过表达SOD1 G41S和G41D的重组腺相关病毒载体构建及其在肌萎缩侧索硬化N2a细胞模型中的作用探究
发布时间：2018-08-11 10:51
【摘要】：肌萎缩侧索硬化(amyotrophic lateral sclerosis,ALS)是一种快速进展、可累及上、下运动神经元的严重致死性神经系统变性病。研究发现,10%ALS为家族性的ALS(familial ALS,fALS),其中约20%fALS发病与超氧化物歧化酶1(superoxide dismutase1,SOD1)基因突变有关。在SOD1基因上存在两种同一氨基酸位点上的不同突变SOD1 G41S和G41D,而携带这两种突变的患者临床表现明显不同,SOD1G41S突变型fALS患者病程进展迅速,平均生存期约为11.6个月;而SOD1 G41D突变型患者病程进展较为缓慢,平均生存期约为17年。自1993年Rosen等人发现SOD1基因突变和ALS发病有关后,研究者对此种ALS致病机制进行了一系列研究,但至今尚不清楚。目前认为,氧化应激、错误折叠蛋白聚集、线粒体功能障碍、谷氨酸盐兴奋毒性、轴索运输损害、非神经元细胞作用等都参与ALS的致病过程。另外,临床研究发现,ALS患者可出现轴突的高兴奋性,表现为持续性的钠电流增加,患者呈现出肌痉挛和肌束震颤;而先前也有研究发现在ALSG93A模型鼠原代脊髓运动神经元、ALS患者诱导多能干细胞来源的运动神经元及G93A模型鼠离体脑片中的运动神经元都出现过度兴奋的现象。电压门控性钠通道(voltage-gatedsodium channel,VGSC)是神经元的兴奋和动作电位产生、传导的重要元件,因此该通道电生理特性如果改变也将会影响神经元的兴奋性,从而导致疾病的发生。先前研究报道ALS SOD1 G93A模型鼠在疾病早期神经元VGSC电流出现异常。但是目前未有研究对SOD1 G41S和G41D突变的VGSC电生理特性进行探究。因此,为进一步探究SOD1 G41S和G41D突变在ALS中的致病机制,将本研究分为两个部分,首先构建过表达野生型(widetype,WT)SOD1、SOD1 G41S及 G41D 重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体;用上述rAAV感染小鼠神经瘤母细胞(N2a)以构建ALS细胞模型,并观察N2a细胞过量表达此两种突变蛋白后,对神经元VGSC电生理特性和凋亡分子表达的影响。第一部分SOD1WT、SOD1 G41S和SOD1 G41D rAAV载体的构建及鉴定目的构建过表达SOD1 WT、SOD1 G41S及SOD1 G41D重组腺相关病毒载体。方法首先,利用PCR扩增各目的基因片段,应用EcoRI和HindⅢ内切酶对pMT121(pAAV-hSyn-bGlobinintron-EGFP-pA)表达载体进行酶切后,回收纯化线性化表达载体。进而在无缝反应液作用下,将各目的片段与回收后的线性化表达载体进行同源重组后转化DH5α感受态细胞。利用菌落PCR鉴定转化子,阳性克隆送测序,测序无误后进行质粒提抽。最后目的质粒与辅助质粒共转染HEK293细胞进行病毒包装,经纯化、浓缩、RT-PCR滴度测定后保存备用。结果PCR结果显示,在约765 bp、792 bp、521 bp、175 bp、367 bp可见特异性 RFP、GFP、SOD1WT、5'SOD1G41S/G41D、3'SOD1G41S/G41D 条带;应用EcoR Ⅰ和Hind Ⅲ内切酶对表达载体pMT121进行双酶切,回收4470 bp的线性化载体片段;菌落PCR转化子鉴定结果显示,SOD1WT重组质粒阳性克隆得到711bp片段、SOD1G415和SOD1G41D阳性克隆得到721bp片段;测序结果正确;72 h后获得3种SOD1 WT、SOD1 G41S和G41D rAAV颗粒,经浓缩、纯化、测定滴度分别为 2.43×1012 v.g./mL、2.28×1012 v.g./mL、2.52×1012 v.g./mL后,分装保存于-80℃备用。结论成功构建过表达SOD1 WT、SOD1 G41S及SOD1 G41D rAAV载体,病毒纯化、浓缩、滴度皆满足后续实验要求。第二部分SOD1WT、SOD1G41S和SOD1G41DrAAV载体在ALS细胞模型中的作用探究目的探究ALS SOD1上G41S和G41D两种突变型所致神经元VGSC电生理特性的差异及其可能的分子机制。方法构建过表达SOD1 WT、SOD1 G41S及G41D rAAV载体后,体外感染N2a细胞,将实验组分为3组:SOD1WT组、SOD1G41S和SOD1G41D组,并以无目的基因rAAV感染的N2a细胞为阴性对照(Control)组。利用全细胞膜片钳技术分别记录四组细胞VGSC电流,绘制通道快速激活和稳态失活曲线后分析相关参数。比较各组细胞凋亡分子Cleaved caspase-3在病毒感染后24、48和72 h不同时间点的表达情况。结果与Control组和SOD1 WT组比较,感染过表达SOD1 G41S、SOD1 G41D rAAV的细胞峰值电流显著增加(P0.05),且SOD1G41S组显著高于SOD1 G41D组(P0.01)。SOD1 G41S组和SOD1 G41D组细胞半数激活电压和半数失活电压显著高于Control组和SOD1 WT组(P0.05);但SOD1 G41S组半数激活电压高于SOD1 G41D组(P0.05);SOD1 G41S与SOD1 G41D组半数失活电压比较差异无显著性(P0.05);各组激活、失活曲线斜率差异无显著性(P0.05)。虽然 24 和 48h,SOD1WT、SOD1G41S、SOD1G41D 组 Cleaved caspase-3表达量差异无显著性,但转染72 h后,SOD1 G41S组和SOD1 G41D组 Cleaved caspase-3 表达量高于 SOD1 WT 组,且 SOD1 G41S 组高于 SOD1 G41D 组。结论SOD1 G41S和SOD1 G41D突变通过加快VGSC快速激活和抑制失活过程提高神经元活性,且SOD1 G41S突变VGSC活性显著高于SOD1 G41D;SOD1 G41S突变型ALS快速进展病程可能与该突变型促进凋亡有关。
[Abstract]:Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal neurodegenerative disease involving the upper and lower motor neurons. It has been found that 10% of ALS are familial ALS (fALS), of which about 20% are associated with superoxide dismutase 1 (SOD1) gene mutations. There are two different mutations of SOD1 G41S and G41D at the same amino acid locus in SOD1 gene. The clinical manifestations of the patients with these two mutations are significantly different. The patients with SOD1 G41S mutation develop rapidly with an average survival time of about 11.6 months, while the patients with SOD1 G41D mutation develop slowly with an average survival time