CRISPR/dCas9技术介导的联合基因修饰人羊膜上皮细胞治疗糖尿病ED的实验研究

More than a half of the patients with diabetic erectile dysfunction (DMED) are not responsive to the treatment of PDE-5 inhibitors. The intracavernous injection of gene modified stem cells for the treatment of DMED has been a hot area of research for several years, while the key is to find the best kind of stem cells and suitable gene targets. Human amniotic epithelial cells (hAECs) are a recently identified type of adult stem cells. However, will hAECs also be an ideal therapeutic cell type for DMED? And will multiple genes modified stem cells be better than single gene modified ones? Previously, we have confirmed hAECs could be induced and differentiated into endothelial cells, smooth muscle cells and nerve cells in vitro, which indicating hAECs may be a suitable carrier of gene therapy for DMED. At the same time, CRISPR/dCas9, which making multiple genes modification come true, has been developed rapidly. Therefore, in this study we plan to simultaneously activate the expression of eNOS and VEGF in hAECs by CRISPR/dCas9 and transplant these cells into the penes of DMED rats. What’s more, the efficacy and possible mechanism will be comprehensively evaluated. If successfully, we will find a suitable therapeutic cell type. In addition, the first use of multiple genes modified stem cells may improve the efficacy of gene modified stem-cell therapy for DMED and lay the root for clinic application.

一半以上的糖尿病ED患者对PDE-5抑制剂不敏感，利用基因修饰的干细胞进行阴茎海绵体移植治疗糖尿病ED是目前研究的热点，其中寻找理想的干细胞类型和合适的基因治疗靶点非常关键。人羊膜上皮细胞hAECs具有干细胞特性，针对ED基因治疗多为靶向单基因的格局，联合基因修饰的hAECs能否应用于糖尿病ED的治疗呢？前期我们已经在体外成功将hAECs诱导分化为内皮细胞、平滑肌细胞及神经细胞，可作为ED基因治疗的合适载体。因此本项目拟应用可实现细胞内多个基因同时修饰的CRISPR/dCas9技术来修饰hAECs，同时激活hAECs内两个成熟的ED基因治疗靶点eNOS和VEGF的表达，分别在体内和体外，细胞和分子水平探索eNOS和VEGF基因修饰的hAECs（hAECs－eNOS-VEGF）阴茎海绵体移植治疗糖尿病ED的效果，打破ED单基因治疗模式，为临床应用基因修饰干细胞移植治疗ED打下基础。

利用基因修饰的干细胞进行阴茎海绵体移植来治疗糖尿病ED是目前研究的热点。人羊膜上皮细胞（hAECs）具有干细胞特性，但实验中发现高质量的hAECs细胞较难获取，且hAECs经过双基因修饰后存活率低，不是治疗糖尿病 ED 的理想干细胞。因此我们改用脂肪来源的干细胞(ADSCs)代替hAECs，运用 CRISPR/dCas9 技术修饰 ADSCs，并在成功建立DMED大鼠模型后，用ADSCs-eNOS-VEGF进行阴茎移植，检测各组细胞移植治疗对阴茎海绵体组织中相关信号通路以及大鼠勃起功能的影响。结果提示使用eNOS和VEGF基因联合修饰ADSCs进行阴茎移植治疗,能减少糖尿病大鼠阴茎组织纤维化并恢复其勃起功能，这些作用可能与NO-cGMP-PKG通路、VEGF通路、RhoA/ROCK激酶通路的激活有关。CRISPR/dCas9技术可同时激活多个基因表达，打破ED单基因治疗模式，有望改善目前糖尿病ED的治疗效果。从实验结果看，虽然基因联合修饰干细胞移植能够改善勃起功能，但外源性移植物在移植后存在干细胞不能在目的器官定植等问题，导致效果不甚理想，我们也尝试激活阴茎内源性干细胞，目前已成功定位和分离阴茎干细胞（PSPCs），下一步实验拟探索内源性干细胞治疗ED的疗效。同时我们也在lncRNA-miRNA-mRNA调控范式角度做了一些拓展性的实验研究，尝试为糖尿病ED的治疗提供新的切入点。