天麻素抑制大鼠脊髓损伤后胶质瘢痕形成及其促进神经功能恢复的研究

发布时间：2018-06-05 23:58

本文选题：天麻素 + 脊髓损伤　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]脊髓损伤(spinal cord injury,SCI)是一种严重的创伤性疾病,目前尚缺乏有效的治疗手段。SCI后星形胶质细胞活化增生形成胶质瘢痕,维持损伤部位脊髓结构的完整性。但过度增生的胶质瘢痕会阻碍轴突生长,同时分泌神经抑制因子影响神经元存活,阻碍神经再生。本研究用天麻素(GAS)对钳夹型急性大鼠脊髓损伤模型进行干预,探讨SCI后天麻素干预对胶质瘢痕形成的影响以及对脊髓神经功能恢复的作用。[方法]1、80只清洁级成年雄性SD大鼠,体重220g-240g,随机分为5组:①假手术组(Sham组,n=16),②损伤组(SCI组,n=16),③低剂量天麻素治疗组(SCI+GAS(L)组,50mg/Kg,n=16),④中剂量天麻素治疗组(SCI+GAS(M)组,100mg/Kg,n=16),⑤高剂量天麻素治疗组(SCI+GAS(H)组,150mg/Kg,n=16)。建立钳夹型大鼠SCI模型。其中Sham组仅切除椎板,SCI组和各治疗组采用70g动脉瘤夹对大鼠T10节段脊髓进行30秒钳夹损伤。术后各治疗组分别给予不同浓度天麻素连续干预7天,Sham组和SCI组给予等量生理盐水。分别于术后1d、3d、7d、14d、21d、28d 采用 Basso Beattie Bresnahan 运动功能评分(BBB 运动功能评分)和Rivlin斜板实验评估脊髓神经功能恢复情况。2、损伤后4w进行灌注、取材。观察损伤节段脊髓形态变化,常规脱水、包埋,进行冰冻切片。免疫荧光染色后,对图像进行定量分析,测定星形胶质细胞特殊标记物胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)表达以及胶质瘢痕面积。3、损伤后4w行HE染色检测脊髓形态和脊髓空洞面积;行尼氏(Nissl)染色检测神经元尼氏小体数量、形态。4、损伤后2w于各组中各随机抽取4只大鼠,立体定位下注射荧光素标记BDA神经纤维示踪剂。损伤后4w行冰冻切片后在荧光显微镜下观察神经轴突数量,评估脊髓神经传导通路恢复情况。[结果]1、术后各组BBB评分和Rivlin斜板实验评分评分均下降,随着术后时间增长各组评分逐渐升高。Sham组评分于各时相点均高于其余各组(P0.05),各GAS治疗组在术后3d及其后各时相点评分均明显均高于SCI组(P0.05),SCI+GAS(M)组评分均高于其余两个天麻素治疗组(P0.05)。2、损伤后4w各GAS治疗组和SCI组GFAP表达较Sham组均显著增高(P0.05),各GAS治疗组GFAP表达明显低于SCI组,SCI+GAS(M)组GFAP抑制最为明显,(P0.05)。各GAS治疗组胶质瘢痕阳性染色面积明显减小,SCI+GAS(M)组胶质瘢痕面积明显低于SCI组,(P0.05)。3、损伤后4w,SCI组脊髓组织结构排列混乱,脊髓空洞形成,各GAS治疗组脊髓结构更为清晰,空洞面积减小,SCI+GAS(M)组脊髓空洞面积明显低于SCI 组,(P0.05)。SCI+GAS(M)组 Nissl 小体数量明显多于 SCI 组,(P0.05)。4、损伤4周后Sham组脊髓BDA阳性染色神经纤维最多,各GAS治疗组损伤平面阳性染色的神经纤维明显多于SCI组,其中SCI+GAS(M)组阳性染色纤维多于其余两个治疗组,(P0.05)。[结论]1、GAS可以下调SCI后损伤区域星形胶质细胞GFAP表达水平,抑制星形胶质细胞活化,减轻胶质瘢痕的形成。2、GAS可以保护SCI后损伤区域神经元,减轻脊髓空洞形成,促进轴突再生,促使脊髓神经传导通路修复。3、GAS可以提高大鼠SCI后行为学评分,促进神经功能恢复,其治疗大鼠SCI效果的最佳给药浓度范围可能在100mg/Kg左右。
[Abstract]:[Objective] spinal cord injury (SCI) is a serious traumatic disease. At present, there is still a lack of effective treatment of astrocytes by activation of astrocytes to form glial scar and maintain the integrity of the spinal structure in the injured part of the spinal cord. However, hyperplastic glial scar will impede the growth of axon and secrete the neuroinhibitory factor. In this study, the effects of gastrodin on the formation of glial scar and the function of spinal nerve function recovery after SCI were investigated with gastrodin (GAS) and the effect of gastrodin intervention on the formation of glial scar after SCI. [methods only 5 groups of male SD rats, 220g-240g, were randomly divided into 5 groups: (1) the sham operation group (group Sham, n=16), the injury group (group SCI, n=16), the low dose gastrodin group (SCI+GAS (L), 50mg/Kg, n=16), (SCI+GAS (M), 100mg/Kg, n=16) in the middle dose group of gastrodin (SCI+GAS (M), 100mg/Kg, n=16). Group and treatment group were treated with 70g aneurysm clips for 30 seconds of clamp injury to the T10 segment of the spinal cord of rats. The treatment groups were given different concentrations of gastrodin for 7 days, and group Sham and group SCI were given equal amount of saline. 