of about 17 years. Since Rosen et al. discovered the mutation of SOD1 gene and the pathogenesis of ALS in 1993, a series of studies have been carried out on the pathogenesis of ALS, but it is still unclear. The pathogenesis of ALS. In addition, clinical studies have shown that patients with ALS exhibit high excitability of axons, characterized by a persistent increase in sodium current, muscle spasm and fascicular tremor. Previous studies have also found that primary spinal motor neurons in ALSG93A model mice, motor neurons from ALS patients and G93A cells derived from induced pluripotent stem cells. Voltage-gated sodium channel (VGSC) is an important component of excitation and action potential generation and conduction of neurons. Therefore, if the electrophysiological characteristics of VGSC are changed, it will also affect the excitability of neurons and lead to disease. Previous studies have reported abnormal VGSC currents in neurons of ALS SOD1 G93A model mice at the early stage of the disease. However, no studies have been conducted to investigate the electrophysiological characteristics of VGSC with SOD1 G41S and G41D mutations. Wild type (WT) SOD1, SOD1 G41S and G41D recombinant adeno-associated virus (rAAV) vectors were used to infect mouse neuroblastoma (N2a) cells to construct ALS cell model, and the electrophysiological characteristics and apoptotic molecule expression of neuronal VGSC after overexpression of these two mutant proteins in N2a cells were observed. Objective To construct recombinant adeno-associated virus vectors overexpressing SOD1 WT, SOD1 G41S and SOD1 G41D. Methods Firstly, the target gene fragments were amplified by PCR and the pMT121 (pAAV-hSyn-bGlobinintron-EGFP-pA) expression vectors were enzymatically expressed by EcoRI and Hind III endonucleases. After being cut, the linearized expression vectors were recovered and purified. Then the target fragments were homologously recombined with the recovered linearized expression vectors and transformed into DH5a receptor cells. The transformants were identified by colony PCR, and the positive clones were sequenced, and the plasmids were extracted after being sequenced correctly. Results The specific RFP, GFP, SOD1WT, 5'SOD1G41S/G41D, 3'SOD1G41S/G41D bands were found in about 765 bp, 792 bp, 521 bp, 175 bp, 367 BP of HEK293 cells by PCR, and the expression vector pMT121 was digested with EcoR I and Hind III endonucleases. Recombinant plasmid positive clones of SOD1WT were 711 BP fragments, and positive clones of SOD1G415 and SOD1 G41D were 721 BP fragments. Sequencing results were correct. Three SOD1WT, SOD1 G41S and G41D rAAV particles were obtained 72 hours later, and the titers were 2.43 *1012.g. / mL after concentration and purification. CONCLUSIONS Overexpressed SOD1 WT, SOD1 G41S and SOD1 G41D rAAV vectors were successfully constructed. The viruses were purified, concentrated and the titer of the vectors met the following experimental requirements. Part two: The role of SOD1 WT, SOD1 G41S and SOD1 G41D rAAV vectors in ALS cell model Methods N2a cells were infected in vitro by overexpressing SOD1 WT, SOD1 G41S and G41D rAAV vectors. The experimental groups were divided into three groups: SOD1WT group, SOD1G41S and SOD1 G41D group, and N2a cells infected by rAAV were negative control group. The