【作者】 赵东旭；

【导师】 朱庆三；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 脊髓损伤(spinal cord injury, SCI)是由直接或间接外力损伤脊髓，在损害相应的阶段出现各种功能障碍为主要临床表现一种称谓。它是一种高致残性疾病，脊髓损伤是一种严重的神经系统创伤，是常见的截瘫原因之一。脊髓缺血再灌注损伤（spinal cord ischemic reperfusioninjury，SCII）是原发性脊髓损伤的继发性损害，由Allen在1911年首先描述。它是指在一些导致脊髓缺血因素去除后，脊髓恢复血供，神经功能不仅得不到改善，反而在原缺血损伤基础上进一步加重，甚至出现不可逆性脊髓神经元迟发性死亡的现象，是外科手术常见并发症之一，特别是主动脉瘤、骶管肿瘤手术后，常需阻断主动脉，从而导致脊髓损伤，术后出现神经功能损伤的发病率高于4%，甚至可高达38%。脊髓缺血再灌注损伤是一个极其复杂的病理过程，确切的分子和细胞机制尚不清楚，目前认为与组织兴奋性氨基酸的释放、自由基的产生、胞内Ca2+的超载、Na+-K+-ATP平衡紊乱、炎症反应、神经细胞的凋亡等因素有关。各种因素共同存在、交互作用，致使组织细胞受损。人参，五加科人参属植物人参（Panax ginseng C.A. Mey.）的根，是一种极具药用和食用价值的名贵中草药，应用距今已有2000余年的历史。人参中含有多种成分如人参皂甙、挥发油、有机酸及脂(软脂酸、硬脂酸、油酸和亚油酸)、甾醇及其甙、蛋白质、多肽、氨基酸及腐胺、精胺等其他许多含氨化合物、维生素类（维生素A、B1、B2和C等）、微量元素类、木质素、黄铜类、糖类、糖蛋白以及其他许多成分。近年来国内外学者对人参化学、药理及临床应用进行了大量的研究，证实人参皂甙是人参生理活性最重要的有效成分，且因糖基的组成和与甙元的连接位置不同，致使不同的人参皂甙所具有的药理学活性存在较大差异。包括抗肿瘤、抗衰老、抗心律失常、抑制细胞凋亡、降糖降脂、改善学习记忆、增强性功能和免疫功能及解酒等多个方面。本研究拟通过建立大鼠脊髓缺血再灌注损伤模型，应用TheBasso Beattie Bresnahan（BBB）评分系统对脊髓神经功能进行评价；HE染色，尼氏体染色，Tunel凋亡检测等方法明确神经细胞形态变化及凋亡情况；应用免疫荧光、western blot等手段检测凋亡相关蛋白Bcl-2、Bax、磷酸化ASK-1及Caspase-3的表达情况。同时给予人参皂甙Rb1进行干预，观察人参皂甙Rb1干预对上述观测指标的影响，明确凋亡相关蛋白Bcl-2、Bax、磷酸化ASK-1及Caspase-3、人参皂甙Rb1和SCII三者间的作用及其可能机制，为临床研发新型治疗SCII的药物提供理论基础。目的：探讨人参皂甙Rb1通过抗细胞凋亡作用治疗大鼠脊髓缺血再灌注损伤的研究。方法：选用成年SD大鼠，雌雄各半，分为空白组、假手术组、治疗组（10mg/kg剂量每日给予人参皂甙Rb1腹腔注射）、模型组。采用夹闭腹主动脉的方法制备SCII模型。分别于再灌注后1d、2d、3d、4d、5d、6d、7d对四组模型进行The Basso Beattie Bresnahan(BBB)运动功能评分。缺血再灌注损伤1、3、5、7天后，分别取大鼠脊髓进行HE染色、尼氏体染色、Tunel法、免疫荧光化学和Westernblot检测，观察各组脊髓组织形态和凋亡情况及凋亡相关蛋白的表达情况。结果：HE染色和尼氏体染色表明治疗组病理组织形态改变明显好于模型组。Tunel结果表明治疗组细胞凋亡率显著低于模型组（P<0.05）。Bax/Bcl-2比值治疗组显著低于模型组（P<0.05），磷酸化ASK-1、Caspase-3蛋白表达治疗组显著低于模型组（P<0.05）。Westernblot检测结果显示空白组和假手术组两组的p-ASK-1和Caspase-3蛋白表达较少；模型组中p-ASK-1和Caspase-3的蛋白表达明显增多，与空白组及假手术组相比有显著性差异（P＜0.05）；治疗组p-ASK-1和Caspase-3蛋白表达随着时间推移逐渐减少，且各个时间点与模型组相比均有显著性差异（P＜0.05）。结论：人参皂甙Rb1可以治疗大鼠脊髓缺血再灌注损伤；人参皂甙Rb1可以促进Bcl-2蛋白的表达，且可以抑制Bax、p-ASK-1和Caspase-3蛋白的表达；抗凋亡作用可能是人参皂甙Rb1治疗大鼠脊髓缺血再灌注损伤的作用机制之一。更多还原

【Abstract】 Spinal cord injury (SCI) is caused by directly or indirectly force,which often result in some clinical manifestations such as neurologicaldysfunction in the injuryed segments. SCI is not only a highly disablingdisease, but a common causes of paraplegia. Spinal cord ischemicreperfusion injury (SCII) is the secondary injury after the spinal cordprimary injury, which was first reported by Allen in1911. SCII refers towhen the spinal cord blood supply was restored after the removal ofspinal ischemic causes, neurological dysfunction not only can not beimproved but further aggravated base on the ischemic injury, and evencause the irreversible spinal neurons delayed death. SCII is a commoncomplication of surgery operation, especially of some operations whichneed to blocking the aorta, such as operations associated with aneurysmaortic and sacral canal tumor. The post operation incidence rate of nerveinjury is more than4%, even up to38%. SCII is a extremely complexpathological process, the precise molecular and cellular mechanism areremain unclear. At present, studies suggest that SCII is related tofollowing factors, such as the release of excitatory amino acids and freeradical, intracellular Ca2+overload, Na pump disorders, inflammation,and neurons apoptosis. Those factors are existing together and areinteracting each other, finally result in the neurological damaged.Ginseng, the root of the Araliaceae plant panax ginseng, is a rareherbal traditional medicine with medical and edible value, it has been utilized more than2000years.Ginseng contains a variety of ingredients, such as ginseng saponin,volatile oils, organic acids and lipids (palmitic acid, stearic acid, oleicacid and linoleic acid), sterols and their glycosides, proteins, peptides,amino