【作者】 施立奇；

【导师】 蔡贤华；

【作者基本信息】 湖北中医药大学 ， 中医骨伤科学， 2014， 博士

【摘要】 目的为减少副作用，确保疗效，建立并评价螺钉压迫法致上颈髓损伤的动物模型，探讨上颈髓损伤后丹参酮替代或部分替代甲基泼尼松龙的可行性及其机制。方法实验一：健康成年雄性大耳兔36只，随机分为造模组24只和对照组12只，造模组在电生理监测下采用螺钉持续压迫枢椎段的脊髓建立上颈髓损伤的动物模型，对照组仅暴露枢椎椎板后即刻缝合，不造成脊髓损伤。术后行体感诱发电位检测验证脊髓损伤是否形成，行CT检查验证螺钉是否进入椎管。术后1d、3d、7d、14d进行改良行为学Tarlov评分，原位末端脱氧核糖核酸转移酶介导的脱氧尿苷三磷酸标记测定法检测脊髓神经细胞的凋亡数量。实验二：健康成年雄性大耳兔60只，随机分为假手术组（A组）、模型组（B组）、丹参酮组（C组）、甲基泼尼松龙组（D组）、丹参酮联合甲基泼尼松龙组（E组），每组各12只。假手术组仅暴露枢椎椎板后立即缝合，不造成脊髓损伤，其余四组同样在电生理监测下采用螺钉压迫法建立兔上颈髓损伤模型，A、B组术后立即注射等量的生理盐水，C组术后注射丹参酮注射液（3mg/kg/d），D组术后立即耳缘静脉注射甲基泼尼松龙（30mg/kg），E组术后同时注射丹参酮注射液（3mg/kg/d）和甲基泼尼松龙（30mg/kg）。术后3、7、14d时用改良Tarlov法评价兔四肢运动功能，检测脊髓损伤节段的超氧化歧化酶活性和丙二醛含量，应用脱氧尿苷三磷酸标记测定法检测实验各组脊髓神经细胞的凋亡水平。术后14d时分别在400倍光镜和8000倍透射电镜下观察脊髓组织病理变化。实验三：实验动物分组同实验二，术后14d时对实验各组应用逆转录-聚合酶链反应方法（RT-PCR）定量观察Caspase-3、Bcl-2和Bax mRNA表达，同时应用蛋白质印迹技术检测Caspase-3蛋白含量。数据统计采用SPSS17.0统计软件分析，计量资料用均值±标准差（X±s）表示，两组间数据比较采用独立样本t检验，多组间数据比较采用单因素方差分析，组内数据两两比较采用SNK－q检验。P<0.05表示差异有统计学意义。结果实验一：使用螺钉持续压迫法建立了上颈髓损伤的动物模型，通过行体感诱发检测验证脊髓损伤形成，行CT检查验证螺钉进入椎管。观察造模组和对照组在1d、3d、7d、14d不同时间点的改良Tarlov评分、Tunel阳性细胞数，造模组的Tarlov评分在各时间点均低于对照组，而阳性细胞数均高于对照组，且具有统计学差异（P<0.05），二者观察指标的表达在时间上具有相关性。实验二：1.在3d时，B组和D组的Tarlov评分和SOD活性分别低于C组和E组，B组和D组的MDA含量和Tunel阳性细胞数分别高于C组和E组，但相比不具有统计学差异（P>0.05）；在7d和14d时，B组和D组的Tarlov评分和SOD活性分别低于C组和E组，B组和D组的MDA含量和Tunel阳性细胞数分别高于C组和E组，且组间比较均有统计学差异（P<0.05）。2.脊髓组织在400倍光镜下表现：A组见正常兔的脊髓结构，神经髓鞘与轴突形态正常；B组见白质区髓鞘变薄，部分溶解、消失，海绵样改变明显，部分空泡相互连接；C组见髓鞘结构破坏程度较B组轻；D组见髓鞘结构基本完整，神经纤维水肿数量较C组少；E组见有髓神经纤维的水肿数量与程度进一步减少、减轻。3.脊髓组织在8000倍透射电镜下表现：A组见神经纤维髓鞘形态正常；B组见板层结构紊乱，呈洋葱皮样改变，轴突内大部分结构溶解，重度水肿；C组见髓鞘分层、变性明显，形态不规则，轴突内结构有局部溶解表现，中度水肿；D组见髓鞘部分分层，轴突内结构基本完整，局部有水肿表现；E组见髓鞘有轻度分层，形态略有不规则。实验三：根据RT-PCR检测结果计算各组Caspase-3mRNA的相对表达量，B组高于C组、高于D组、高于E组、高于A组，比较时具有统计学差异（P<0.05），Bcl-2mRNA/Bax mRNA比值，B组低于C组、低于D组、低于E组、低于A组；Western blot检测Caspase-3蛋白含量，B组高于C组、D组高于E组、E组高于A组，比较时均有统计学差异（P<0.05）。结论1.本研究在电生理辅助下建立了螺钉持续压迫的上颈髓损伤动物模型，并通过CT检查验证，造模后实验动物出现了相应的四肢运动功能障碍及细胞凋亡异常表达。此模型能够基本反映上颈髓损伤时所表现的病理和生理过程，且具有良好的操作性及重复性，可用于上颈髓损伤的基础实验研究。2.丹参酮注射液可提高兔上颈髓损伤后的SOD活性，降低MDA含量，抑制继发性损伤时的过氧化反应。通过光镜和电镜的观察，丹参酮减轻了脊髓损伤时过氧化物对神经髓鞘、轴突结构的破坏及细胞水肿的程度，联合应用甲基泼尼松龙时具有协同保护神经细胞的作用。3.丹参酮注射液可以减少神经细胞的凋亡数量，RT-PCR和Westernblot检测结果表明抑制了Caspase-3过量表达，提高Bcl-2mRNA/Bax mRNA比值，从而减轻氧自由基损伤所介导的细胞凋亡作用，可以在首次甲基泼尼松龙冲击治疗后长期使用，减少副作用，但不能替代它首次使用时的治疗作用，对脊髓早期继发性损伤有协同保护的作用。更多还原

【Abstract】 ObjectiveTo explore feasibility and its mechanisms of tanshinone totalor partial substitute for methylprednisolone on the animal modelof screw compressive upper cervical spinal cord injury to reduceside effects and ensure the efficacy of the drugs.MethodsPart one:36healthy male rabbits were randomly divided into twogroups,24in the model group and12in the control group. Therabbits in model group were subjected to contusion of upper cervicalspinal cord injury with continuing screw compression mothed inelectrophysiological monitoring, and the rabbits in control groupwere stitched immediately after exposing axis lamina without spinalcord injury. Postoperative somatosensory evoked was measured toverify whether the spinal cord injury had been formed or not, andthe CT examination was made to verify whether the screws had enteredinto the spinal canal. Behavioral evaluation