外可分泌含miRNAs的外泌体，将USC移植入DED大鼠阴茎内可恢复部分miRNAs的表达。据此我们提出“USC通过外泌体中的miRNAs调控修复DED大鼠阴茎海绵窦内皮功能而改善其勃起功能”的假设。为验证此科学假设，本项目优化了USCs的培养、分离、体外增殖调控体系，改进了USCs-exo的提取方法，使其提取效率较常规方法提升近百倍。成功建立了糖尿病勃起功能障碍（DED）大鼠模型，并成功分离鉴定DED大鼠阴茎海绵窦内皮细胞。通过体外实验证明USC-exo可以旁分泌的方式转运进入DED大鼠阴茎海绵窦内皮细胞，USC-exo可通过Spred-1/VEGF/VEGFR1/eNOS通路提高细胞增殖及存活能力。进行RNA-seq分析显示USCs-exo中表达量最高的15种miRNA中，11种miRNA具有促血管生成作用。体内实验中，我们对糖尿病勃起功能大鼠模型进行USCs及USCs-exo注射，证明USCs-exo可以miRNAs通过改善DED大鼠的阴茎海绵窦内皮功能以及海绵体平滑肌功能，最终治疗勃起功能障碍。此外，我们还发现USCs-exo除能通过miRNAs改善DED大鼠的阴茎海绵窦内皮功能和勃起功能障碍以外，还能通过抑制内皮细胞自噬发挥作用。总之，通过本项目的实施，己获得USC通过外泌体调控修复DED大鼠内皮功能及勃起功能的直接证据，更深刻地理解USC治疗DED的作用机制与调控节点，将有助于糖尿病性勃起功能障碍这一大类难治性勃起功能障碍患者得到有效治疗，也为推动干细胞疗法成为勃起功能障碍治疗的新手段提供理论依据。并己发表SCI论文10篇，申请专利2项，培养博士研究生2名，硕士研究生2名，非常圆满地完成了本项目的所有研究目标。