骶上脊髓完全性损伤后神经源性膀胱的光修复

Urinary dysfunction after spinal cord injury is the main reason of advance patient death. Neurogenic bladder resulted from supersacral spinal cord injury is very common in clinical practice. The effects of previous treatment measures such as intermittent catheterization, medication, conventional surgery were not good enough and had some problems. Sacral anterior root electrical stimulation with sacral posterior root deafferentation have been widely used and obtained good results. There are still some problems, which cannot be overcome, such as stimulator fault resulting in treatment failure, complications of long-term foreign body implantation, physical discomfort caused by sacral nerve root stimulation, higher cost, etc. Light-sensitive gene ChR2 encodes cell membrane cationic channel protein which could be activated by photostimulation of blue light. Activated ChR2 cationic channel protein may open and result in cations internal flow that could lead to cell action potential and excitation. Therefore, this study intends to transfect sacral parasympathetic nucleus which dominate movement of bladder detrusor in sacral cord of complete supersacral spinal cord injury rats with light-sensitive gene ChR2. At the same time, sacral posterior root deafferentation will be performed to release detrusor excessive reflection and detrusor-sphincter dyssynergia. Then, sacral parasympathetic nucleus expressing ChR2 in sacral cord will be exposed in blue light photostimulation and generate the action potential and nervous excitation. The sacral parasympathetic motor nerve will be stimulated selectively and bladder detrusor contraction will be activated with optogenetic activation technology. This method can reconstruct urinary dysfunction by promotion of bladder detrusor contraction and detrusor- sphincter synergia. Maybe this study will provide a new idea for functional reconstruction of neurogenic bladder after complete supersacral spinal cord injury.

脊髓损伤后排尿功能障碍是晚期患者死亡的主要原因。骶上脊髓损伤所致神经源性膀胱临床较常见，既往治疗措施如间歇导尿、药物、常规手术等效果欠佳且存在一定不足。骶神经前根电刺激联合后根切断去传入虽获得广泛应用和较好效果，但仍有问题无法克服，如刺激器故障导致失败、异物长期植入并发症、前根刺激引发躯体不适、费用较高等。光感基因ChR2编码胞膜阳离子通道蛋白，蓝光照射可引起通道开放致阳离子内流，引发细胞兴奋。因此，本研究拟将ChR2基因转染骶上脊髓完全性损伤大鼠骶髓内支配膀胱逼尿肌运动的骶副交感核，使其表达ChR2通道，同时行骶神经后根切断去传入抑制逼尿肌过度反射及逼尿肌/尿道外括约肌协同失调，骶髓行蓝光照射，利用光感基因活化技术选择性刺激支配膀胱逼尿肌的骶副交感核产生兴奋，引起膀胱逼尿肌收缩，促进逼尿肌/括约肌协调，从而修复排尿功能，以期为骶上脊髓完全性损伤后神经源性膀胱的功能重建提供一种崭新的思路。

脊髓损伤后排尿功能障碍是晚期患者死亡的主要原因。骶上脊髓损伤所致神经源性膀胱临床较常见，既往治疗措施如间歇导尿、药物、常规手术等效果欠佳且存在一定不足。光感基因ChR2 编码胞膜阳离子通道蛋白，蓝光照射可引起通道开放致阳离子内流，引发细胞兴奋。本研究将ChR2 基因转染骶上脊髓完全性损伤大鼠骶髓内支配膀胱逼尿肌运动的骶副交感核，骶髓行蓝光照射，利用光感基因活化技术选择性刺激支配膀胱逼尿肌的骶副交感核产生兴奋，引起膀胱逼尿肌收缩，从而为骶上脊髓完全性损伤后神经源性膀胱的功能重建提供一种新的思路。本研究首先通过光感基因活化OEG及光照系统设制的体外研究，为脊髓损伤后膀胱功能重建提供前期研究基础。本研究构建光感基因ChR2和大鼠Lingo-1-Fc基因的慢病毒载体，体外转染大鼠OEG细胞，使用RT-PCR、ELISA、细胞内蛋白定位技术验证转染成功，即光感基因ChR2及Lingo-1-Fc蛋白的表达。根据LED闪光电路，自行设计制备LED蓝光光照装置，蓝光LED光源波长为470nm，辐射照度能够达到光感基因调控刺激细胞的条件。OEG细胞可被光感基因技术调控和活化；体外光刺激增强了OEG细胞的生理功能；.本研究采用脊髓完全横断建立动物模型， ChR2光感基因载体精确注射于大鼠骶髓双侧SPN内，使其表达ChR2通道蛋白，骶髓行蓝光照射，检测膀胱尿动力和肌电图变化，观察休克期之后逼尿肌收缩排尿功能，利用HE染色技术对骶髓行蓝光照射前后膀胱的形态学变化进行观察，并且通过Real time PCR技术检测毒蕈碱型乙酰胆碱受体M2、M3在大鼠膀胱逼尿肌中的表达情况，进一步验证光感基因对骶上脊髓完全性损伤引起的神经源性膀胱修复的可行性。尿动力结果发现损伤蓝光刺激组的大鼠膀胱出现规律性的反射性收缩，与损伤无蓝光刺激组比较，膀胱压力降低，膀胱容量增加，顺应性升高，趋近于假手术组。肌电图结果显示膀胱逼尿肌肌条舒缩曲线可见大部分呈规律性变化，提示光感基因调控技术可调节骶上脊髓损伤后膀胱逼尿肌的收缩功能。另外，病理结果显示经过光刺激后膀胱壁炎性细胞数目减少，逼尿肌的排列更加规整；光刺激后膀胱壁减少了逼尿肌M2、M3 受体mRNA 的表达，减少逼尿肌异常收缩频率。影响逼尿肌细胞β3‐AR 介导的cAMP ‐PKA 信号转导途径，提高膀胱储尿能力，为脊髓损伤后膀胱功能修复提供新的方法。