过表达SOD_1 G41S和G41D的重组腺相关病毒载体构建及其在体外N2a细胞中的作用研究

Construction of Overexpressed SOD_1 G41S and G41D Recombinant Adeno-associated Virus Vectors and Pathogenesis of the Two Mutations in N2a Cells

目的研究家族性肌萎缩侧索硬化(fALS)超氧化物歧化酶1(SOD_1)上G41S和G41D两种突变型所致神经元电压门控性钠通道(Na_v)电生理特性的差异及其可能的分子机制。方法构建过表达SOD_1野生型(WT)、SOD_1G41S及SOD_1G41D重组腺相关病毒(rAAV)载体后,体外感染小鼠神经瘤母细胞(N2a),并分为3个实验组:SOD_1WT组、SOD_1G41S和SOD_1G41D组,并以无目的基因rAAV感染的N2a细胞为阴性对照组。利用全细胞膜片钳技术分别记录4组细胞Na_v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在感染后24、48和72 h不同时间点的表达情况。结果与对照组和SOD_1WT组比较,感染过表达SOD_1G41S组、SOD_1G41D组rAAV的细胞峰值电流显著增加(P<0.05),且SOD_1G41S组显著高于SOD_1G41D组(P<0.01)。SOD_1G41S组和SOD_1G41D组细胞半数激活电压和半数失活电压显著高于对照组和SOD_1WT组(P<0.05);但SOD_1G41S组半数激活电压高于SOD_1G41D组(P<0.05);SOD_1G41S与SOD_1G41D组半数失活电压比较差异无显著性(P>0.05);各组激活、失活曲线斜率差异无显著性(P>0.05)。虽然24和48h,SOD_1WT、SOD_1G41 S、SOD_1G41D组cleaved caspase-3表达量差异无显著性;但转染72 h后,SOD_1G41S组和SOD_1G41D组cleaved caspase-3表达量高于SOD1WT组,且SOD_1G41S组高于SOD_1G41D组。结论SOD_1G41S和SOD_1G41D突变通过加快Na_v快速激活和抑制失活过程提高神经元活性,SOD_1G41S突变Na_v活性显著高于SOD_1G41D。SOD_1G41S突变导致cleaved caspase-3表达水平高于SOD_1G41D突变。

Aim To explore the differences of voltage-gated sodium channels （Nay） electrophysiological characteristics and the possible molecular mechanisms between two mutations G41S and G41D in superoxide dismutase 1 （SOD1） in familial amyotrophic lateral sclerosis （fALS）. Methods Overexpressed wild type （WT） SOD1, SOD1G41S and G41D recombinant adeno-associated virus （rAAV） vectors were constructed firstly, then mouse neuroblastoma N2a cells （N2a） were infected by constructed vectors in vitro. Nay currents of cells in the control （N2a without expression of target genes）, SOD1WT, G41S and G41D groups were recorded using the whole-cell patch clamp technique, then the curves of channel fast activation and steady-state inactivation were drawn and related parameters were analyzed. Moreover, the expression of apoptotic molecular cleaved caspase-3 was compared after 24, 48, 72 h of virus infection. Results Compared with the control and SOD1WT cells, the peak currents of infected SOD1G41S and G41D cells were increased significantly （P〈0.05）, and G41S were significantly higher than G41D （P〈0.01）. Potentials for half-maximal activation and inactivation of G41S and G41D were significantly higher than other two groups （P〈0.05）, but potentials for half-maximal activation of G41S were higher than G41D （P〈0.05）, while there were no differences of potentials for half-maximal inactivation between G41S and G41D groups （P〉0.05）. And there were no differences of slope factors of activation and inactivation curves in each group （P〉0.05）. Although there were no differences of the expression of cleaved caspase-3 after 24, 48 h of infection in WT, G41S, G41D groups, but the expression of cleaved caspase-3 in G41 S, G41D groups higher than that of WT group after 72 h, and G41S group were higher than G41D group. Conclusion SOD1G41S and G41D mutations improved the activity of neurons by accelerating the process of Nav fast activation and inhibiting the process of Nay inactivation, and Nay activity of G41D was significantly higher than that of G41S mutation. The expression of apoptotic molecular cleaved caspase-3 in G41S were higher than that of G41D mutation.