【作者】 王俊华；

【导师】 陈邦国；

【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2009， 博士

【摘要】 [目的]随着现代交通业、建筑业的飞速发展便利了人们生活、生产的同时，健康与安全隐患也日益突出，外伤等事故所造成的脊髓损伤的高发病率现象就是其中表现之一。脊髓损伤具有终生致残率高、治疗困难等特点，其并发症尿潴留更加使得脊髓损伤患者生活质量下降，甚至威胁到生命，故脊髓损伤后尿潴留的治疗已成为医学研究者高度重视的问题。目前诸多临床研究证实，中医针灸疗法对其疗效肯定。相对于西医疗法，针灸疗法对本病有其独特的优势，但其作用机制尚未明确，本实验旨在研究电针关元穴对脊髓损伤后尿潴留模型大鼠逼尿肌超微结构、逼尿肌细胞凋亡及相关因子（bax、bcl-2）、逼尿肌兴奋性及α-平滑肌肌动蛋白（α-SMA）的表达以及对脊髓中脑源性神经营养因子（BDNF）及其酪氨酸受体激酶B（TrkB）表达的影响，并探讨穴位电针治疗本病的可能作用机制。[方法]实验Ⅰ将健康成年雌性SD大鼠随机分为正常组、模型组、电针治疗组，每组5只；实验Ⅱ、实验Ⅲ和实验Ⅳ均随机分为正常组、假手术组、模型组、电针治疗组、电针对照组，每组20只。按照经典的重物坠击法建立脊髓损伤后尿潴留大鼠模型。造模的大鼠被重物撞击脊髓时，身体痉挛性颤动，尾巴痉挛性摆动，双下肢及躯体回缩样扑动，大鼠脊髓损伤后立即处于脊髓休克期，膀胱逼尿肌呈现低反射或无反射状态，膀胱排尿无力，造模手术后24小时内可在其耻骨联合上缘触及明显胀大的膀胱，即造模成功。电针治疗组给予电针“关元”穴治疗，电针对照组给予电针“水道”穴治疗；假手术组大鼠在被暴露T₆-T₁₀椎体后，不做任何损伤处理，5分钟后直接缝合。电针治疗时使用MP150WSW型16导生理记录仪刺激输出模块，采用疏密波，频率2Hz／15Hz，强度1mA。造模手术24小时后进行第一次电针，每隔24小时电针一次，电针治疗共10次，每次持续刺激20分钟。分别运用电镜观察逼尿肌细胞和组织的超微结构；应用末端脱氧核糖核苷酸转移酶介导的缺口末端标记技术（TUNEL）及免疫组织化学SP法，观察膀胱逼尿肌细胞凋亡情况和凋亡相关因子bcl-2和bax的表达；应用逼尿肌肌条实验行离体逼尿肌最小收缩张力和收缩频率的测定，观察逼尿肌兴奋性，并用免疫组织化学SP法检测逼尿肌肌层组织中α-SMA的表达，观察其收缩能力；免疫组化SP法检测损伤段脊髓组织中BDNF及TrkB的表达。所得数据输入计算机，经SPSS13．0统计分析软件进行处理，多组数据间的比较，采用单因素方差分析或者R×C表X²检验，检验标准α值取0．05，检验结果P＜0．05为有统计学意义，实验结果以均数土标准差（（?）±S）表示。[结果]（1）与正常组比较，模型组大鼠逼尿肌超微结构变化主要为细胞排列紊乱，细胞核内染色质边集、浓缩，大量线粒体水肿，粗面内质网明显扩张、水肿、局部空泡化；平滑肌细胞之间胶原纤维大量增多。电针关元穴治疗后逼尿肌超微结构较模型组有较大变化，以线粒体、粗面内质网和胶原纤维的改变最明显，表现为线粒体的水肿程度与粗面内质网的扩张程度明显减轻，平滑肌细胞之间的胶原纤维的数量明显减少，细胞排列变规则。（2）在细胞凋亡的观察中，正常组和假手术组细胞凋亡呈较低表达，两组无明显差异（P＞0．05），模型组细胞凋亡指数（AI）高于正常组（P＜0．05）；关元组AI低于模型组及水道组（P＜0．05）。关元组bax蛋白表达较水道组及模型组减少（P＜0．05）。关元组bcl-2蛋白表达较水道组及模型组无明显差异（P＞0．05）。（3）与正常组和假手术组相比，模型组逼尿肌最小收缩张力增高，收缩频率减慢（P＜0．05）；正常组和假手术组比较，无明显统计学差异（P＞0．05）；与模型组相比，电针组最小收缩张力均明显降低，收缩频率增加（P＜0．05）。关元组与水道组之间的差异有统计学意义（P＜0．05）。在光镜下任一视野内对逼尿肌α-SMA表达进行观察，发现各组均呈阳性表达，正常组和假手术组呈强阳性表达，模型组呈弱阳性表达，低于正常组，模型组散在有少量斑块状强阳性表达区域，电针水道组和电针关元组任一视野内的大部分区域均呈中度阳性表达，散在有少量斑块状强阳性表达区域，而电针关元组斑块状强阳性区域大于电针水道组和模型组。（4）在各组脊髓中BDNF及其受体TrkB表达水平观察中，模型组BDNF及TrkB的表达均比正常组和假手术组高，关元组和水道组均明显高于其它组，差异有显著性（P＜0．05），且关元组高于水道组（P＜0．05）。[结论]逼尿肌超微结构的变化、细胞凋亡的发生、细胞凋亡因子（bcl-2、bax）、逼尿肌兴奋性的改变、α-SMA、BDNF、TrkB均参与了脊髓损伤后尿潴留的发生发展过程，电针关元穴疗法对脊髓损伤后尿潴留大鼠逼尿肌的病理状态具有良好的调整作用，推测这一作用可能通过以下途径实现的：（1）改善了逼尿肌细胞和组织的超微结构，肌细胞正常功能得到恢复；（2）降低了促凋亡因子在逼尿肌中的表达，从而减少了逼尿肌的细胞凋亡；（3）逼尿肌的兴奋性得到有效提高，膀胱收缩排尿能力增强；（4）促进了损伤段脊髓神经元和具有传导排尿反射冲动的脊髓神经通路的修复。穴位电针疗法从多环节、多水平改善了脊髓损伤后尿潴留大鼠膀胱逼尿肌低反射或无反射的病理状态。更多还原

【Abstract】 Objective: Accompanied by the rapid development of modern transport industry and construction industry,which facilitate the people’s life and manufacture, On the other hand, the hidden trouble of health and safety stand out increasingly. The high attack rate phenomenon of spinal cord injury caused by trauma accident is one of the performances.Spinal cord injury has the peculiarity of high rate of disability in lifetime and difficult treatment.Its complication, urinary retention, makes the life quality of spinal cord injury patient descend more,even threaten their lives.So,the treatment of urinary retention with spinal cord injury has become a problem paid highly attention by medicine researcher.Now, a good many of clinical research has approved that acupuncture treatment has the affirmative curative effect on this disease. Relative to western medicine, acupuncture treatment has unique advantage on the disease, but the mechanism have not been made clear. The purpose of our experiment is to study the effect of electroacupuncture at Guanyuan acupoint on the ultrastructure of detrusor urinae,apoptosis and related gene（bax、bcl-2）, excitability of detrusor urinae and expression level ofα-smooth muscle actin（α-SMA） in detrusor urinae and brain-derived neuro-trophiefactor（BDNF） and tropomyosine receptor kinase B（TrkB） in spinal cord of rats of urinary