The studies with adipose-derived stem/stromal cells therapeutic development of erectile dysfunction

前言与目的：糖尿病患者由于海绵体严重受损，对磷酸二酯酶5抑制剂(PDE-5I)的治疗更难治。我们以前曾报道，海绵体内急性注射肾上腺髓质素(AM)可增强大鼠的勃起功能。在对脂肪组织源干细胞(ASC)产生的细胞因子进行筛选的过程中，我们发现ASC在含有血管内皮细胞(VEC)生长因子的培养液中培养时，尤其能产生AM。方法：从Wistar大鼠皮下脂肪组织中分离ASC，在含有血管内皮细胞生长因子的EBM或EGM+5%胎牛血清中培养1周，观察AM是否参与ASC诱导的勃起功能恢复。给雄性Wistar大鼠注射链脲佐菌素(STZ)。将用上述两种方法培养的干细胞注射到CorpU…中注射链脲佐菌素后第9周，模型大鼠和正常大鼠的阴茎S海绵体增多。测量平均动脉压(MAP)和海绵体内压(ICP)。我们使用慢病毒(LV)表达大鼠AM siRNA和ADAM，并检测ASC产生的AM是否在ASC诱导的勃起功能恢复中起关键作用。结果：1.给予ASC可使ICP恢复，给予ASC可显著提高ICP/MAP和AUC。2.ASC可恢复海绵体结构，注射ASC后，海绵体小梁变大，分布更致密。3.ASC产生AM，ASC以时间依赖的方式在培养液中分泌AM。当ASC感染表达AM siRNA的LV并培养1周后，ASC分泌AM的能力受到明显抑制。4.AM基因敲除及强迫表达的作用：将表达AM基因的LV siRNA(LV_AMsiRNA)或其阴性对照NC siRNA(LV_NC SiRNA)感染ASC，培养1周后注射入海绵体。经ICPMAP和AUC检测，感染LV_NC siRNA后，ASC勃起功能明显恢复。反之，当ASC感染LV_AM siRNA时，ASC诱导的ICP/MAP和AUC的恢复明显减弱。结论：ASC具有恢复勃起功能的潜能，ASC在培养过程中产生的AM在勃起功能恢复中的作用较小。

INTRODUCTION AND OBJECTIVES : Diabetic patients are more refractory to the treatment with phosphodiesterase type 5 inhibitors(PDE-5I) because of severe damage in their cavernous body. We have previously reported that acute administration of Adrenomedullin(AM) into the cavernous body enhances erectile function in rats. During the screening of cytokines that adipose tissue-derived stem cells(ASC) produce, we found that ASC produced AM especially when ASC were cultured in medium containing growth factors for vascular endothelial cells(VEC). In this study, we examined whether AM would be implicated in ASCinduced restoration of erectile function.METHODS : ASC isolated from the subcutaneous adipose tissue of a Wister rat was cultured for one week in either EBM or EGM medium which contains vascular endothelial cell growth factor such as VEGF-A, IGF with 5% FBS. Streptozotocin(STZ) was injected into a male Wister rat. Stem cells cultured using the two methods above were injected into the corpu … More s spongiosum penis of the model rat and a normal rat in the ninth week after STZ administration. The mean arterial blood pressure(MAP) and the intracavernosal pressure(ICP) were measured. We used lentivirus(LV) expressing rat AM siRNA and AdAM, and examined whether AM produced by ASC played pivotal roles in ASC-induced restoration of erectile function. RESULTS : 1. ASC administration restores ICP ; Administration of ASC significantly increased ICP/MAP and AUC. 2. ASC administration restores the structure of cavernous body ; When ASC were injected, the trabeculae of cavernous body became larger and were distributed more densely. 3. ASC produce AM ; The ASC secreted AM in the culture medium in a time-dependent fashion. When ASC were infected with LV that expresses AM siRNA and cultured for 1 week, AM secretion by ASC was significantly suppressed. 4. Effects of AM knockdown and forced expression of AM ; ASC were infected with LV expressing AM siRNA(LV_AMsiRNA) or its negtive control NC siRNA(LV_NC siRNA), cultured for 1 week, and injected in the cavernous body. When ASC were infected with LV_NC siRNA, ASC significantly restored erectile function, as assessed by ICP/MAP and AUC. In contrast, when ASC were infected with LV_AM siRNA, ASC-induced restoration of ICP/MAP and AUC was significantly diminished. CONCLUSIONS : These results suggested that ASC have potentials to restore erectile function and that AM produced by ASC when cultured especially in EGM plays a major role in the restoration of erectile function Less