【作者】 陈剑峰；

【导师】 唐天驷；

【作者基本信息】 苏州大学 ， 骨外科（专业学位）， 2014， 博士

【摘要】 第一部分红花对大鼠急性脊髓损伤后急性期水肿、组织结构的影响目的：采用重物下降打击法，制备相当于中度大鼠脊髓损伤的模型，探讨红花注射液对大鼠脊髓损伤后运动功能、组织病理学及组织含水量的影响。方法：将24只SD大鼠随机分为假手术组(A组)，脊髓打击损伤组(B组)、甲基强的松龙组(C组)、红花溶液组(D组)，每组6只。B、C、D组根据改良的重物撞击装置制备脊髓急性打击损伤动物模型，C、D组在脊髓打击损伤后即刻进行腹腔注射MPSS30mg/kg、红花溶液100mg/kg，观察并检测4组大鼠SCI后1h、24h、48h3个不同时间点后肢运动功能评分变化以及脊髓组织病理学、组织含水量改变。结果：1、A组大鼠功能完全恢复。C组、D组与B组相比较24h，48h2个时间点大鼠后肢运动功能均有所改善，但24h时已出现明显差异(P<0.05)，48h时差异更为显著(P<0.01)；C组和D组相比各时间点相差不大。2、大鼠脊髓组织HE染色光镜下可见A组脊髓组织结构正常,神经元轮廓清楚，核仁清晰可见，胞质均匀深染。SCI后48h B组神经组织多灶性出血并有大量神经元坏死、尼氏体溶解消失、核固缩变小。C组神经组织结构损伤轻微，细胞轻度肿胀，胞质均匀，D组与C组相似。3、大鼠脊髓组织透射电镜下可见A组髓鞘组织结构正常。B组髓鞘板层结构紊乱、断裂分层，轴浆外溢，甚至出现空泡。C组神经组织结构损伤轻微，轴索轻微水肿，个别神经髓鞘变薄或缺失，D组与C组相似4、SCI后B、C、D各组的损伤段组织含水量均与A组有明显差异(P<0.05)。SCI后24h时D组与B组相比明显较低(P<0.05)，48h差异更显著(P<0.01)。结论：1、红花和MPSS可抑制SCI后神经细胞的凋亡，减轻神经元坏死，挽救部分未直接受损的神经元，从而为神经功能的恢复提供组织学的基础。2、红花可以促进运动功能的恢复，消除SCI所造成的组织水肿，减缓组织水肿引起的炎症反应，作用优于MPSS。第二部分红花对大鼠急性脊髓损伤后受损部位局部能量代谢的影响目的：采用重物下降打击法，制备相当于中度大鼠脊髓损伤的模型，探讨红花注射液对大鼠脊髓损伤后乳酸（LD）含量、乳酸脱氢酶(LDH)活性以及Na+/K+-ATPase活性的影响。方法：将24只SD大鼠随机分为假手术组(A组)，脊髓打击损伤组(B组)、甲基强的松龙组(C组)、红花溶液组(D组)，每组6只。A、B组根据改良的重物撞击装置制备脊髓急性打击损伤动物模型，C、D组在脊髓打击损伤后即刻进行腹腔注射MPSS30mg/kg、红花溶液100mg/kg，观察并检测4组大鼠SCI后1h、24h、48h3个不同时间点LD含量、LDH活性以及Na+/K+-ATPase活性改变。结果：1、B、C、D各组的LD含量均明显高于A组(P<0.01)。SCI后24h时C组、D组与B组相比明显较低(P<0.05)，在48h差异更显著(P<0.01)；而D组与C组相比则没有统计学意义。2、SCI后B组的损伤段LDH活性均明显高于A组(P<0.01)。SCI后24h时C组与B组相比明显降低(P<0.05)，D组与B组相比降低更为显著(P<0.01)，SCI后48h C组、D组与B组差异都显著(P<0.01)；而D组与C组相比没有统计学意义。3、SCI后B组的损伤段Na+/K+-ATPase活性均明显低于A组(P<0.01)。SCI后24h、48h时C组、D组与B组相比明显较高(P<0.05)；而D组与C组相比则没有统计学意义。结论：1、红花注射液可以有效改善SCI后脊髓组织的局部组织糖代谢，减少组织水肿，防止SCI进一步恶化。2、红花注射液可以有效改善SCI后脊髓组织的局部能量代谢，减少组织水肿，防止SCI进一步恶化。第三部分红花对大鼠急性脊髓损伤后受损部位Ca2+、Mg2+微环境的影响目的：采用重物下降打击法，制备相当于中度大鼠脊髓损伤的模型，探讨红花注射液对大鼠脊髓损伤后电解质Ca2+、Mg2+含量以及Ca2+-ATPase、 Mg2+-ATPase、Ca2+/Mg2+-ATPase活性影响。方法：将24只SD大鼠随机分为假手术组(A组)，脊髓打击损伤组(B组)、甲基强的松龙组(C组)、红花溶液组(D组)，每组6只。A、B组根据改良的重物撞击装置制备脊髓急性打击损伤动物模型，C、D组在脊髓打击损伤后即刻进行腹腔注射MPSS30mg/kg、红花溶液100mg/kg，观察并检测4组大鼠SCI后1h、24h、48h3个不同时间点Ca2+、Mg2+含量以及Ca2+-ATPase、Mg2+-ATPase、Ca2+/Mg2+-ATPase活性改变。结果：1、SCI后B组的损伤段Ca2+含量均明显高于A组(P<0.01)。SCI后24h、48h时C组、D组与B组相比明显较低(P<0.01)；而D组与C组相比则没有统计学意义。2、SCI后B组的损伤段Mg2+含量均明显低于A组(P<0.01)。SCI后24h、48h时C组、D组与B组相比明显较高(P<0.01)；而D组与C组相比则没有统计学意义。3、SCI后B组的损伤段Ca2+-ATPase活性均明显低于A组(P<0.01)。SCI后1h、24h时C、D组与B组相比明显升高，差异显著(P<0.01)， SCI后48h时C组与B组相比无差异；而D组与B组相比仍有明显升高（P<0.05）。4、SCI后B组的损伤段Mg2+-ATPase活性均明显低于A组(P<0.01)。SCI后1h、24h时C、D组与B组相比明显升高(P<0.05)， SCI后48h时C组、D组与B组相比仍有明显升高（P<0.05），且D组优于C组。5、SCI后B组的损伤段Ca2+/Mg2+-ATPase活性均明显低于A组(P<0.01)。SCI后1h、24h时C组、D组与B组相比明显较高(P<0.05)；而48h时D组差异更明显（P<0.01）。结论：1、红花注射液可有效抑制SCI后的Ca2+-ATPase、Ca2+/Mg2+-ATPase活性下降，从而可阻止Ca2+局部集聚致超载引起的SCI对神经细胞继发性损伤。2、红花注射液可有效抑制SCI后的Mg2+-ATP酶活性下降，从而可阻止Mg2+浓度下降，有效改善局部Ca2+超载以及能量代谢状态，延缓SCI后神经细胞继发性损伤的发生。第四部分红花对大鼠急性脊髓损伤后受损部位脂质过氧化反应和活性氧影响目的：采用重物下降打击法，制备相当于中度大鼠脊髓损伤的模型，探讨红花注射液对大鼠脊髓损伤后丙二醛(MDA)、超氧化物歧化酶(SOD)和活性氧(ROS)的影响。方法：将24只SD大鼠随机分为假手术组(A组)，脊髓打击损伤组(B组)、甲基强的松龙组(C组)、红花溶液组(D组)，每组6只。A、B组根据改良的重物撞击装置制备脊髓急性打击损伤动物模型，C、D组在脊髓打击损伤后即刻进行腹腔注射MPSS30mg/kg、红花溶液100mg/kg，观察并检测4组大鼠SCI后1h、24h、48h3个不同时间点MDA、SOD和ROS改变。结果：1、SCI后B组的损伤段MDA含量均明显高于A组(P<0.01)。SCI后1h、24h时C组、D组与B组相比明显较低(P<0.05)，D组在48h差异更显著(P<0.01)；而D组与C组相比则没有统计学意义。2、SCI后B组的损伤段ROS含量均明显高于A组(P<0.01)。SCI后1h、24h时C组、D组与B组相比明显较低(P<0.05)，D组在48h差异更显著(P<0.01)；而D组与C组相比则没有统计学意义。3、SCI后B组的损伤段SOD活性均明显低于A组(P<0.01)。SCI后1h D组与B组相比已有明显升高(P<0.05)，24h时C组、D组与B组相比明显升高(P<0.05)，在48h差异更显著(P<0.01)；而D组与C组相比则没有统计学意义。结论：1、红花注射液可以明显降低MDA、ROS含量，提高SOD活性，从而抑制SCI后受损部位氧自由基生成，同时有效清除氧自由基等，从而阻断脂质过氧化反应。2、红花注射液抑制MDA、ROS生成的能力优于MPSS。更多还原

