過表達SOD1 G41S和G41D的重組腺相關(guān)病毒載體構(gòu)建及其在肌萎縮側(cè)索硬化N2a細胞模型中的作用探究
發(fā)布時間：2018-08-11 10:51
【摘要】：肌萎縮側(cè)索硬化(amyotrophic lateral sclerosis,ALS)是一種快速進展、可累及上、下運動神經(jīng)元的嚴(yán)重致死性神經(jīng)系統(tǒng)變性病。研究發(fā)現(xiàn),10%ALS為家族性的ALS(familial ALS,fALS),其中約20%fALS發(fā)病與超氧化物歧化酶1(superoxide dismutase1,SOD1)基因突變有關(guān)。在SOD1基因上存在兩種同一氨基酸位點上的不同突變SOD1 G41S和G41D,而攜帶這兩種突變的患者臨床表現(xiàn)明顯不同,SOD1G41S突變型fALS患者病程進展迅速,平均生存期約為11.6個月;而SOD1 G41D突變型患者病程進展較為緩慢,平均生存期約為17年。自1993年Rosen等人發(fā)現(xiàn)SOD1基因突變和ALS發(fā)病有關(guān)后,研究者對此種ALS致病機制進行了一系列研究,但至今尚不清楚。目前認為,氧化應(yīng)激、錯誤折疊蛋白聚集、線粒體功能障礙、谷氨酸鹽興奮毒性、軸索運輸損害、非神經(jīng)元細胞作用等都參與ALS的致病過程。另外,臨床研究發(fā)現(xiàn),ALS患者可出現(xiàn)軸突的高興奮性,表現(xiàn)為持續(xù)性的鈉電流增加,患者呈現(xiàn)出肌痙攣和肌束震顫;而先前也有研究發(fā)現(xiàn)在ALSG93A模型鼠原代脊髓運動神經(jīng)元、ALS患者誘導(dǎo)多能干細胞來源的運動神經(jīng)元及G93A模型鼠離體腦片中的運動神經(jīng)元都出現(xiàn)過度興奮的現(xiàn)象。電壓門控性鈉通道(voltage-gatedsodium channel,VGSC)是神經(jīng)元的興奮和動作電位產(chǎn)生、傳導(dǎo)的重要元件,因此該通道電生理特性如果改變也將會影響神經(jīng)元的興奮性,從而導(dǎo)致疾病的發(fā)生。先前研究報道ALS SOD1 G93A模型鼠在疾病早期神經(jīng)元VGSC電流出現(xiàn)異常。但是目前未有研究對SOD1 G41S和G41D突變的VGSC電生理特性進行探究。因此,為進一步探究SOD1 G41S和G41D突變在ALS中的致病機制,將本研究分為兩個部分,首先構(gòu)建過表達野生型(widetype,WT)SOD1、SOD1 G41S及 G41D 重組腺相關(guān)病毒(recombinant adeno-associated virus,rAAV)載體;用上述rAAV感染小鼠神經(jīng)瘤母細胞(N2a)以構(gòu)建ALS細胞模型,并觀察N2a細胞過量表達此兩種突變蛋白后,對神經(jīng)元VGSC電生理特性和凋亡分子表達的影響。第一部分SOD1WT、SOD1 G41S和SOD1 G41D rAAV載體的構(gòu)建及鑒定目的構(gòu)建過表達SOD1 WT、SOD1 G41S及SOD1 G41D重組腺相關(guān)病毒載體。方法首先,利用PCR擴增各目的基因片段,應(yīng)用EcoRI和HindⅢ內(nèi)切酶對pMT121(pAAV-hSyn-bGlobinintron-EGFP-pA)表達載體進行酶切后,回收純化線性化表達載體。進而在無縫反應(yīng)液作用下,將各目的片段與回收后的線性化表達載體進行同源重組后轉(zhuǎn)化DH5α感受態(tài)細胞。利用菌落PCR鑒定轉(zhuǎn)化子,陽性克隆送測序,測序無誤后進行質(zhì)粒提抽。最后目的質(zhì)粒與輔助質(zhì)粒共轉(zhuǎn)染HEK293細胞進行病毒包裝,經(jīng)純化、濃縮、RT-PCR滴度測定后保存?zhèn)溆�。