【作者】 周若南；

【导师】 夏磊；

【作者基本信息】 郑州大学 ， 外科学， 2010， 硕士

【摘要】 背景与目的脊髓是中枢神经的一部分,不仅为支配肢体运动、感觉、内脏活动的低级中枢,而且是联系大脑和外周神经系统的关键结构,上、下行神经传导束密集。局部的损伤也会有严重的后果,如永久性的瘫痪、感觉功能丧失、大小便功能障碍以及植物神经功能紊乱等症状。因此脊髓损伤是致死率和致残率非常高的疾病,给患者及其家庭带来极大地经济和心理负担。不过脊髓损伤后神经功能的保护和恢复对于医学界仍然是一大世界性难题,在过去几十年国内外广大科研工作者孜孜不倦努力,虽然没有彻底攻克它,但也取得了一些进展。已有研究表明神经细胞在受到应激性刺激后会马上产生热休克蛋白(heat shock proteins, HSPs),其中热休克蛋白27(HSP27)能够通过各种机制对神经细胞起保护作用。有一些因素能促使神经细胞产生HSP27,但这种方法目前还无法应用于临床。如果通过药物诱导HSP27在脊髓内大量地表达,对临床脊髓损伤治疗则具有重要的现实意义。甲基强的松龙(MethylprednisoloneMP)是美国FDA批准的唯一一个用于治疗脊髓损伤的药物。研究表明它能够诱导HSP27表达量的上调来发挥对脊髓神经细胞的保护作用。经过美国3次全国急性脊髓损伤研究,其标准治疗方案已经在世界各地广泛应用于急性脊髓损伤。但是临床上缺血再灌注脊髓损伤(spinal cord ischemic reperfusion injury SCⅡ)非常多见,如严重的脊髓性颈椎病、胸椎管狭窄等手术减压术后都会导致脊髓缺血再灌注损伤。如果能够应用MP有效减轻缺血再灌注脊髓损伤,对于脊柱疾病的治疗将是个非常大的进步。不过对于MP治疗缺血再灌注脊髓损伤的用法尚没有统一的标准,临床和科研工作者也都是在探索性应用。已经有人研究发现应用MP治疗缺血再灌注的比较合适时间点是损伤前30分钟,但是应用MP的比较好的剂量目前尚未见相关报道。本课题通过制作缺血再灌注脊髓损伤动物模型,在损伤前30分钟静脉给于不同剂量的甲基强的松龙,免疫组织化学法检测脊髓组织细胞内HSP27的表达,来探讨甲基强的松龙通过诱导脊髓组织细胞内HSP27的表达而起到神经保护作用的比较好的剂量。材料与方法1.动物模型制作和分组健康普通级成年大鼠(郑州大学实验动物中心提供)100只,雌雄不分,质量200±20g。随机分成5组：A组(正常组)20只；B组(缺血再灌注模型组)20只大鼠,三个治疗组C组、D组及E组各20只大鼠。采用夹闭左右肾动脉间腹主动脉30分钟再灌注3个小时的方法制作SCⅡ模型；C组、D组及E组在脊髓缺血前30分钟分别给予MP剂量为10mg/kg、20mg/kg、30mg/kg,用生理盐水稀释至0.5ml从大鼠尾部静脉注射,B组给于相同剂量的生理盐水。2.标本取材实验动物麻醉后用4%多聚甲醛全身灌注,咬除T11、T12、L1棘突和椎板后从圆锥开始向上取脊髓约1cm,常规甲醛固定,脱水,石蜡包埋,切片,HE染色和免疫组织化学染色。3.免疫组织化学染色和图像分析切片免疫组织化学染色后采用图像分析系统测量阳性染色物质吸光度,每张片随机选取6个不同视野,400高倍镜下用计算机计算出平均吸光度值A,A反应染色强度,代表相对含量。4.统计学处理用SPSS13.0统计分析软件处理实验数据,所有数据采用均数加减标准差(x±s)表示,按α=0.05检验水准,五个样本均数组间采用单因素方差分析(ANOVA)进行数据处理,组间两两比较采用LSD法。结果1.大鼠脊髓缺血再灌注损伤后,损伤处有明显水肿、变性、坏死等病理变化。2.正常组脊髓内HSP27的表达量呈弱阳性,缺血再灌注组脊髓神经细胞内HSP27表达量明显增加。3.缺血再灌注组损伤前使用MP,脊髓的继发性损伤减轻,HSP27的表达量明显增强,显示为强阳性。4.HSP27的表达水平上,五组之间总体上差异有统计学意义(P<0.05),B组与A组差异性有统计学意义(P<0.05),C组与B组差异性无统计学意义(P>0.05),D组和C组差异性有统计学意义(P<0.05),E组和D组差异性有统计学意义(P<0.05)。结论1.在缺血再灌注损伤前预防性使用MP可明显改善损伤脊髓的病理形态,表明脊髓缺血再灌注损伤前早期使用MP对损伤脊髓有一定的保护作用。2.正常脊髓神经细胞内HSP27的表达呈弱阳性,大鼠脊髓缺血再灌注损伤后脊髓神经细胞内HSP27的表达明显上调,而在脊髓损伤前使用MP可使大鼠脊髓神经细胞HSP27的表达显著提高。3.脊髓缺血再灌注损伤前预防性应用不同剂量MP使脊髓神经细胞内HSP27表达量上调程度不同,30mg/kgMP治疗组HSP27表达上调最明显。更多还原

