外泌体对电针诱导归巢的干细胞修复损伤肛门括约肌的影响

Anal incontinence (FI) is a kind of refractory disease caused by various reasons, which can not control liquid and fecal excretion, and seriously affect the life treatment of patients. In previous studies, it has been proved that mesenchymal stem cell transplantation and electroacupuncture have some effect on sphincter injury. Recently, more and more researchers believe that stem cells mainly through paracrine mechanisms, exocrine secretion that paracrine system as complete components mainly involved in cell - cell communication, exosomes and target cell membrane protein binding, through receptor dependent regulation of receptor cells directly or, by the transfer of specific protein, mRNA, miRNA and other regulatory receptor cell function, thereby affecting the target cell or tissue characteristics. Therefore, this project will mainly explore the effect of the exocrine secreted by bone marrow mesenchymal stem cells (MSCs) induced by Electroacupuncture on the repair of the anal sphincter. To study the effects of the characteristic components of the extracellular secretory of stem cells on the proliferation, differentiation and fibrosis of fibroblasts. To further improve the theoretical basis of electroacupuncture combined with bone marrow mesenchymal stem cell transplantation in the treatment of sphincter injury type fecal incontinence, and lay a theoretical foundation for clinical treatment of fecal incontinence.

肛门失禁(FI)是指各种原因引起的不能随意控制液体和固体粪便排出，严重影响患者生活治疗，是一种难治性疾病。在前期的研究中，已证实应用间充质干细胞移植及电针对括约肌损伤具有一定作用。近来，越来越多的研究者认为干细胞主要通过旁分泌机制发挥作用，发现旁分泌系统分泌的外泌体作为完整的组分主要参与细胞-细胞间的信息传递，外泌体与靶细胞膜蛋白结合后，通过受体依赖性调控机制直接作用受体细胞，或通过转运特定蛋白、mRNA、miRNA等调控受体细胞功能，进而影响靶细胞或组织特性。因此，本项目将主要探索电针诱导归巢的骨髓间充质干细胞分泌的外泌体对损伤肛门括约肌修复的影响。研究干细胞源外泌体的特性成分对成纤维细胞增殖、分化及纤维化的影响。进一步完善电针联合骨髓间充质干细胞移植治疗括约肌损伤型大便失禁的理论依据，为临床治疗大便失禁奠定理论基础。

[目的]：C2C12成肌细胞作为骨骼肌组织损伤后重建的肌组织前体细胞，对于肌组织损伤快速修复的意义极大。肛门括约肌是由骨骼肌与平滑肌构成，共同维持肛门的括约功能，但其中骨骼肌在其中发挥更为重要的作用。因此，本研究拟探究骨髓间充质干细胞源性外泌体对C2C12成肌细胞增殖与凋亡的影响及相关机制，从侧面探索BMSC源性外泌体对括约肌损伤修复的潜力。[方法]：分离培养小鼠骨髓间充质干细胞及其源性外泌体后，将等体积的PBS（对照组）与外泌体（实验组）分别与C2C12细胞共培养48h。采用EdU染色和流式细胞术检测C2C12细胞的增殖或凋亡情况，进行外泌体高通量测序分析来自骨髓间充质干细胞的外泌体中的miRNAs，结合文献和qPCR验证，筛选具有潜在作用的miRNA靶分子mmu-miR-10a-5p，并利用mmu-miR-10a-5p模拟物作用C2C12细胞，从而研究外泌体功能及作用机制。动物实验中，随机分组，将大鼠分为电针联合骨髓间充质干细胞组(EA+BMSC 组)、电针联合骨髓间充质干细胞外泌体组(EA+BMSC-Exo)、PBS 组、无处理组。每组实验大鼠 12 只，12 只细分成三个疗程组，共计 48 只大鼠。各组大鼠肛门括约肌损伤均采用Zutshi模型，在腹腔麻醉（舒泰50：30mg/kg）后，于解剖显微镜下进行大鼠腹侧纵行切除25%的肛门括约肌复合体，即切除10点位到2点位之间的括约肌复合体，术后采用压迫止血及布比卡因（0.5mg/kg）镇痛。在各个时间节点进行肛压测量以助判断肛门功能恢复情况。[结果]：与对照组相比，实验组骨髓间充质干细胞源性外泌体可通过促进C2C12成肌细胞的增殖，抑制凋亡以促进肛门括约肌损伤的修复，证实是通过mmu-miR-10a-5p/P21调控轴发挥这种疗效的。与对照组（5.46±1.01cmH2O）相比，EA+BMSC 组（6.18±0.87cmH2O）和EA+BMSC-Exo（8.84±1.15cmH2O）组肛压升高。[结论]：骨髓间充质干细胞源性外泌体可以通过mmu-miR-10a-5p靶向P21促进C2C12成肌细胞的增殖、抑制凋亡。