expression of cell apoptosis molecule Cleaved caspase-3 at different time points 24, 48 and 72 h after virus infection was compared between control group and SOD1 WT group. The peak cell current of SOD 1 G41S and SOD 1 G41D rAAV increased significantly (P 0.05), and SOD 1 G41S group was significantly higher than SOD 1 G41D group (P 0.01). The half activation voltage and half inactivation voltage of SOD 1 G41S group and SOD 1 G41D group were significantly higher than those of Control group and SOD 1 WT group (P 0.05), but the half activation voltage of SOD 1 G41S group was higher than that of SOD 1 G41D group (P 0.05). There was no significant difference between SOD1 G41D group and SOD1 G41D group in half inactivation voltage (P 0.05); there was no significant difference in activation and slope of inactivation curve (P 0.05). Conclusion SOD1 G41S and SOD1 G41D mutations can increase neuronal activity by accelerating the rapid activation and inhibiting the inactivation of VGSC, and the VGSC activity of SOD1 G41S mutation is significantly higher than that of SOD1 G41D. The rapid progression of SOD1 G41S mutation may be related to the apoptosis-promoting effect of this mutation.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8
本文编号：2176784
[Abstract]:Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal neurodegenerative disease involving the upper and lower motor neurons. It has been found that 10% of ALS are familial ALS (fALS), of which about 20% are associated with superoxide dismutase 1 (SOD1) gene mutations. There are two different mutations of SOD1 G41S and G41D at the same amino acid locus in SOD1 gene. The clinical manifestations of the patients with these two mutations are significantly different. The patients with SOD1 G41S mutation develop rapidly with an average survival time of about 11.6 months, while the patients with SOD1 G41D mutation develop slowly with an average survival time of about 17 years. Since Rosen et al. discovered the mutation of SOD1 gene and the pathogenesis of ALS in 1993, a series of studies have been carried out on the pathogenesis of ALS, but it is still unclear. The pathogenesis of ALS. In addition, clinical studies have shown that patients with ALS exhibit high excitability of axons, characterized by a persistent increase in sodium current, muscle spasm and fascicular tremor. Previous studies have also found that primary spinal motor neurons in ALSG93A model mice, motor neurons from ALS patients and G93A cells derived from induced pluripotent stem cells. Voltage-gated sodium channel (VGSC) is an important component of excitation and action potential generation and conduction of neurons. Therefore, if the electrophysiological characteristics of VGSC are changed, it will also affect the excitability of neurons and lead to disease. Previous studies have reported abnormal VGSC currents in neurons of ALS SOD1 G93A model mice at the early stage of the disease. However, no studies have been conducted to investigate the electrophysiological characteristics of VGSC with SOD1 G41S and G41D mutations. Wild type (WT) SOD1, SOD1 G41S and G41D recombinant adeno-associated virus (rAAV) vectors were used to infect mouse neuroblastoma (N2a) cells to construct ALS cell model, and the electrophysiological characteristics and apoptotic molecule