AAV9-IGF1对SOD1G93A小鼠运动神经元阈电位的影响

发布时间：2018-01-03 19:00

本文关键词：AAV9-IGF1对SOD1G93A小鼠运动神经元阈电位的影响　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目前已识别了超氧化物歧化酶1(superoxide dismutase 1,SOD1)基因100多种突变,占家族型ALS患者的~20%。人SOD1鼠模型的临床表型及病理基础与ALS散发性及家族性患者最为相近。其中,有三种SOD1G85R、SOD1G37R及SOD1G93A已被广泛的应用于ALS的转基因鼠模型。SOD1是表达153个氨基酸多肽的同型二聚体酶,每个亚单元结合一个铜及一个锌离子。铜负责酶的催化活性,锌负责稳定蛋白的结构。SOD1的主要功能包括自由基的清除,通过催化超氧化物阴离子转化成分子氧和过氧化氢,通过谷胱甘肽过氧化物酶及过氧化氢酶分解成水。SOD1分布广泛,主要分布于细胞质中,线粒体及其他亚细胞器中也能被检测到。腺相关病毒(adeno-associated virus,AAV)是一种小的,无包膜病毒,已经成为基因治疗领域研究的主题。它介导的基因转导是安全有效的,也是作为临床研究的一种治疗工具。大量的动物模型显著的证据表明了重组腺相关病毒(recombinant adeno-associated virus,r AAVs)的有效性及安全性。其中,将携带编码人胰岛素生长因子1基因重组腺相关病毒(insulin like growth factor 1 delivery by recombinant adeno-associated virus,r AAV-IGF1)注射到SOD1G93A模型小鼠的呼吸及后肢肌肉中,这延长了生存期及延缓了运动的削减。AAV带菌者具有逆向传递的特性,如脊髓神经元是选择性的靶组织,允许局部分泌的人胰岛素生长因子1(insulin like growth factor 1,IGF1)对周围细胞有广泛的作用,并不仅仅局限于传递病毒的运动神经元细胞。中枢神经系统中,AAV研究最多的血清型是1,2,5,8,9,及重组。血清型的效果取决于脑分布、物种及靶细胞类型。这些血清型有效地转导给神经元,星形胶质细胞、少突胶质细胞及小胶质细胞转导受限,使用特定细胞激活子能够得到改善。研究证实,腺相关病毒血清型9(adeno-associated virus 9,AAV9)能通过血脑屏障且有效靶向脑及脊髓中的神经元、星形胶质细胞。小鼠中,AAV9介导的中枢神经系统通过小脑延髓池途径的靶向基因传递,与其它AAV血清型相比,AAV9是在中枢神经系统中传递更好的载体,能更好的透过血脑屏障。为了探究肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)症状前期运动神经元电生理的改变及IGF1对电生理的影响,本实验采用侧脑室及脑实质两种方法注射AAV9-IGF1及AAV9-GFP(green fluorescent protein,GFP)于SOD1G93A新生鼠中,观察运动皮层M2区的表达情况,选用IGF1表达量多的方法注射AAV9-IGF/AAV-GFP,并记录20-40天SOD1G93A小鼠运动皮层2区(motor cortex 2 area,M2)的动作电位,分析AAV9-IGF1对SOD1G93A小鼠症状前期运动神经元动作电位的影响。目的:1记录SOD1G93A转基因及非转基因小鼠的动作电位,并比较动作电位的阈电位、频率、动作电位幅度、潜伏期及上升速率是否存在差别;2通过侧脑室及脑实质注射AAV9-IGF1/AAV-GFP于SOD1G93A新生鼠(出生24h内)中,分别观察其在运动皮层M2区的表达效果;3分别记录脑实质注射AAV9-IGF1/AAV-GFP的20-40天SOD1G93A转基因小鼠M2区的动作电位,并比较阈电位、频率、动作电位幅度、潜伏期及上升速率是否存在差别。方法:本实验应用C57/6小鼠及SOD1G93A转基因及非转基因同窝对照小鼠,SOD1G93A转基因小鼠的鉴定通过PCR扩增的方法。采用脑组织及侧脑室两种方法注射AAV9-IGF1/AAV-GFP,选用20-40天C57/6、SOD1G93A转基因和非转基因小鼠的脑片,用免疫组化及免疫荧光方法观察AAV9-IGF1及AAV-GFP在运动皮层M2区的表达,选用表达量多的方法注射AAV9-IGF1/AAV-GFP于SOD1G93A转基因新生鼠中,采用全细胞膜片钳技术记录20-40天小鼠脑片M2的动作电位,并统计分析。结果:1应用全细胞膜片钳技术,记录20-40天SOD1G93A转基因(h SOD1G93A)组及非转基因(m SOD1WT)组小鼠脑片运动皮层M2区动作电位:两组阈电位有区别,但差异无统计学意义;两组频率中,SOD1G93A转基因组高于同窝对照组,150p A电流刺激时差异有统计学意义(150p A,P=0.040.05),200-500p A电流刺激时,差异无统计学意义;动作电位幅度,两组之间差异无统计学意义;两组潜伏期比较,差异无统计学意义;两组上升速率,SOD1G93A转基因组高于同窝对照组(450p A除外),250p A电流刺激时差异有统计学意义(250p A,P=0.0320.05)。2通过侧脑室及脑实质两种方法注射AAV9-IGF1/AAV-GFP,应用免疫组化及免疫荧光方法观察20-40天小鼠脑片M2区IGF1的表达情况。在小鼠脑片运动皮层M2区中,与侧脑室注射相比,脑实质注射的方法IGF1表达量更高,且差异有统计学意义。3应用全细胞膜片钳技术,记录脑实质注射AAV9-IGF1/AAV-GFP20-40天SOD1G93A转基因组小鼠脑片M2区的动作电位:与AAV9-GFP组相比,AAV9-IGF1组阈电位明显减小,且差异有统计学意义;两组频率,差异无统计学意义;与AAV9-GFP组相比,AAV9-IGF1组动作电位幅度降低,但差异无统计学意义;与AAV9-GFP组相比,AAV9-IGF1组动作电位的潜伏期缩短,且在150p A、500p A电流刺激时,差异有统计学意义(P0.05);两组上升速率比较,差异无统计学意义。结论:1与同窝对照组相比,SOD1G93A转基因鼠症状前期运动神经元有兴奋性增高的趋势。2与侧脑室注射相比,脑实质注射在20-40天小鼠M2区的IGF1表达量更高。3 IGF1降低了SOD1G93A转基因鼠症状前期运动神经元的阈电位,发挥了神经保护作用。
[Abstract]:Has identified the superoxide dismutase 1 (superoxide dismutase 1, SOD1) 100 kinds of gene mutations, ~20%. patients accounted for SOD1 rat model of familial ALS clinical phenotypes and pathologic basis and ALS in sporadic and familial patients were mostly similar. Among them, there are three kinds of SOD1G85R, SOD1G37R and.SOD1 transgenic mouse model SOD1G93A has been widely used in ALS is the same two dimeric enzyme expression polypeptide of 153 amino acids, each sub unit with a copper and a zinc ion. The catalytic activity of the enzyme responsible for the removal of copper, the main function structure of.SOD1 is responsible for the zinc stable