电针影响干细胞外泌体治疗肛门括约肌损伤型大便失禁的机理研究

The incidence of fecal incontinence is high and the clinical efficacy is poor. It is necessary to explore new treatment methods. The applicant's previous study found that electroacupuncture and stem cells can treat fecal incontinence, electroacupuncture can also induce stem cell homing, and electroacupuncture combined with stem cell transplantation is satisfactory for fecal incontinence caused by sphincter injury. Moreover, the study found that stem cells play a role mainly through the paracrine mechanism, that is, by exchanging the information between exosomes and recipient cells, can induce differentiation, migration, proliferation, etc. of the original cells, and promote wound repair. In this study, fluorescent staining is used to observe the fluorescence intensity of stem cells and exosomes and whether the both co-localization occurre. On the basis of electroacupuncture affecting the homing of stem cells, it is explored whether electroacupuncture also affecte the secretion of exosomes; By HE, Masson Dyeing and other observations of damaged anal sphincter tissue fibrosis and tissue hyperplasia, observe the relationship between exocrine secretion and tissue repair, detecte the differentiation of internal and external sphincters by immunohistochemistry, indicating that exosomes affect tissue differentiation. It will be confirmed by the above method that electroacupuncture can affect the secretion of exosomes, exosomes can affect the repair of damaged sphincters, and provide further theoretical support for electroacupuncture combined with stem cells for the treatment of sphincter-induced fecal incontinence.

大便失禁发病率较高，临床疗效欠佳，有必要探寻新的治疗方法。申请人前期研究发现，电针和干细胞均可治疗大便失禁，电针亦可诱导干细胞归巢，且电针联合干细胞移植对于括约肌损伤导致的大便失禁疗效满意。有研究发现干细胞主要通过旁分泌机制发挥作用，即通过外泌体与受体细胞之间进行信息交流，诱导原始细胞分化、迁移、增殖等，促进创面修复。本研究主要通过荧光染色，观察干细胞与外泌体的荧光强度及是否产生共定位现象，在电针影响干细胞归巢的基础上，探索电针是否同样影响外泌体的分泌；通过HE、Masson染色等观察受损的肛门括约肌组织纤维化及组织增生情况，分析外泌体的分泌情况与组织修复的关系；通过免疫组化技术观察内、外括约肌的分化情况，分析外泌体是否影响组织的分化。通过以上方法证实电针可影响外泌体分泌，外泌体可影响受损括约肌的修复，为电针联合干细胞治疗括约肌损伤型大便失禁提供进一步的理论支持。

大便失禁发病率较高，临床疗效欠佳；前期研究发现，电针和干细胞均可治疗大便失禁，且两者联合对于括约肌损伤导致的大便失禁疗效满意。研究发现干细胞主要通过分泌的外泌体与受体细胞之间进行信息交流，诱导原始细胞分化、增殖等，促进创面修复。本研究主要在前期研究的基础上，观察干细胞来源的外泌体是否影响大鼠成肌细胞的增值，进而影响损伤的括约肌修复，为治疗括约肌损伤型大便失禁提供进一步的理论支持。.研究内容：.（1）重复前期实验：造模、干细胞提取及制备、分组及治疗方法同前期实验。.（2）分离提取干细胞来源的外泌体（BMSC-Exo）并鉴定，培养大鼠成肌细胞（L6）并鉴定。.（3）共培养BMSC-Exo及L6细胞48小时，每12小时观察BMSC-Exo对L6细胞增值的影响。.（4）通过芯片技术，对共培养的（L6增值最明显时间段）生物物质进行多条信号通路筛选和比较，通过磷酸化等表达调变情况分析信号通路与特异性蛋白之间的关系。.结果数据: .1..同前期研究，HE染色观察发现EA联合BMSC组较EA、BMSC组能明显缩小造模的手术切口，尤其是治疗后第7天及第14天。.2..各组收缩压比较发现，组间差异有统计学意义，各实验组间疗效不同，各组第3天、第7天的有统计学差异（p＜0.05），表明随着时间的增加，肌肉的收缩力明显上升，与HE染色结果相一致。.3..培养BMSC，提取干细胞来源的外泌体，通过蛋白质印迹（WB）、纳米透射电镜检测（TEM）、纳米微粒追踪检测（NTA）成功鉴定。.4..培养大鼠L6细胞，分别于不同倍数镜下观察并进行种鼠鉴定。.5..将BMSC-Exo及L6细胞共培养发现，24小时时外泌体能明显促进L6细胞增值。.6..将24小时共培养细胞分组对比（BMSC-L6、control-L6），通过芯片分析发现，16条信号通路主要参与调控的通路为P13K-AKT、mToR、MAPK、P53等，通过磷酸化表达调变情况分析发现，EIK1、HSP90B、GSK3等因子有磷酸化作用，而P53等因子有去磷酸化作用。

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