was observed by Tarlovscore(1day,3days,7days and14days after operation), and thelevel of the neuronal apoptotic cells was detected by the terminal deoxynucleotidyl transferase mediated dUTP nick end labelingmethods.Part two:60healthy male rabbits were randomly divided into thesham operation group (group A), model group (group B), Tanshinonegroup (group C), methylprednisolone group (group D), Tanshinonecombined with methylprednisolone group (group E), and each groupwas12rabbits. The rabbits in sham operation group were stitchedimmediately after exposing axis lamina without spinal cord injury,and other ones in the rest groups were subjected to contusion ofupper cervical spinal cord injury (CSCI) with screw compressionmethod in electrophysiological monitoring. The rabbits in group A,Bwere received equal volume of normal saline, ones in group C wereadministered with Tanshinone injection (3mg/kg/d) after CSCI, onesin group D were injected methylprednisolone (30mg/kg) via ear veinimmediately after surgery and ones in group E were injectedTanshinone combined with methylprednisolone. Tarlov score was usedto assess the motor function of limbs at3,7and14days after CSCI,while superoxide dismutase (SOD) and malondialdehyde (MDA) changeswere observed at the same time, as well as it was deleted the levelof the neuronal apoptotic cells by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling methods. Histologicalchanges of spinal cord were observed by light microscopy with400times and electron microscopy with8000times at14days afteroperation.Part three: Experimental animal groups were the same asexperiment two. All experimental groups were assessed theexpression of Caspase－3, Bcl-2and Bax mRNA by reverse transcription-polymerase chain reaction and the Caspase－3proteinby Western blotting.SPSS17.0statistical software was used to analyze databases.Measurement data was recorded by terms of mean±standarddeviation (X±s). The two groups were compared with independentsamples t test, data of multiple groups was compared with univariateanalysis of variance, the SNK-q test was used to pairwise comparisonof data within the group. It indicated a significant difference forP<0.05.ResultsPart one: The usage of sustain screw compression method was aimedto establish the animal model of upper cervical spinal cord injury,which was verified by somatosensory evoked detection. The CT scanwas used to exam whether the screws was entered the spinal canal.Compared the model group with the control group at the time of1day,3days,7days and14days after operation, the Tarlov scoreand Tunel positive cells were significantly difference(P<0.05),and the expression of the two indicators was correlated in time.Part two:1. At the time of3days after operation, Tarlov scoreand the SOD activity in group B and group D were lower than thoseof group C and group E respectively, and the content of MDA, Tunelpositive cells in group B and group D were higher than that in groupC and group E respectively, while all of them didn’t have significantdifference (P>0.05). Tarlov score, SOD activity, MDA content