【Abstract】 Part Ⅰ The effectsof Safflower injection on the acute edema andorganizational structure after acute spinal cord injury in adult ratsObjective:To study the effects of safflower on locomotor function, histopathology and tissuewater content of the experimental spinal cord injury model with weight-dropping methodin adult rats.Methods:Twenty-four SD rats were randomly divided into4groups: Sham-operationgroup(group A), spinal cord injury group(group B), Methylprednisolone SodiumSuccinate(MPSS) group(group C), and safflower group(group D). Animal models of spinalcord injury were induced with weight-dropping method. In C and D group, MPSS of30mg/kg and safflower of100mg/kg was respectively injected by intraperitonealimmediately after modeling. The locomotor function of the hind limbs were evaluated at1h,24h,48h after SCI, and the histopathological study, tissue water content changes in thespinal cord were observed at the1h,24h and48h after modeling.Results:1. The hind limb locomotor function of the rats in group A was completely recovered.Compared with that in group B, the hind limb locomotor function of rats in group C andgroup D all improved at24h and48h, with a little significant improvement at24h(P<0.05)and more significant difference at48h (P<0.01).2. Spinal cord tissues after rat spinal cord injury were observed by HE staining andlight microscopy. In group A, the tissue structure was well preserved, and some neurons were clear, in which cytoplasm is homogeneous and nucleoli were evident. In group B, thenerve tissue showed multifocal leukoencephalopathy bleeding and a number of nerve cellsand nissl bodies disappeared. The nucleus became small because of pycnosis. In group C,the injury of the nerve tissue was mild, the cells appeared swelling and the cytoplasm washomogeneous. The tissue situation in group D is similar to those in group C.3. Spinal cord tissues after rat spinal cord injury were observed by transmissionelectron microscopy. In group A, the structure of myelin sheath was in integrity. In group B,lamellar structures were disordered with fractured layers, axoplasms were out of axilemmaand even some vacuoles appeared in myelin sheath. In group C, the injury of the nervetissue was mild, the axons appeared swelling and individual myelin sheath was thinningand missing. The tissue situation in group D is similar to those in group C.4. Tissue water content in group B,C and D all are significantly higher than that ingroup A(P<0.05) after modeling. And tissue water content in group D is lower than that ingroup B at24h(P<0.05), even more significantly lower at48h(P<0.01)。Conclusions:1. Safflower injection and MPSS inhibits apoptosis and alleviate necrosis on nervecell after SCI, even protect the parts of directly undamaged neurons, which providehistological basis for recovery of neurological function.2. Safflower injection can promote the recovery of locomotor function, eliminatetissue edema from SCI, and reduce inflammation caused by tissue edema, better than thatof MPSS. Part Ⅱ The effectsof Safflower injection on energy metabolism ofdamaged section after acute spinal cord injury in adult ratsObjective:To study the effects of safflower on lactic acid(LD), lactic dehydrogenase(LDH) andNa+/K+-ATPase of the experimental spinal cord injury model with weight-dropping methodin adult rats.Methods:Twenty-four SD rats were randomly divided