低能量体外冲击波治疗糖尿病膀胱功能障碍机制的研究

Diabetic bladder dysfunction (DBD) refers to a range of voiding complaints that are prevalent in men and women with diabetes mellitus. There are many treatment options for voiding symptoms that may be applied to patients with DBD; however, patients with diabetes are often refractory to management due to low response to therapy or high recurrence rates. Low-energy shock wave (LESW) therapy is a relative new treatment modality in regeneration medicine, especially in promoting the healing of bone fractures and diabetic/nondiabetic skin ulcers. Although LESW has been applied in some pilot studies and even clinical trials, its precise mechanisms remain unclear. Revascularization and anti-inflammatory are considered as the main pathophysiological changes of the wound tissues. We previously found LESW could enhance the recruitment of stem cells to the bladder, and researchers found LESW could enhance the recruitment of endothelial progenitor cells by up-regulating stromal cell-derived factor-1 (SDF-1) in wound tissues. Hence, we assume that stem cells play important roles in the healing process of LESW. We hypothesize that the over-expression of SDF-1 induced by LESW activates stem cells to recruit to the wound tissues. Then the stem cells will proliferate and secrete many growth factors and trophic factors to promote the wound regenerations. To address these issues, in this proposed study, we aim 1) to study whether LESW could ameliorate DBD; 2）to study whether LESW could enhance the recruitment of stem cells to the bladder; 3) to study whether LESW could promote the proliferation and secretion of adipose tissue-derived stem cells (ADSCs); 4) to study the molecular signaling pathways in ADSCs activated by LESW.

糖尿病膀胱功能障碍（DBD）是糖尿病常见并发症，尚无有效治疗措施。本项目利用低能量体外冲击波（LESW）治疗DBD并研究其治疗机制。我们前期实验发现LESW可诱导外源性间充质干细胞在膀胱组织中定植，并能在体外促进间充质干细胞的分泌。本项目提出了关于LESW治疗DBD机制的新假说，即LESW通过促进膀胱组织分泌基质细胞衍生因子1，诱导间充质干细胞向膀胱趋化，干细胞被激活后分泌大量细胞因子，促进损伤组织血管和神经修复。本项目拟利用LESW体外冲击脂肪来源干细胞（ADSCs），检验能否激活ADSCs，促进其增殖和分泌；拟利用LESW治疗DBD大鼠，检验LESW能否促进膀胱功能恢复，能否促进内源性干细胞向膀胱趋化；拟利用LESW与ADSCs联合治疗DBD，检验LESW能否促进外源性干细胞向膀胱趋化。本项目提出了治疗DBD的新措施，探讨了LESW治疗的新机制，为后续临床实验提供了研究基础。

1.项目背景. 目前尚无针对糖尿病膀胱功能障碍(DBD)有效的治疗方法。我们既往发现脂肪来源间充质干细胞（ADSCs）能促进DBD功能的恢复。低能量体外冲击波（DLSW）是修复医学领域中新兴治疗方式，近年来对于DLSW的基础研究越来越多，甚至已经开展临床试验，然而其治疗机制仍然不明。迄今为止，DLSW治疗过程中是否存在干细胞的激活，激活后的干细胞怎样发挥其作用尚无明确结论。.2.主要研究内容. 在体外实验中，研究DLSW能否影响间充质干细胞表面标记物，能否促进充质干细胞的分泌分裂增殖。间充质干细胞内哪些分子信号通路被激活。在体内，研究DLSW能否促进DBD大鼠膀胱排尿功能的恢复及大鼠膀胱组织学的改善情况，研究DLSW能否诱导内源性干细胞向膀胱趋化募集，并确定募集的干细胞的特性。.3.重要结果及关键数据. (1)骨髓间充质干细胞（BMSCs）冲击后分泌更多的血管内皮生长因子（VEGF）和CXC配体-5（CXCL5）。CD166，CD44和CD34没有明显的表达区别。表达更多的PCNA和Ki67。其条件培养基更能促进盆腔神经节（MPG）神经纤维的生长及毛细血管的生成。. (2) DLSW不改变ADCSs细胞形态和细胞骨架。MAPK通路，PI3K通路和NF-kB通路磷酸化水平明显增高，但是JAK/STAT通路无明显改变。. (3) DLSW能明显改善DBD大鼠的膀胱功能，膀胱组织学发现DLSW冲击后膀胱组织内的神经，血管及肌肉修复速度加快。膀胱内5-乙炔基-2'-脱氧尿苷（EdU）标记的细胞明显增多，膀胱组织内基质细胞衍生因子-1（SDF-1）的表达增多，某些EdU阳性的细胞同时表达SDF-1，Stro-1，但不表达CD34。膀胱组织中VEGF和神经生长因子（NGF）表达明显增多，EdU阳性的细胞中仅有少部分分化成为膀胱平滑肌细胞。.4.科学意义. 我们提出了DLSW治疗机制的新理论学说：DLSW首先激活受损组织局部的干细胞，使其分泌SDF-1，诱导体内干细胞向损伤处募集，被募集的干细胞被DLSW激活后分泌大量的生长因子和营养因子，促进损伤组织的血管及神经修复。我们首次将DLSW用于治疗DBD，将减轻社会及患者个人治疗DBD的经济或心理负担，为后续提高DLSW治疗效果的研究及开展DLSW治疗DBD临床实验奠定了研究基础。