1D, 3D, 7d, 14d, 21d and 28d were evaluated by Basso Beattie exercise function after the operation. Scores) and Rivlin oblique plate test were used to evaluate the recovery of nerve function of the spinal cord,.2, and 4W was perfused after injury. The morphological changes of the spinal cord were observed. Dehydration, embedding, and frozen section were observed. After immunofluorescence staining, the images were quantified and the glial fibrillary acidic protein (Glial Fibri), a special marker of astrocytes, was measured. The expression of llary Acidic Protein, GFAP) and the area of glial scar were.3. The morphology of spinal cord and the cavity area of spinal cord were detected by HE staining after injury. Nissl's (Nissl) staining was used to detect the number of Nissl corpuscles and.4. 4 rats were randomly selected from each group after injury, and the fluorescein labeled BDA nerve tracer was injected under stereotyping. After the frozen section of 4W, the number of axon was observed under the fluorescence microscope, and the recovery of spinal nerve conduction pathway was evaluated. [results]1, the BBB score and the Rivlin oblique plate test score were all decreased after the operation. With the increase of postoperative time, the scores of each group were gradually higher than those of the other groups (P0.05), each G was higher than the other groups (P0.05), each G The score of AS in the treatment group was higher than that of the SCI group (P0.05), and the score of SCI+GAS (M) group was higher than that of the other two gastrodin group (P0.05).2, and the GFAP expression of 4W each GAS treatment group and the SCI group increased significantly after the injury, and the expression of the GFAP group was significantly lower than that of the group. (P0.05). The positive staining area of glial scar in each GAS treatment group decreased obviously, and the area of glial scar in SCI+GAS (M) group was obviously lower than that of group SCI, (P0.05).3, 4W in the group after injury and the formation of spinal cord in SCI group, the formation of spinal cord cavity, the spinal cord structure of each GAS treatment group was clearer, the cavity area decreased, and the cavity area of the SCI+GAS (M) group was clear. The number of Nissl corpuscles in group.SCI+GAS (P0.05).SCI+GAS (M) was significantly more than that of group SCI, (P0.05).4. The majority of BDA positive staining nerve fibers in the spinal cord of Sham group after 4 weeks of injury were more than those in the SCI group, and the positive staining fibers in the group were more than the other two groups. On]1, GAS can reduce the GFAP expression level of astrocytes in the injured region of SCI, inhibit astrocyte activation and reduce the formation of.2 in glial scar. GAS can protect the injured region neurons after SCI, reduce the formation of the spinal cord, promote the regeneration of axon, promote the spinal nerve conduction pathway to repair.3, GAS can improve the behavior of rats after SCI. The score could promote the recovery of nerve function, and the optimal concentration range of SCI for treating rats could be around 100mg/Kg.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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