Nestin通过Nrf2调控间充质干细胞活性并逆转神经损伤性ED的机制研究

The incidence of erectile dysfunction (ED) caused by nerve injury during rectal cancer surgery remains high, and thus leads to a worldwide problem. Mesenchymal stem cells (MSCs) have been proved to restore the erectile function of cavernous nerve injury (CNI) rats through paracrine mechanism, while the disadvantage of insufficient in vivo activity has limited the clinical application of MSCs. In the previous study, we proved that the MSCs with higher expression level of Nestin revealed more survival time in vivo, stronger paracrine ability and better restoration of erectile function in CNI rats. However, the exact mechanism is still largely elusive. Our subsequent study revealed that Nestin was effective in inhibition of ubiquitination of Nrf2 caused by Keap1, promoted phosphorylation and nuclear admission of Nrf2, thus regulated cell viability. On this basis, we plan to establish the Nestin over-expressed MSCs, and prove its enhanced viability and better effect on restoration of erectile function in CNI rats. In addition, we detect the Nestin, Keap1-Nrf2 signal pathway and targeted gene expression, to investigate the mechanism of enhanced viability of MSCs mediated by Nestin, and molecular mechanism of restoration of nerve injury related ED through paracrine effect. This study aims to illuminate the molecular mechanism of MSCs viability and nerve restoration regulated with Nestin, and provide theoretical value and practical significance to improve the therapeutic effect of nerve injury related ED.

直肠癌术中神经损伤导致勃起功能障碍（ED）高发，其治疗是世界难题。间充质干细胞（MSCs）能通过旁分泌作用改善阴茎海绵体神经损伤（CNI）大鼠勃起功能，但存在体内活性低下的短板。我们发现高表达Nestin的MSCs在CNI大鼠体内存活时间更长，旁分泌作用及勃起功能修复效果更强，然具体调控机制未明。进一步研究发现Nestin可抑制Keap1对核因子相关因子（Nrf2）的泛素化降解，促进Nrf2磷酸化及入核，调控细胞活性。在此基础上，本项目拟构建Nestin过表达的MSCs，体内外实验证实其活性增强并高效修复CNI大鼠勃起功能；通过Nestin、Keap1-Nrf2通路及靶基因检测，阐述Nestin通过Nrf2调控MSCs活性，及通过旁分泌作用逆转神经损伤性ED的分子机制。本研究对阐明Nestin调控MSCs活性及神经修复的分子机制，提高神经损伤性ED的治疗水平具有重要的理论价值和现实意义。

直肠癌术后勃起功能障碍（Erectile dysfunction, ED）高达50%，根源在于术中难以完全避免的盆腔自主神经（PAN）损伤。课题组利用神经电刺激技术辨认保护PAN，显著降低ED发生率，但仍有20%男性直肠癌患者无法避免。对神经损伤性ED患者，目前临床无有效治疗方法。利用脂肪干细胞（Adipose stem cells, ADSC）修复神经损伤的基础研究有望解决这一临床难题，然而当前缺乏直观的体外盆腔神经节（major pelvic ganglion, MPG）损伤模型，ADSC在修复神经损伤的过程中亦存在活性低下的缺点。本研究对成年SD大鼠MPG进行体外培养，并通过不同浓度过氧化氢(Hydrogen peroxide, H2O2 )诱导MPG损伤，有效模拟直肠癌术后神经损伤状态，以期为基础研究提供有效模型基础。进一步，通过药物干预ADSC，调节Nrf2 (Nuclear factor erythroid-2 related factor) 因子表达状态后，探讨对神经损伤性勃起功能障碍修复效果的影响。结果证实：使用RPMI-1640培养基培养，并以200μmol/L H2O2诱导后，可以构建稳定的成年SD大鼠MPG体外损伤模型。Nrf2通过提高ADSC的抗氧化应激能力，提高ADSC的活性，发挥更好的神经组织及勃起功能修复效果。.本研究通过构建体外稳定的MPG损伤模型，并获取体内高效活性的MSCs，探明其修复神经损伤性ED的作用机制及高效性，对提高神经损伤性ED的治疗水平具有重要的理论价值和现实意义。目前已发表文章3篇，其中1篇在IF 13.787的顶尖期刊Annals of Surgery上发表，并有实用新型专利1项；参与本课题的2名博士研究生及1名硕士研究生均顺利毕业。