【Abstract】 Background and ObjectiveAs a part of the central nervous system, the spinal cord is not only a junior centrum controling body movement, feeling, internal activity,but also but also is a key structure communicating the brain with peripheral nervous system, where there are dense fasciculuses. So even there is a focal injury to the spinal cord, it can induce a severe result such as permanent paralysis, feeling function losing, defecation function disturbance and vegetative nerve functional disturbance. So the morbidity of death and deformity in spinal cord injury is very high, which bring great economy and psychological burden. But protection and restoration of nerve function after spinal cord injury for medicine is still a big problem in the world. Although it is not totally conquered, some progress has been also made through tireless efforts of scientists in the past few decades. Studies have shown that nerve cells will quickly generate heat shock protein after stimulation and HSP27 of nerve cells can play protective effect through various mechanisms. Many factors can cause nerve cells to produce HSP27, but these methods are still unable to be used in clinical applications.It will have important practical significance in clinical treatment of spinal cord injury if HSP27 is induced by drugs in the spinal cord of expression.Methylprednisolone is only medicine used in clinic approved by FDA. Research shows that it can raise HSP27 expression to protect spinal nerve cell. After U.S. three national acute spinal cord injury study, the standard therapy in the world has been widely applied in acute spinal cord injury.But there is much SCII(Spinal Cord ischemia-reperfusion Injury) phenomenon in clinic, spinal cord ischemia-reperfusion injury can appear after decompression surgery such as severe spinal cord of cervical spondylosis, thoracic spinal stenosis. If the application of MP can effectively reduce ischemia-reperfusion for spinal cord injury, the treatment of spinal column diseases is a very big progress.But there is not a unified standard for MP usage in treatment of spinal cord ischemic reperfusion injury, clinical and scientific researchers are also exploring the application. Some people has found that the best time of using MP in treatment of SCⅡ, but the best dose has not been reported.This study is to discover the better MP dose of protecting spinal cord by inducing the expression of HSP27 through such methods as making animal models of spinal cord ischemia-reperfusion injury, using different doses of Methylprednisolone within 30 minutes before SCⅡ,detecting the expression of the HSP27 of rats’spinal cord by immunohistochemstryMethods100 ordinary rats were randomly divided into five groups (n=20):Group A、B、C、D and E, normal group、SCⅡgroup、MP treatment groups (three different doses ).we obstructed the abdominal aorta between the left and the right renal artery for 30min and let it receive reperfusion after 3h to make the SCⅡmodel.At 30min prior to injury, the rats in C, D and E group received 10mg/kg、20mg/kg、30mg/kgMP respectively.And the rats in group B received the same dose of normal saline as control before injury. The spinal cord specimen from group A 3h after surgery and from other groups 3h after injury were taken out and cut ,then the pathological changes of spinal cord cells with HE staining and the expression of Hsp27 in spinal cord cells with immunohistochemical staining were observed respectively. The data were analysed with SPSS 13.0 soft ware.Results1. There were significantly edema, degeneration, necrosis and so on in spinal cord after SCⅡin rats2. The expression of HSP27 showed weak positive in the normal group and obviouly increased in SCⅡgroup.3. The secondary injury of spinal cord was significantly improved in MP group. The expression of HSP27 significantly increased and showed strong positive.4. In the expression of HSP27,there was a significant difference among each group in total (P<0.05); There was significant statistical difference between group B and group A(P<0.05). NO significant statistical difference was found in the expression between group B and group C (P>0.05). There was significant statistical difference between group D and group C (P<0.05) and also statistical significance was found between group E and group D (P<0.05).Conclucions1. The pathological morphology of spinal cord can be obviously improved when preventive using MP before SCⅡ, and it appears that early using MP before SCⅡhas certain protective effect to some protective effect.2. HSP27 expression is weak positive in normal spinal nerve cells and is up-regulated after SCⅡ,but its expression will be up-regulated significantly when using MP at 30min prior to injury.3. HSP27 expression is up-regulated at different levels when using different doses MP prior to injury, and 30mg/kg MP is the best dose.更多还原