acids and putrescine, spermine ammonia, and many othercompounds, vitamins (vitamin A, B1, B2and C), trace elements, lignin,brass, sugars, glycoproteins. Recently, a lot of research have been appliedto study the chemistry characteristics, pharmacology mechanism andclinical application, which confirm that ginsenosides is the mostimportant active ingredient in ginseng. In addition, because of thedifferent components and connections of the aglycone glycosylationposition, there is a big difference between the pharmacologically activevarious ginsenosides. Such as anti-tumor, anti-aging, anti-arrhythmic,inhibition of apoptosis, reducing blood glucose and lipids, improvelearning and memory, enhance sexual function and immune function.In this study, through the establishment of the rat spinal cordischemia-reperfusion injury model and the Basso Beattie Bresnahan(BBB) scoring system, we could evaluate the spinal cord neurologicalfunction. By using the HE staining, nissl body staining and the Tunelapoptosis detection, we could figure out the apoptosis status and themorphological modification of neuron. By using the Immunofluorescencetechnique and Western blot, we could detect some apoptosis associatedproteins, such as Bcl-2, Bax, P-ASK-1and Caspase-3. We useginsenosides Rb1to perform the intervention experiment, and detect themodification of those mentioned index after the Rb1application. Fromthose experiments, we can not only confirm the functions andrelationships among apoptosis associated proteins (Bcl-2, Bax,P-ASK-1and Caspase-3), ginsenosides Rb1and SCII, but also provide the theoretical basis for the development of novel SCII treatment medicine.Objectives: To discuss the anti-apoptosis mechanism of the SCIItherapeutical effect of ginsenosides Rb1.Methods:Taking adult SD rats, both male and female, divided intocontrol group, sham group, treatment group (10mg/kg per day,intraperitoneal injection of ginsenosides Rb1), SCII model group (Byoccluding the abdominal aorta). Assessing the Basso Beattie Bresnahan(BBB) motor function score in1,2,3,4,5,6,7days after the reperfusion offour groups respectively.1,3,5,7days after reperfusion, to observe the tissuemorphology, apoptosis status of spinal cord and apoptosis associated proteins,spinal cord from each groups were taken out and perform HE staining, Nisslstaining, Tunel dectection, immunofluorescence detection and Westren blot.Results: HE and Nissl body staining results suggest that thepathological and morphological changes of treatment group is better thanSCII group. Tunel results suggest the apoptosis rate of treatment group islower than the SCII model group significantly (P<0.05). Bax/Bcl-2valueof treatment group is still lower than the SCII model group significantly(P<0.05). The P-ASK-1and Caspase-3expression level of treatmentgroup is lower than the SCII model group significantly (P<0.05). Westernblot results indicate that the P-ASK-1and Caspase-3expression level ofcontrol group and sham group is lower than the other two operationgroups. In SCII model group, the P-ASK-1and Caspase-3expressionlevel is higher than control group and sham group (P<0.05). In Rb1treatment group, the P-ASK-1and Caspase-3expression level aredecreased gradually as time goes by, compare with the SCII model group,the expression level in each time point are all different significantly(P<0.05). Conclusion: Ginsenoside Rb1can treat spinal cord ischemia-reperfusion injury. Ginsenosides Rb1can promote the expression ofBcl-2, and inhibit the expression of Bax, p-ASK-1and Caspase-3. Theanti-apoptotic effects of ginsenosides Rb1is one of the probably treatmentmechanisms of SCII.更多还原