A型肉毒素重链对大鼠脊髓损伤后总体蛋白表达、髓鞘相关糖蛋白及其受体表达的影响

发布时间：2018-01-23 22:54

本文关键词： 脊髓损伤模型 A型肉毒杆菌神经毒素重链神经损伤与再生髓磷脂相关糖蛋白 Nogo-receptor　出处：《山西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.在大鼠腰段脊髓损伤模型基础上局部给予小剂量A型肉毒杆菌神经毒素重链(botulinum neurotoxin serotype A heavy chain,BoNT/A HC),观察其对脊髓损伤后局部总体蛋白表达谱的影响;2.观察BoNT/A HC对脊髓损伤局部髓磷脂相关糖蛋白及其受体表达的影响,为探讨BoNT/A重链促进神经突起再生的机制提供实验依据。方法:1.采用1ml注射器针头改制的脊髓损伤器械复制大鼠腰段脊髓半离断性损伤模型;在模型复制同时由针头所连接微量注射器向髓腔内注射不同剂量BoNT/A重链(2μg、4μg、6μg和8μg);2.于大鼠脊髓损伤后2天和20天提取损伤局部及损伤近端2~3cm组织蛋白进行SDS-PAGE+考马氏亮蓝染色确定蛋白表达有无差异,同时采用BoNT/A重链6mg剂量组进行双向电泳+硝酸银染色进一步确定蛋白表达差异的点数或点群分布;3.根据双向电泳所出现的点群变化的分子量水平,采用western blot主要检测BoNT/A重链对脊髓组织髓磷脂相关抑制因子—髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)及其受体(nogo-receptor,N gR)的表达的干预作用。结果:1.单侧腰段脊髓损伤模型显微镜下呈现明显的一侧脊髓结构破坏,局部神经细胞坏死、白质神经传导束崩解不可辨认;2.SDS-PAGE+考马斯亮蓝染色显示,单纯脊髓损伤后,损伤局部蛋白表达与正常相比有明显不同,有些分子量水平的蛋白表达明显增多;BoNT/A重链用药后2天及20天时,局部蛋白的表达与单纯损伤组相比明显不同,但与正常对照组类同、趋向正常化;3.双向电泳+硝酸银染色的结果进一步确定了这些蛋白表达变异的现象出现在多个分子量水平,呈现点或点群的表达变异。有10余种蛋白表达与单纯损伤组明显不同,呈向正常转化的趋势;主要的特点是:单纯损伤时,某些蛋白点或点群表达增加,而BoNT/A重链干预后,这些变异又趋向于与正常对照组相似;4.根据蛋白表达变异的分子量特点,选取MAG和NgR作为目标蛋白的wesetrb-blot结果显示:单纯脊髓损伤后MAG及NgR表达皆呈现增多,而髓腔内注射BoNT/A重链后这两种蛋白的表达均减少,差异具有统计学意义(P0.05)。结论:1.BoNT/A重链可干预大鼠脊髓损伤后局部蛋白表达谱变化;2.BoNT/A重链可逆转脊髓损伤后局部大量髓磷脂抑制因子的产生及其受体的表达。
[Abstract]:Objective 1. To give a small dose of botulinum A neurotoxin heavy chain on the basis of lumbar spinal cord injury model in rats. Botulinum neurotoxin serotype A heavy chain. To observe the effect of BoNT/A on the expression profile of local total protein after spinal cord injury. 2. To observe the effect of BoNT/A HC on the expression of myelin associated glycoprotein and its receptor in spinal cord injury. To explore the mechanism of BoNT/A heavy chain promoting neurite regeneration. Methods 1: 1. Spinal cord injury model was established by using 1 ml syringe needle modified spinal cord injury instrument in rats. At the same time, the microinjector connected with the needle was injected into the intramedullary cavity with different doses of BoNT/A 2 渭 g, 4 渭 g, 6 渭 g and 8 渭 g / g respectively. 2. Two and twenty days after spinal cord injury, 3 cm tissue proteins were extracted from the injured area and the proximal end of the injury to determine whether there was a difference in the expression of the protein by SDS-PAGE Coombe bright blue staining. At the same time, the BoNT/A heavy chain 6mg dose group was used to determine the point or point group distribution of protein expression difference by two dimensional electrophoresis silver nitrate staining. 3.According to the molecular weight level of point group changes in two-dimensional electrophoresis. Western blot was used to detect myelin associated inhibitor (myelin sheath associated glycoprotein) of BoNT/A heavy chain in spinal cord tissue. Myelin associated glycoprotein. Results: 1. Unilateral spinal cord injury model showed obvious unilateral spinal cord structure damage under microscope. Local nerve cell necrosis, white matter nerve conduction bundle disintegration unrecognizable; 2. SDS-PAGE Coomassie brilliant blue staining showed that after spinal cord injury alone, the expression of local protein was significantly different from that of normal, and the expression of some proteins with molecular weight increased obviously. On the 2nd and 20th day after BoNT/A heavy chain administration, the expression of local protein was significantly different from that of the simple injury group, but similar to that of the normal control group, and tended to normalize. 3. The results of silver nitrate staining further confirmed that the variation of these proteins appeared at multiple molecular weight levels. The expression of more than 10 proteins was significantly different from that of the simple injury group and showed a trend of normal transformation. The main characteristics were as follows: the expression of some protein points or groups was increased in simple injury, but after the intervention of BoNT/A heavy chain, these variations tended to be similar to those of the normal control group. 4.According to the molecular weight characteristics of protein expression variation. The results of wesetrb-blot with MAG and NgR as target protein showed that the expression of MAG and NgR increased after spinal cord injury alone. However, the expression of these two proteins decreased after intramedullary injection of BoNT/A heavy chain. Conclusion: 1. BoNT / A heavy chain can interfere with the changes of local protein expression profile after spinal cord injury in rats. 2. BoNT-A heavy chain could reverse the production of a large amount of myelin inhibitor and the expression of its receptor after spinal cord injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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