expression of neuronal VGSC after overexpression of these two mutant proteins in N2a cells were observed. Objective To construct recombinant adeno-associated virus vectors overexpressing SOD1 WT, SOD1 G41S and SOD1 G41D. Methods Firstly, the target gene fragments were amplified by PCR and the pMT121 (pAAV-hSyn-bGlobinintron-EGFP-pA) expression vectors were enzymatically expressed by EcoRI and Hind III endonucleases. After being cut, the linearized expression vectors were recovered and purified. Then the target fragments were homologously recombined with the recovered linearized expression vectors and transformed into DH5a receptor cells. The transformants were identified by colony PCR, and the positive clones were sequenced, and the plasmids were extracted after being sequenced correctly. Results The specific RFP, GFP, SOD1WT, 5'SOD1G41S/G41D, 3'SOD1G41S/G41D bands were found in about 765 bp, 792 bp, 521 bp, 175 bp, 367 BP of HEK293 cells by PCR, and the expression vector pMT121 was digested with EcoR I and Hind III endonucleases. Recombinant plasmid positive clones of SOD1WT were 711 BP fragments, and positive clones of SOD1G415 and SOD1 G41D were 721 BP fragments. Sequencing results were correct. Three SOD1WT, SOD1 G41S and G41D rAAV particles were obtained 72 hours later, and the titers were 2.43 *1012.g. / mL after concentration and purification. CONCLUSIONS Overexpressed SOD1 WT, SOD1 G41S and SOD1 G41D rAAV vectors were successfully constructed. The viruses were purified, concentrated and the titer of the vectors met the following experimental requirements. Part two: The role of SOD1 WT, SOD1 G41S and SOD1 G41D rAAV vectors in ALS cell model Methods N2a cells were infected in vitro by overexpressing SOD1 WT, SOD1 G41S and G41D rAAV vectors. The experimental groups were divided into three groups: SOD1WT group, SOD1G41S and SOD1 G41D group, and N2a cells infected by rAAV were negative control group. The expression of cell apoptosis molecule Cleaved caspase-3 at different time points 24, 48 and 72 h after virus infection was compared between control group and SOD1 WT group. The peak cell current of SOD 1 G41S and SOD 1 G41D rAAV increased significantly (P 0.05), and SOD 1 G41S group was significantly higher than SOD 1 G41D group (P 0.01). The half activation voltage and half inactivation voltage of SOD 1 G41S group and SOD 1 G41D group were significantly higher than those of Control group and SOD 1 WT group (P 0.05), but the half activation voltage of SOD 1 G41S group was higher than that of SOD 1 G41D group (P 0.05). There was no significant difference between SOD1 G41D group and SOD1 G41D group in half inactivation voltage (P 0.05); there was no significant difference in activation and slope of inactivation curve (P 0.05). Conclusion SOD1 G41S and SOD1 G41D mutations can increase neuronal activity by accelerating the rapid activation and inhibiting the inactivation of VGSC, and the VGSC activity of SOD1 G41S mutation is significantly higher than that of SOD1 G41D. The rapid progression of SOD1 G41S mutation may be related to the apoptosis-promoting effect of this mutation.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.8
【参考文献】
相关期刊论文 前1条
1 张华纲;唐璐;张楠;樊东升;;中国家族性肌萎缩侧索硬化患者超氧化物歧化酶1基因突变与临床表型[J];中华神经科杂志;2012年07期
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