proteins including free radicals, superoxide anion catalyzed by into molecular oxygen and hydrogen peroxide, decomposed into water.SOD1 widely distributed through glutathione peroxidase and catalase, mainly distributed in the cytoplasm, mitochondria and other subcellular organelles can also be detected by adenovirus. Close the virus (adeno-associated virus, AAV) is a small, non enveloped virus, has become the field of gene therapy. Gene transduction mediated by theme it is safe and effective, but also as a therapeutic tool for clinical research. A large number of animal models significant evidences to prove that the recombinant adeno-associated virus (recombinant adeno-associated virus, R, AAVs) efficacy and safety. Among them, the encoding human insulin-like growth factor 1 gene recombinant adeno-associated virus (insulin like growth factor 1 delivery by recombinant adeno-associated virus, R AAV-IGF1) was injected into the SOD1G93A mouse model of respiratory and hindlimb muscles, the prolonged survival and slow motion cut the.AAV carrier has the characteristics of reverse transfer, such as spinal cord neurons are the target tissue selective, allowing local secretion of human insulin-like growth factor 1 (insulin like Growth factor 1, IGF1) has a wide effect on the surrounding cells, motor neurons is not limited to transfer the virus. The central nervous system, AAV of serotype 1,2,5,8,9 is the most, and depends on the effect of serum type. The recombinant brain distribution, species and target cell types. These serotypes efficiently transduced for neurons, astrocytes, oligodendrocytes and microglial cell transduction is limited, the use of specific cell activator can be improved. The research confirmed that adeno-associated virus serotype 9 (adeno-associated virus 9, AAV9) can pass through the blood-brain barrier and effective targeting to the brain and spinal cord neurons, astrocytes in mice. In the central nervous system AAV9 mediated gene transfer to the cisterna magna pathway target, compared with other AAV serotypes, AAV9 is the better carrier in the central nervous system, through the blood better Brain barrier. In order to explore the amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS) changes of electrophysiological pre motor neuron symptoms and IGF1 on electrophysiology, the lateral ventricle and brain parenchyma in two methods of injection of AAV9-IGF1 and AAV9-GFP (green fluorescent protein, GFP SOD1G93A) in neonatal rats, observe the expression of the motor cortex area M2, the expression of IGF1 AAV9-IGF/AAV-GFP injection quantity, and record the 20-40 days SOD1G93A mice cortex area 2 (motor cortex 2 area, M2) of the action potential, analysis on the effect of AAV9-IGF1 on early symptoms of SOD1G93A mice motor neuron action potentials. Objective: the records of 1 SOD1G93A transgenic and non transgenic mouse action potential, threshold potential, and to compare the frequency of action potential, action potential amplitude, latency and the rising rate of whether there exists difference; 2 by intracerebroventricular injection of AA and brain parenchyma V9-IGF1/AAV-GFP SOD1G93A in neonatal rats (born in 24h), respectively, to observe the expression effect in motor cortex area M2; 3 of the action potential were recorded in brain parenchyma injection of AAV9-IGF1/AAV-GFP 20-40 day SOD1G93A M2 transgenic mice, and compare the threshold potential, action potential