RNA干扰与基因修饰BM-MSCs移植联合应用治疗老年ED的作用及机制研究

Erectile dysfunction (ED) in the eldly is a common disorder. There are currently not much optimal therapy options for it even if inhibitors of phosphodiesterase-5 (PDE5) have been clinically medicated. It has been demonstrated that the major cause of ED in the eldly is a diffuse and progressive corporal fibrosis in the penis associated with aging. Granted by NSFC, animal models of ED with the aging were succefully established, and our preliminary study showed that the recovery of erectile function in the aged rats could be improved by intracavernous injection of genetically modified mesenchymal stem cell (BM-MSCs) with a lentiviral vector constructs targeting PDE5 (PDE5-RNAi-LV) by interference. However, its mechanism remains still obscure. We hypothesize that BM-MSCs modified with PDE5-RNAi-LV could differentiate into smooth muscle cells (SMCs) and might help to regenerate damaged penile smooth musculature after BM-MSCs are transplated. This study aims to elucidate the molecular mechanisms by which the inhibition of PDE5 with a silencing vector in BM-MSCs transplanted into the penis of the aged rat. In vivo MR imaging is used to track delivered BM-MSCs after superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells are intracavernously injected. Erectile function is assessed by intracavernous pressure (ICP) measurement during electrostimulation of the cavernous nerve. The content of smooth muscle in corporal tissue sections will be detected by histopathological examination and immunohistofluorescence. PDE5 expression in cavernous smooth muscle will be examined by RT-PCR and Werstern blot. The down-regulation of PDE5 by RNA interference will be measured by cGMP Direct Immuno-ssay Kit which provided a direct competitive immuno-assay for sensitive and quantitative determination of cGMP. It is envisaged that stem-cell-based gene therapy for age-associated ED may serve as a new strategy in the near future.

老年ED常见，其重要原因是阴茎海绵体平滑肌变性纤维化，因此高选择性PDE5i治疗效果仍欠佳。我们前期在NSFC资助下已成功建立老年ED动物模型，并初步研究证实RNAi靶向沉默PDE5与基因修饰BM-MSCs移植联合应用可增强老龄大鼠阴茎勃起功能。那么其时效性和可能的分子机制是什么？进而预实验研究表明基因修饰BM-MSCs移植可修复老龄大鼠变性纤维化阴茎海绵体。下一步拟深入探讨PDE5-RNAi-LV与基因修饰BM-MSCs联合移植治疗老年ED的时效性和BM-MSCs移植后对阴茎海绵体的修复程度及其对PDE5的靶向抑制效应。本项目拟采用基因转染、ICP测压、SPIO标记MR成像活体动态示踪、双标荧光鉴定BM-MSCs分化和PDE5活性检测等方法，旨在阐明RNAi靶向沉默PDE5与基因修饰BM-MSCs移植联合应用治疗老年ED的分子机制，从而为二者联合应用于基因治疗老年ED提供充分的科学依据。

在该基金资助下，我们成功建立了老龄ED动物模型，并在BMSC作为细胞载体进行基因治疗ED的系列实验研究中得到了应用。前期我们已熟练掌握胶原酶融骨法分离培养SD大鼠BMSC及诱导分化鉴定等，本项目采用RNA干扰技术构建靶向沉默PDE5基因表达的PDE5-RNAi-Lv，转染SD大鼠BMSC并注射移植于老龄ED大鼠阴茎海绵体内，结果证实RNA干扰联合基因修饰BMSC移植可改善老龄ED大鼠阴茎勃起功能；随后通过留取各组不同时间点阴茎海绵体组织标本在形态学和分子水平深入研究了PDE5-RNAi-Lv与基因修饰BMSC联合移植治疗老年ED的时效性和BMSC对变性纤维化阴茎海绵体修复程度以及对PDE5表达的靶向抑制效应，结果表明在注射移植后的2-4周，基因修饰BMSC治疗组的老龄大鼠阴茎勃起功能均得到不同程度改善，且效果优于单纯BMSC治疗组；同时，RNA干扰联合基因修饰BMSC注射移植后可增加阴茎海绵体组织中平滑肌细胞和内皮细胞的含量，并通过抑制目的基因PDE5表达使得BMSC更多地向平滑肌细胞分化而发挥作用。本项目在阐明PDE5-RNAi与基因修饰BMSC移植联合应用可改善老龄ED大鼠勃起功能的同时，也通过分子生物学技术揭示了靶向沉默PDE5表达的基因修饰BMSC可增加阴茎海绵体组织中平滑肌细胞标志物SMA、Smoothelin和内皮细胞标志物CD31、eNOS的表达而发挥作用的分子机制，该项目系列研究成果详见结题正文成果部分。