結(jié)果PCR結(jié)果顯示,在約765 bp、792 bp、521 bp、175 bp、367 bp可見特異性 RFP、GFP、SOD1WT、5'SOD1G41S/G41D、3'SOD1G41S/G41D 條帶;應(yīng)用EcoR Ⅰ和Hind Ⅲ內(nèi)切酶對表達載體pMT121進行雙酶切,回收4470 bp的線性化載體片段;菌落PCR轉(zhuǎn)化子鑒定結(jié)果顯示,SOD1WT重組質(zhì)粒陽性克隆得到711bp片段、SOD1G415和SOD1G41D陽性克隆得到721bp片段;測序結(jié)果正確;72 h后獲得3種SOD1 WT、SOD1 G41S和G41D rAAV顆粒,經(jīng)濃縮、純化、測定滴度分別為 2.43×1012 v.g./mL、2.28×1012 v.g./mL、2.52×1012 v.g./mL后,分裝保存于-80℃?zhèn)溆谩＝Y(jié)論成功構(gòu)建過表達SOD1 WT、SOD1 G41S及SOD1 G41D rAAV載體,病毒純化、濃縮、滴度皆滿足后續(xù)實驗要求。第二部分SOD1WT、SOD1G41S和SOD1G41DrAAV載體在ALS細胞模型中的作用探究目的探究ALS SOD1上G41S和G41D兩種突變型所致神經(jīng)元VGSC電生理特性的差異及其可能的分子機制。方法構(gòu)建過表達SOD1 WT、SOD1 G41S及G41D rAAV載體后,體外感染N2a細胞,將實驗組分為3組:SOD1WT組、SOD1G41S和SOD1G41D組,并以無目的基因rAAV感染的N2a細胞為陰性對照(Control)組。利用全細胞膜片鉗技術(shù)分別記錄四組細胞VGSC電流,繪制通道快速激活和穩(wěn)態(tài)失活曲線后分析相關(guān)參數(shù)。比較各組細胞凋亡分子Cleaved caspase-3在病毒感染后24、48和72 h不同時間點的表達情況。結(jié)果與Control組和SOD1 WT組比較,感染過表達SOD1 G41S、SOD1 G41D rAAV的細胞峰值電流顯著增加(P0.05),且SOD1G41S組顯著高于SOD1 G41D組(P0.01)。SOD1 G41S組和SOD1 G41D組細胞半數(shù)激活電壓和半數(shù)失活電壓顯著高于Control組和SOD1 WT組(P0.05);但SOD1 G41S組半數(shù)激活電壓高于SOD1 G41D組(P0.05);SOD1 G41S與SOD1 G41D組半數(shù)失活電壓比較差異無顯著性(P0.05);各組激活、失活曲線斜率差異無顯著性(P0.05)。雖然 24 和 48h,SOD1WT、SOD1G41S、SOD1G41D 組 Cleaved caspase-3表達量差異無顯著性,但轉(zhuǎn)染72 h后,SOD1 G41S組和SOD1 G41D組 Cleaved caspase-3 表達量高于 SOD1 WT 組,且 SOD1 G41S 組高于 SOD1 G41D 組。結(jié)論SOD1 G41S和SOD1 G41D突變通過加快VGSC快速激活和抑制失活過程提高神經(jīng)元活性,且SOD1 G41S突變VGSC活性顯著高于SOD1 G41D;SOD1 G41S突變型ALS快速進展病程可能與該突變型促進凋亡有關(guān)。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.8
本文編號：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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.8
【參考文獻】
相關(guān)期刊論文 前1條
1 張華綱;唐璐;張楠;樊東升;;中國家族性肌萎縮側(cè)索硬化患者超氧化物歧化酶1基因突變與臨床表型[J];中華神經(jīng)科雜志;2012年07期
，本文編號：2176784
本文鏈接：http://sikaile.net/yixuelunwen/shenjingyixue/2176784.html
最近更新
教材專著
