【作者】 林翔；

【导师】 张俐；

【作者基本信息】 福建中医药大学 ， 中医骨伤科学， 2010， 博士

【摘要】 实验目的研究脊髓缺血再灌注损伤模型的建立及鉴定,观察丹参酮对脊髓缺血再灌注损伤大鼠EAAs-NMDA-Ca2+通路的影响。实验方法1.运用血管铸型技术制作SD大鼠动脉铸型标本,观察脊髓血供来源,确立脊髓缺血再灌注损伤模型制作血管夹闭的最佳部位；采用Zivin法改进复制模型,于最佳夹闭部位夹闭腹主动脉制作脊髓缺血再灌注损伤模型,使用数字减影心血管机观察确定脊髓缺血完全,鉴定模型成立；观察血管夹闭20分钟、30分钟、40分钟及60分钟对大鼠脊髓神经功能及脊髓组织学的影响,以确定脊髓缺血再灌注损伤模型动脉夹闭的最佳时限。2.选用SD大鼠128只按随机数字表法将动物分为假手术组(n=8)、模型组(n=40)、尼莫地平组(n=40)及丹参酮组(n=40)。假手术组大鼠打开腹腔,不夹闭血管,抽取下腔静脉血及切取脊髓；其它三组采用Zivin法改进复制模型,随机在脊髓缺血再灌注0.5h、1h、4h、8h、12h的相应时间点,抽取下腔静脉血,切取损伤L2-4节段脊髓,观察大鼠脊髓缺血再灌注损伤时脊髓Na+-K+-ATP酶、谷氨酸转运体、脊髓及血清谷氨酸含量、大鼠神经功能、脊髓前角细胞NMDAR1蛋白表达、脊髓含水量、神经细胞内Ca2+浓度及脊髓组织形态学的改变,并观察丹参酮对它们的作用。实验结果1.大鼠脊髓缺血再灌注损伤模型制作时动脉夹闭的最佳部位是腹主动脉在右肾动脉起始部的近心端,血供阻断20分钟不能造成大鼠截瘫；阻断30分钟截瘫率为70%,且神经功能有一定的恢复；阻断40分钟截瘫率达90%,而神经功能无明显恢复；而阻断60分钟,造成大鼠神经功能不可逆损伤。2.模型组脊髓Na+-K+-ATP酶活性及谷氨酸转运体功能在再灌注0.5h开始下降,再灌注4h达最低点,然后其活性逐渐增强,再灌注12h未能达到正常水平(P<0.05),各观测点脊髓Na+-K+-ATP酶活性及谷氨酸转运体功能：尼莫地平组与模型组无明显差别(P>0.05),丹参酮组均高于模型组(P<0.05)。模型组血清谷氨酸含量、脊髓谷氨酸含量及脊髓前角细胞NMDAR1蛋白表达在再灌注0.5h开始升高,再灌注4h达最高点,然后逐渐下降,再灌注12h未能达到正常水平。各观测点血清及脊髓谷氨酸含量、脊髓前角细胞NMDAR1蛋白表达：尼莫地平组与模型组无明显差别(P<0.05),丹参酮组均低于模型组(P<0.05)。模型组神经细胞内Ca2+、脊髓含水量在再灌注1h后开始升高,并于再灌注4h达到高峰后逐渐降低,再灌注12h时未能恢复正常。再灌注1h、4h、8h及12h,尼莫地平组、丹参酮组神经细胞内Ca2+均低于模型组(P<0.05)；再灌注1h及再灌注4h丹参酮组神经细胞内Ca2+浓度均低于尼莫地平组(P<0.05),再灌注8h、12h两组无明显差别(P>0.05)。再灌注4h、8h、12h,尼莫地平组、丹参酮组脊髓含水量均低于模型组(P<0.05)；再灌注4小时丹参酮组脊髓含水量低于尼莫地平组(P<0.05),再灌注8h、12h两组无明显差别(P>0.05)。3.脊髓缺血再灌注大鼠模型血清谷氨酸与脊髓谷氨酸在0.01水平显著正相关(相关系数为0.998),同时脊髓谷氨酸含量、血清谷氨酸含量分别与大鼠神经功能在0.01水平显著负相关(相关系数分别为：-0.439及-0.451)。结论1.大鼠脊髓缺血再灌注损伤模型制作动脉夹闭的最佳部位是腹主动脉在右肾动脉起始部的近心端。最佳的阻断时限为30分钟。2.丹参酮能影响EAAs-NMDA-Ca2+通路上游因素,从而减少神经细胞Ca2+超载,对脊髓缺血再灌注损伤具有保护作用；3.血清谷氨酸可能成为临床脊髓缺血再灌注损伤诊断、治疗及疗效监测评估的一个微创、方便、有效的指标。更多还原