retention with spinal cord injury, and discuss the possible mechanism of point electroacupuncture treat this disease.Methods: In experiment one, adult healthy SD female rats were randomly divided into normal group, model group, electroacupuncture treatment group, 5 rats in each; In experiment two,three and four, adult healthy SD female rats were randomly divided into normal group, sham operation group, model group, electroacupuncture treatment group and electroacupuncture control group,20 rats in each.Established model rats of urinary retention after spinal cord injury by the classic weight drop method.When the spinal cord was stricken, the rat’s body occurred spastic shiver and the tail spastic swing,and more the body and both lower extremities occurred retraction flutter-like. The rat’s body was in the spinal shock stage immediately after spinal cord injury,when the detrusor urinae was in the state of no reflex or low reflex,and bladder micturate feebly. Above the pubic symphysis in abdomen area,the puffy bladder could be touched in 24 hours after the operation of model making, namely the model was successful. Electroacupuncture treatment group chose Guanyuan point and electroacupuncture control group chose Shuidao point.when the rats in sham operation group were exposed the centrum of T₆-T₁₀,didn’t make injure any more and seam directly after 5 minites.Use MP150WSW 16 channel physiologic recorder stimulate output module when treating.Adopted density-wave,2Hz/15Hz frequency and 1mA intensity. When the model making operation was finished and after 24 hours, gave the first electroacupuncture treatment and another every 24 hours, 10 times totally,and stimulated 20 minites constantly every time. Respectively applied electron microscope to observe the ultrastructure of cell and tissue of detrusor urinae; Applied terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） and immunohistochemistry SP method to observe the rats detrusor urinae apoptosis and apoptosis related gene bcl-2 and bax expression; Used the invitro detrusor urinae muscle strips experiment to determine the minimum contraction tension and frequency of contraction of invitro detrusor urinae,observed excitability of detrusor urinae,and by the immunohistochemistry SP method, determined the expression ofα-SMA in muscular layer of detrusor urina, observed its contraction ability; By immunohistochemistry SP method, determined the expression of BDNF and TrkB in spinal cord injured.All the data were input into computer and dealt with SPSS13.0 statistical analysis software.Variance analysis of one way or R×C form chi-square test was used in the comparison among multi-group data.Examination standard,namely a was 0.05,and P<0.05 meant its had statistical significance.Experiment results were showed by mean±standard deviation（（x|-）±S）.Results: （1） Compared with normal group,the main change of ultrastructure of detrusor urinae in model group was that cell arrangement was in disorder, the chromatin in the cell nucleus centralized side and concentrate,and many mitochondrion had oedema, rough surfaced endoplasmic reticulum had obvious dilatation,oedema and local vacuolization, and what’s more, collagenous fibers increased a lot among cell of smooth muscle. After electroacupuncture Guanyuan treatment, ultrastructure of detrusor urinae had much more change in comparison with model group,and the change of the mitochondrion, rough surfaced endoplasmic reticulum and collagenous fibers were the most distinct.The manifestation was that the degree of oedema in mitochondrion and dilatation in rough surfaced endoplasmic