c-kit信号系统介导的电针治疗脊髓损伤后尿潴留的分子机制研究

This project is based on the latest progress research on of pathogenesis mechanism of urinary retention caused by spinal cord injury. Combining with our the clinical and animal experimental practice research of detrusor excitability and bladder ICCs is assumed to affect bladder ICCs with benign regulation of SCF/c-kit signal change, and ultimately play effect of improving the lower reflex state and no reflex state of bladder detrusor after spinal cord injury. The project intends to establish the model of urinary retention of complete spinal cord injury in rats and use specific inhibitor of c-kit, randomized comparison with the normal group, sham operation group and model group, inhibitor group, and electroacupuncture with inhibitor group. In this trial, we can observe the effect of function and histmorphology of rat bladder treated with electroacupuncture. And at the same time, we can observe the effect of immunofluorescence distribution of bladder c-kit protein by transmission and laser scanning confocal microscope. We will find out the influence to expression of SCF and c-kit at the of protein and mRNA level by Western blot, RT-PCR. Then explore the effect of electroacupuncture therapy on urinary retention caused by spinal cord injury. The purpose of our project is to reveal the cellular action and molecular mechanism, so as to guide clinical practice.

根据国内外研究的新进展，结合课题组的前期的临床研究以及有关逼尿肌兴奋性与膀胱ICC细胞的实验研究，提出针刺通过对c-kit信号系统的良性调节，对膀胱ICC细胞进行调控，是其最终改善脊髓损伤后尿潴留逼尿肌的低反射或无反射状态的重要机制之一。本项目拟建立完全性骶髓损伤尿潴留大鼠模型，并引入工具药物c-kit特异性阻断剂，通过与正常组、假手术组、模型组、阻断剂组、阻断+电针剂组的随机可比对照，深入观察电针对脊髓损伤尿潴留大鼠膀胱功能的影响及组织形态学的影响。并通过应用激光共聚焦显微镜及免疫荧光染色、Western blot、RT-PCR等先进而成熟分子生物学技术，从电针对膀胱c-kit蛋白的分布，c-kit和SCF在蛋白及基因水平的表达的影响，来探讨c-kit信号系统介导的针刺治疗作用机制，以期为针刺治疗脊髓损伤后尿潴留的分子机制提出新阐释，为针刺治疗脊髓损伤后尿潴留的临床应用提供新的科学依据。

背景：膀胱cajal间质细胞（interstitial cells of cajal，ICCs）是膀胱逼尿肌收缩的自主起搏细胞。c-kit信号系统与膀胱ICCs的增生、分化和表型维持密切相关。骶髓损伤导致排尿失去中枢支配，电针治疗依然有效。.方法：建立完全性骶髓损伤尿潴留模型。将造模成功大鼠随机分为模型组、电针组、抑制剂组、抑制剂加电针组，与假手术组进行对照比较。电针组、抑制剂加电针组均予关元、三阴交穴电针干预；抑制剂组、抑制剂加电针组均予甲磺酸伊马替尼腹腔注射。干预14天后，根据膀胱最大容量、残余尿量、膀胱顺应性、排尿率评价大鼠排尿功能。光镜观察膀胱组织形态改变；透射电镜观察观察膀胱ICCs数量和超微结构；免疫组化法观察膀胱c-kit免疫染色分布表达；Real time-PCR和Western Blot检测膀胱组织c-kit、SCF基因和蛋白表达；Western Blot检测膀胱c-kit蛋白磷酸化水平。.结果：电针能减少膀胱最大容量、残余尿量和膀胱顺应性，提高排尿率，改善膀胱组织形态。抑制剂伊马替尼影响了电针对尿动力学指标和膀胱组织形态的改善。电针能明显改善膀胱ICCs的数量和超微结构，上调c-kit和SCF mRNA和蛋白的相对表达，提高c-kit蛋白磷酸化水平。抑制剂伊马替尼对c-kit和SCF mRNA和蛋白表达没有显著改变，但进一步降低了c-kit蛋白磷酸化水平，损害膀胱ICCs的数量和超微结构。.结论：电针能改善脊髓损伤大鼠的排尿功能。电针通过上调脊髓损伤后尿潴留大鼠膀胱c-kit、SCF的基因及蛋白表达、提高c-kit蛋白磷酸化水平，良性调控膀胱ICCs，发挥其自主起搏的功能，促进逼尿肌的收缩。电针对c-kit信号系统的良性调节，可能是其最终改善脊髓损伤后尿潴留逼尿肌的低反射状态的重要机制之一。.科学意义：本研究探索了c-kit信号系统介导的电针治疗脊髓损伤后尿潴留的分子机制研究，为针刺治疗脊髓损伤后尿潴留的临床推广应用提供了新的科学依据。