amplitude, frequency, latency and the rising rate of whether there is a difference. The experimental application of C57/6 mice and SOD1G93A transgenic and non transgenic littermate control mice, method of identification of SOD1G93A transgenic mice by PCR amplification. The brain tissue and the lateral ventricle injection of AAV9-IGF1/AAV-GFP with two methods, 20-40 day C57/6, SOD1G93A transgenic and non transgenic mice, to observe the expression of AAV9-IGF1 and AAV-GFP in motor cortex M2 the use of immunohistochemistry and immunofluorescence method, the expression method of quantity of injection of AAV9-IGF1/AAV-GFP in transgenic SOD1G93A in neonatal rats, the The action potential of the whole cell patch clamp techniques 20-40 days M2 mouse brain slices, and statistical analysis. Results: 1 using whole cell patch clamp technique to record 20-40 days (H SOD1G93A) SOD1G93A transgenic and non transgenic group (m SOD1WT) mice brain slices of motor cortex area M2 action potential threshold potential: the two groups are different, but no statistically significant difference between the two groups; frequency in SOD1G93A group was higher than that of transgenic littermate control group, was statistically significant 150P A current stimulation (150P A, P=0.040.05) between 200-500p, A current stimulation, the difference was not statistically significant; the amplitude of action potential, no significant difference between the two groups were compared between the two groups; latency difference the two groups had no statistical significance; the rising rate of SOD1G93A group was higher than that of transgenic littermate control group (450p A), 250p was statistically significant difference (250p A stimulated A, P= 0.0320.05).2 by lateral ventricle and brain parenchyma in two ways Injection of AAV9-IGF1/AAV-GFP, immunohistochemistry and immunofluorescence method to observe the expression of 20-40 in mouse brain slices of M2 days IGF1. In the movement of mouse brain slices M2 cortex, compared with intracerebroventricular injection method, cerebral parenchyma injection the expression of IGF1 is higher, and the difference was statistically significant.3 using the whole cell patch clamp technique recording, action potential brain parenchyma injection AAV9-IGF1/AAV-GFP20-40 day SOD1G93A transgenic mice brain slices M2 region: compared with AAV9-GFP group, AAV9-IGF1 group significantly reduced the threshold potential, and the difference was statistically significant; two groups of frequencies, no significant difference; compared with group AAV9-GFP, group AAV9-IGF1 decreased the action potential amplitude, but without statistical significance. Differences; compared with AAV9-GFP group, AAV9-IGF1 group, action potential latency, and at 150P A, 500p A current stimulation, the difference was statistically significant (P0.05); group two increased rate comparison, difference There is no statistical significance. Conclusion: 1 littermates were compared with the control group, SOD1G93A transgenic mice compared with early symptoms of motor neuron excitability increased.2 and intracerebroventricular injection, cerebral parenchyma injection at 20-40 days of mouse M2 region of IGF1 is more highly expressed.3 IGF1 lowered the threshold potential of SOD1G93A transgenic mice early symptoms of motor neurons and play a neuroprotective effect.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8

【参考文献】