into4groups: Sham-operationgroup(group A), spinal cord injury group(group B), Methylprednisolone SodiumSuccinate(MPSS) group(group C), and safflower group(group D). Animal models of spinalcord injury were induced with weight-dropping method. In C and D group, MPSS of30mg/kg and safflower of100mg/kg was respectively injected by intraperitonealimmediately after modeling. Changes on lactic acid, lactic dehydrogenase andNa+/K+-ATPase in the spinal cord were observed at the1h,24h and48h after modeling.Results:1. LD content in group B,C and D all are significantly higher than that in group A(P<0.01) after modeling. LD content in group C and D are lower than those in group B at24h(P<0.05), even more significantly lower at48h(P<0.01). There are no significantdifference between group C and D.2. Compared with those in group A, LDH activity at1h,24h,48h are obviouslyhigher in group B after SCI(P<0.01). LDH activity in group C are lower than those ingroup B(P<0.05), even more significantly lower in group D at24h(P<0.01). LDHactivity in group C and D are both significantly lower than those in group B(P<0.01),while no significant difference between group C and D.3. Compared with those in group A, Na+/K+-ATPase activity at1h,24h,48h areobviously lower in group B after SCI(P<0.01). Na+/K+-ATPase activity in group C and D are higher than those in group B at24h and48h(P<0.05), while no significant differencbetween group C and D.Conclusions:1. Safflower injection can effectively improve glucose metabolism and reduce edemin damadged section of the spinal cord tissue after SCI, which prevent further deterioratioof SCI.2. Safflower injection can effectively improve energy metabolism and reduce edemin damadged section of the spinal cord tissue after SCI, which prevent further deterioratioof SCI. Part Ⅲ The effects of Safflower injection on energy metabolism ofdamaged section after acute spinal cord injury in adult ratsObjective:To study the effects of safflower on Ca2+, Mg2+and Ca2+-ATPase, Mg2+-ATPase,Ca2+/Mg2+-ATPase of the experimental spinal cord injury model with weight-droppingmethod in adult rats.Methods:Twenty-four SD rats were randomly divided into4groups: Sham-operationgroup(group A), spinal cord injury group(group B), Methylprednisolone SodiumSuccinate(MPSS) group(group C), and safflower group(group D). Animal models of spinalcord injury were induced with weight-dropping method. In C and D group, MPSS of30mg/kg and safflower of100mg/kg was respectively injected by intraperitonealimmediately after modeling. Changes on Ca2+, Mg2+and Ca2+-ATPase, Mg2+-ATPase, Ca2+/Mg2+-ATPase in the spinal cord were observed at the1h,24h and48h aftermodeling.Results:1. Compared with those in group A, Ca2+content at1h,24h,48h are obviouslyhigher in group B after SCI(P<0.01). Ca2+content in group C and D are lower than thosein group B at24h and48h (P<0.01). There are no significant difference between group Cand D.2. Compared with those in group A, Mg2+content at1h,24h,48h are obviouslylower in group B after SCI(P<0.01). Mg2+content in group C and D are higher than thosein group B at24h and48h (P<0.01), while no significant difference between group C andD.3. Compared with those in group A, Ca2+-ATPase activity at1h,24h,48h areobviously lower in group B after SCI(P<0.01). Ca2+-ATPase activity in group C and D arehigher than those in group B at24h after SCI (P<0.01), while compared with