微泡介导超声结合序贯缓释SDF-1α/BDNF促海绵体神经再生的机制研究

Erectile dysfunction associated with injury of cavernous nerves (CN) is difficult to manage. In our previous study, we had constructed Lentiviral vectors expressing Brain-derived neurotrophic factor（BDNF）, and transfected the human umbilical cord blood mesenchymal stem cells (hUCBMSCs), and transplant the hUCBMSCs with over expressing BDNF into the corpus cavernosum penis of rat. The results demonstrated that hUCBMSCs, genetically modified to express BDNF facilitates the neural regeneration and erectile function recovery after CN injury. However, the exogenous stem cells transplantation has the risk of being tumorigenic, teratogenic and infected, and its clinical feasibility is poor. Stem cells, resides in various tissues, play an important role in tissue maintenance and repair. Human body has a robust self-healing capability to repair the damaged organs through endogenous stem cells（ESCs）. Mobilization of the body’s innate ability to heal CN injury by activating ESCs is likely a meritorious approach in future ED treatment. .The reparative capability of ESCs is limited by the low levels of targeted homing. Previously, a substantial body of studies has demonstrated that stromal-derived factor-1 alpha (SDF-1α) and its specific receptor CXCR-4 played pivotal roles on ESCs migration and survival. Recently, as a biological effect of ultrasound, acoustic cavitation refers to an action that microbubbles in a liquid, insonated by ultrasound, generate a series of dynamic processes, such as oscillation, expansion, shrinkage, implosion, etc., with multifarious patterns of energy release. When cavitation takes place at the acoustics interface of tissue, physical mechanical energy, accompanied by microstreaming, shock wave, etc., can destruct tight junction between cells, and enhance vascular endothelium permeability, namely “sonoporation”. Micro-bubble-mediated ultrasound exposure (MBMUE) has become one of the most promising strategies for the targeted delivery of drugs, genes and cells. Also, MBMUE was able to enhance SDF-1α expression in the local micro-environment. We tested the hypothesis MBMUE can enhance vascular endothelium permeability, thereby subsequently resulting in ESCs oriented migration and improve ESCs survival at the site of CN injury..Based on the results of our previous studies, the present project is to study the effects of MBMUE and SDF-1α/BDNF on ESCs’ mobilization, migration, and homing to the target site after CN injury. The aligned electrospun silk nanofibers(AESNF) will be made according to our previous methods to sequential slow-release SDF-1α and BDNF, and then the cultured major pelvic ganglion (MPG) is used to test the pleiotropic effects of MBMUE and SDF-1α/BDNF in vitro..For the in vivo study, the Sprague-Dawley rats underwent CN crush injury, MBMUE will be given by combined with SDF-1α/BDNF loaded AESNF implantation into the CN injury site. After that, the continuous observation of ESCs by EdU cell tracking technology will be done at multiple time points, the ESCs will be tracked from the bone marrow to MPG. The relation and interaction among MBMUE and SDF-1α/BDNF loaded AESNF will be studied in detail. We also will investigate the signal transduction mechanisms of MBMUE combined with SDF-1α/BDNF loaded AESNF for the CN regeneration, erectile function recovery and cavernous tissue remodeling; thus provide a feasible and effective way on the treatment of CN injured ED.

海绵体神经（CN）损伤所导致的ED是临床上常见的治疗难题。本课题组前期研究显示高表达BDNF的外源性干细胞移植可促进CN 再生。然而，外源性干细胞存在免疫、致瘤等诸多问题。内源性干细胞 (ESCs) 是体内丰富的“宝藏”，动员ESCs靶向迁移，参与CN再生，极具研究前景。新近发现微泡介导低频超声（MBMUE）可开放血管内皮间隙，促进ESCs归巢。故，本项目拟建立大鼠CN损伤模型及盆大神经节培养体系，研究MBMUE对ESCs的动员、跨内皮迁移和归巢的作用及其对CN再生局部微环境的影响。在此基础上，通过前期建立的载药丝素纳米序贯缓释技术，提高CN损伤局部的SDF-1α和BDNF水平，诱导周围组织内ESCs的“变形虫”式迁移，使“MBMUE”和“SDF-1α/BDNF序贯缓释”发挥协同作用，强化募集ESCs，探索其参与CN再生、阴茎组织重构的相关信号通路，从而为CN损伤性ED的治疗提供新思路。

海绵体神经（CN）损伤所导致的ED是临床上常见的治疗难题。本课题以三维培养体系中的大鼠盆大神经节（MPG）为模型，围绕低强度脉冲超声（LIPUS）、丝素蛋白纤维材料和脂肪干细胞来源外泌体，探索促进CN再生的方式及其机制。采用正交实验法获取CN再生的最适LIPUS参数，并对比LIPUS治疗组与对照组神经再生情况。分别使用LIPUS处理后的脂肪干细胞（ADSCs）和未经LIPUS处理的ADSCs提取外泌体，观察两组外泌体对CN再生的影响。发现CN损伤后，本身即具有一定的轴突再生能力。使用强度为2.0 W/cm2的LIPUS直接作用于MPG，或在培养体系中加入浓度为1012/mL的ADSCs来源外泌体能够明显促进CN轴突的生长。ADSCs经LIPUS预处理后，所提取的外泌体中BDNF和SDF-α表达上调，对CN再生的促进作用增强。通过注射EdU标记的外源性干细胞，发现海绵体注射法能够提高干细胞在阴茎海绵体中的驻留比例；通过标记内源性干细胞发现，对于无损伤的阴茎海绵体，随着时间的推移，干细胞数量逐渐减少。.研究中先后制备了用于预血管化的膀胱脱细胞基质水凝胶/静电纺丝素纤维复合支架、细胞相容性较好的中等孔径丝素蛋白纤维支架以及可缓释SDF-1α且适合神经纤维生长及雪旺氏细胞迁移的有序静电纺丝素纳米纤维。并对其进行改进，以丝素蛋白（SF）、膀胱脱细胞基质（BAM）和透明质酸（HA）三者的混合水溶液为纺丝液进行静电纺丝,制备了SF/BAM/HA复合纤维支架,利用BAM中的内源性生长因子,以期提高复合纤维支架的生物活性。在此基础上，制备了壳芯结构的双因子缓释支架。动物实验证明，该支架不仅能够促进损伤CN的再生，还能够促进周围组织的血管化，且在植入8周后能够大部分降解。.综上所述，本研究为探讨CN损伤后的再生策略积累了经验。通过多维度的探讨，摸索出了促进CN再生的多种途径，并分析了其可能的机制。