andTunel positive cells in each group at the time of7and14days afterCSCI were statistically significant (P <0.05).2. The performance of spinal cord tissue by light microscopy (×400): Group A showed the normal structure of rabbit spinal cord, the myelin and axons were normal; Group B showed the myelinationwas thin and partly dissolved and disappeared in white matter, andit had a sponge-like change obviously, while a part of the vacuoleswere interconnected; Compared with Group B, the extent ofstructural damage to the myelin sheath in Group C was lighter; themyelin structure in group D was intact basically, and the nervefiber edema number in group D was less than that of group C; thenumber and the extent of myelinated nerve fiber edema in Group Ereduced futher.3. The performance of spinal cord tissue by transmissionelectron microscope (×8000): the morphology of myelin sheath ingroup A was normal; Group B showed lamellar structure disordered,onion-like change, most of the structure within the axon wasdissolved and severely edema; Group C showed layered anddegenerated myelin obviously, irregular shapes, partialdissolution of the axonal structure performance, and moderate edema;Group D showed partly layering of myelin, the basic structure intactaxons, and localized edema; Group E showed mild myelin layers andslightly irregular shapes.Part three: RT-PCR test results showed the amount of relativeexpression of Caspase-3from high to low was B,C,D,E and Asuccessively. while the results of the ratio of Bcl-2mRNA and BaxmRNA was on the contrary,and had significant differences (P<0.05).Conclusions1. This study that the animal model of upper cervical spinal cordinjury is established through sustain screw compression is assistedby electrophysiological monitoring and verified by CT scan. Thereis a corresponding animal limb movement dysfunction and abnormal apoptosis after modeling. This model could basically reflect thepathophysiology of spinal cord injury, and have better operabilityand repeatability. The research may be used on the basis ofexperimental spinal cord injury.2. Tanshinone injection improves the SOD activity, decreases thecontent of MDA, inhibits the peroxidation of the rabbits after uppercervical cord secondary injury. By the scanning of light andelectron microscopy, tanshinone reduces the degree of peroxide onmyelin, axon structure damage and edema of spinal cord injury. Ithas a synergistic protective effect on nerve cells when it combineswith methylprednisolone.3. Tanshinone injection reduces the number of apoptotic cells.The RT-PCR and Western blot test results show that tanshinoneinhibits the overexpression of Caspase-3, and raises the ratio ofBcl-2mRNA/Bax mRNA, thus it reduces the apoptosis effect of oxygenfree radical injury mediated. Tanshinone injection can be long-termused after the first time in the methylprednisolone pulse therapyto reduce side effects. It has a synergistic protective effect onearly secondary injury of spinal cord, but it is not a substitutefor therapeutic effect when methylprednisolone is used at the firsttime.更多还原