those ingroup B at48h, Ca2+-ATPase activity only are higher in group D(P<0.05) and notsignificantly different in group C.4. Compared with those in group A, Mg2+-ATPase activity at1h,24h,48h areobviously lower in group B after SCI(P<0.01). Compared with those in group B,Mg2+-ATPase activity are higher in group C and D at1h and24h (P<0.05), while evenmore significantly higher at48h(P<0.01) and those in group D are better than in group C.5. Compared with those in group A, Ca2+/Mg2+-ATPase activity at1h,24h,48h areobviously lower in group B after SCI(P<0.01). Compared with those in group B,Mg2+-ATPase activity are higher in group C and D at1h and24h (P<0.05), while evenmore significantly higher in group D at48h(P<0.01).Conclusions:1. Safflower injection can effectively inhibit the decrease of Ca2+-ATPase, Ca2+Mg2+-ATPase activity after SCI, which prevent the secondary damage caused by calciumoverload in nerve cells.2. Safflower injection can effectively inhibit the decrease of Mg2+-ATPase after SCI, which prevent Mg2+concentration decrease. Consequently,these effectively improvedlocal calcium overload as well as the state of energy metabolism, and delayed theoccurrence of the secondary damage in nerve cells after SCI. Part Ⅳ The effects of Safflower injection on Lipid peroxidation andand ROS after acute spinal cord injury in adult ratsObjective:To study the effects of safflower on malondialdehyde(MDA), superoxidedismutase(SOD) and reactive oxygen species(ROS) of the experimental spinal cord injurymodel with weight-dropping method in adult rats.Methods:Twenty-four SD rats were randomly divided into4groups: Sham-operationgroup(group A), spinal cord injury group(group B), Methylprednisolone SodiumSuccinate(MPSS) group(group C), and safflower group(group D). Animal models of spinalcord injury were induced with weight-dropping method. In C and D group, MPSS of30mg/kg and safflower of100mg/kg was respectively injected by intraperitonealimmediately after modeling. Changes on MDA, SOD and ROS in the spinal cord wereobserved at the1h,24h and48h after modeling.Results:1. Compared with those in group A, MDA content at1h,24h,48h are obviouslyhigher in group B after SCI(P<0.01). Compared with those in group B, MDA are lower ingroup C and D at1h and24h (P<0.05), while even more significantly higher in group Dat48h(P<0.01).There are no significant difference between group C and D. 2. Compared with those in group A, ROS content at1h,24h,48h are obviouslyhigher in group B after SCI(P<0.01). Compared with those in group B, ROS are lower ingroup C and D at1h and24h (P<0.05), while even more significantly higher in group Dat48h(P<0.01).There are no significant difference between group C and D.3. Compared with those in group A, SOD activity at1h,24h,48h are obviouslylower in group B after SCI(P<0.01). Compared with those in group B, SOD activity arehigher in group D at1h, then higher in group C and D at24h (P<0.05), and even moresignificantly higher in group C and D at48h(P<0.01). There are no significant differencebetween group C and D.Conclusions:1. Safflower injection can obviously reduce the content of MDA and ROS and raiseSOD activity, which inhibited oxygen free radicals in damage section after SCI andeffective removed oxygen free radicals and so on, consequently blocked lipid peroxidation.2. Safflower injection can inhibit MDA and ROS, better than that of MPSS.更多还原