reticulum lessened obviously,and the amount of collagenous fibers among cell of smooth muscle decreased distinctly, and cell arrangement become regular. （ 2 ） In the observation of apoptosis, the normal group and sham operation group showed low expression of apoptosis,and there were no significant differentce in the two group（P>0.05）. Apoptosis index （AI） of model control group was higher than the normal group（P<0.05）.The AI of electroacupuncture at the Guanyuan treatment group was lower than the model control group and electroacupuncture at Shuidao control group（P<0.05）.Guanyuan group bax protein expression was less than the Shuidao group and the model control group（P<0.05）. Guanyuan Group bcl-2 protein expression had no significant differentce in comparison with the Shuidao group and model control group（P>0.05）. （3 ） Compared to normal group and sham operation group,the minimum contraction tension in model group increased, and the frequency of contraction decreased（P<0.05）; Compared to normal group and sham operation group, there was no significant difference（P>0.05）.Compared to model group,the minimum contraction tension in electroacupuncture group reduced significantly, and the frequency of contraction increased （p<0.05）. The difference between Guanyuan group and Shuidao group had statistical significance（P<0.05）.In the observation ofα-SMA expression of detrusor urinae in any field of vision under optical microscope, all groups showed positive expression. The normal group and sham operation group displayed strong positive expression, and model control group showed weak positive expression, which was lower than the normal group and had a spot of scattered plaquelike area of strong positive expression.Both the electroacupuncture at Guanyuan group and electroacupuncture at Shuidao control group showed medium positive expression in major area of any field of vision,which had a spot of scattered plaquelike area of strong positive expression,and these areas of Guanyuan group were more than Shuidao control group and model control group. （4） In the observation of the expression level of BDNF and TrkB in spinal cord in each group,in both BDNF and TrkB expression,model group exceeded the normal and sham group, both of the Guanyuan and Shuidao group obviously exceeded the other groups,which had significant difference（P<0.05）,and Guanyuan group was higher than the Shuidao group（P<0.05）.Conclusion: These factors,which are the change of ultrastructure of detrusor urinae, the happen of apoptosis, related genes of apoptosis（bcl-2 and bax）, the change of detrusor urinae excitability, a-SMA,BDNF and TrkB,all take part in the happen and development process of urinary retention with spinal cord injury. To the pathological state of detrusor urinae of rats of urinary retention with spinal cord injury, the treatment of electro-acupuncture at Guanyuan point has a good effect of adjustment. The effect realizes presumably by the path: （1）ultrastructure of cell and tissue of detrusor urinae is improved; （2）reduce the expression of accelerated apoptosis gene, thus the apoptosis of detrusor urinae decreases; （3）the excitability of detrusor urinae is enhanced effectively,and the ability of contraction emiction of bladder is reenforced; （4）Accelerate the repair of nerve-cell of spinal cord injured and nerve tract of spinal cord which can conduct micturition reflex impulse. Point electro-acupuncture treatment can improve the pathological state of low-reflex or non-reflex bladder detrusor urinae of rats of urinary retention with spinal cord injury by multi-link and multi- level.更多还原