【Abstract】 ObjectiveStudy on establishing a new spinal cord ischemia reperfusion injury model of rat and exploring the interventional effect of tanshinone on EAAs-NMDA-Ca2+ path in a rat model of spinal cord ischemia reperfusion injury.Methods1.Used the casting mould technology of blood vessel to make the casting sample of artery of rat, observed the blood support of spinal cord and established best position to block blood flow of abdominal aorta. Reconstructed model by blocking blood flow of abdominal aorta at the best position according to method of Zivin.Used the digital subtraction angiography cardiovascularly machine to observe and confirm the spinal was ischemic completely and the model was established. Observed the ischemia time of twenty, thirty, fourty and sixty minutes influence on spinal cord function and histopathology, in order to confirm best time of blocking blood flow of abdominal aorta for reconstructing model.2. A total of 128 Sprague Dawley rats were randomly divided into sham operation group (n=8),model group (n=40),tanshinone group (n=40) and Nimodipine(n=40) group. Rats in sham operation group were opened abdominal cavity without spinal cord ischemia and collected inferior vena cava blood and damaged L2-4 segment spinal cord.At hour 0.5,1,4,8,and 12 following perfusion, inferior vena cava blood and damaged L2-4 segment spinal cord of rats in the model, tanshinone and Nimodipine groups were collected to observe the change of Na+-K+-ATPase, glutamate transporters, glutamic acid content in blood serum and spinal cord, spinal cord function, NMDAR1 protein expression in the anterior horn cells of the spinal cord, moisture capacity of spinal cord,intraneuronal Ca2+ concentration and histopathology of spinal cord. Found out the impact of tanshinone.Results 1.The best position to block blood flow of abdominal aorta for reconstructing model was in the initial of right renal artery proximal part. It couldn’t couse rat’s paraplegia for blocking blood flow for 20 minutes. Blocking blood flow for 30 minutes, the paraplegia rate was 70% and the neural function has some recovery. Blocking blood flow for 40 minutes, the paraplegia rate was 90%, but neural function has little recovery.Blocking blood flow for 60 minutes, the neural function could not recovery.2.In model group Na+-K+-ATPase activity and glutamate transporters function began to drop 30 minutes after reperfusion, reached the low ebb 4 hours after reperfusion and then began to increase but it couldn’t reach the normal level 12 hours after reperfusion(P<0.05). There was no difference between Nimodipine group and model group(P>0.05),Those of Tanshinone group were higher than model group(P<0.05). In model group content of glutamic acid in blood serum and spinal cord and NMDAR1 protein expression in the anterior horn cells of the spinal cord increased following 30-minute ischemia/30 minutes reperfusion injury to the spinal cord, reached a peak 4 hours after reperfusion, and then dropped but it couldn’t reach the normal level 12 hours after reperfusion. There was no difference between Nimodipine group and model group(P>0.05),Those of Tanshinone group were lower than model group(P<0.05). In model group intraneuronal Ca2+ concentration and moisture capacity of spinal cord increased following 30-minute ischemia/1 hour reperfusion injury to the spinal cord, reached a peak 4 hours after reperfusion, and then dropped but it couldn’t reach the normal level 12 hours after reperfusion.1,4,8,12 hours after reperfusion intraneuronal Ca2+ concentration of rat in tanshinone and Nimodipine group was lower than that of rat in model group(P<0.05).1 hour and 4 hours after reperfusion, intraneuronal Ca2+ concentration of rat in tanshinone group was lower than that of rat in Nimodipine group(P<0.05) but there was no difference between two groups 8 hours and 12 hours after reperfusion(P>0.05).4,8,12 hours after reperfusion moisture capacity of spinal cord of rat in tanshinone and Nimodipine group was lower than that of rat in model group(P<0.05).4 hours after reperfusion, moisture capacity of spinal cord of rat in tanshinone group was lower than that of rat in Nimodipine group(P<0.05) but there was no difference between two groups 8 hours and 12 hours after reperfusion(P>0.05).Tanshinone and Nimodipine could lessen edema of spinal cord both but effectiveness of tanshinone was better than that of Nimodipine 4 hours after reperfusion.3.Content of glutamic acid in blood serum and that in spinal cord was prominent positive correlation at 0.01 level. Content of glutamic acid in both blood serum and spinal cord and neural function were prominent negatively correlation at 0.01 level.Conclusions1.The best position to block blood flow of abdominal aorta for reconstructing model was in the initial of right renal artery proximal part. The time of best blocking is 30 minutes.2.Tanshinone could influence the anterior factors of EAAs-NMDA-Ca2+ path, thus reduced the nerve cell Ca2+ overload to protect neural function in SCII.3.Content of glutamic acid in blood serum might become a convenience, valid and micro-trauma index for diagnosis, treatment and evaluate curative effect of spinal cord ischemic reperfusion injury in clinic.更多还原