肌萎缩性脊髓侧索硬化症细胞模型中mRAGE和esRAGE选择性剪接变化研究
|
摘要
目的探讨肌萎缩性脊髓侧索硬化症(ALS)细胞模型中晚期糖基化终末产物受体(RAGE)两种亚型mRAGE和esRAGE选择性剪接变化。方法选取鼠运动元样细胞系NSC-34细胞为研究对象,以SH-SY5Y细胞c DNA为模板,构建铜/锌超氧化物歧化酶(SOD1)突变载体。将NSC-34细胞分为:空质粒组、正常SOD1蛋白表达质粒组、G93A质粒组,分别将空质粒、正常SOD1蛋白表达质粒、SOD1突变蛋白表达质粒(G93A质粒组)转染入细胞,另以正常NSC-34细胞组作为对照,不转染。转染后48 h,观察各组细胞形态学变化,采用实时聚合酶链式反应(RT-PCR)检测SOD1、mRAGE、esRAGE表达情况。结果 G93A质粒组与正常NSC-34细胞组细胞形态学比较,细胞胞体变圆,轴突变短且突起减少,空质粒组、正常SOD1蛋白表达质粒组与正常NSC-34细胞组比较无明显变化;qRT-PCR结果显示,正常SOD1蛋白表达质粒组、G93A质粒组细胞中SOD1为阳性表达,空质粒组及正常NSC-34细胞组SOD1为阴性表达,提示成功构建ALS细胞模型;与正常SOD1蛋白表达质粒组比较,G93A质粒组细胞中mRAGE表达明显增加(P<0.05),esRAGE表达明显降低(P<0.05)。结论在ALS病理过程中,胶质细胞中RAGE的选择性剪接调控异常改变,导致mRAGE亚型增加,esRAGE亚型减少。
Objective To explore alternative splicing changes in mRAGE and esRAGE two subtypes of advanced glycation end products receptor(RAGE) in amyotrophic lateral sclerosis(ALS) cell model. Methods NSC-34 cell of rat motor like cell line was selected as the study objects,and SH-SY5 Y cell c DNA was used as template,and copper/zinc superoxide dismutase(SOD1) mutant vector was structured. NSC-34 cells were divided into empty plasmid group,normal SOD1 protein expression plasmid group and SOD1 mutant protein expression plasmid(G93 A plasmid group). The empty plasmid,the normal SOD1 protein expression plasmid,and the G93 A plasmid were transfected into the cells,respectively. What's more,the normal NSC-34 cell group without transfection was used as a control. 48 hours after transfection,the morphological changes of each group were observed,and the expression of SOD1,mRAGE and esRAGE was detected by real-time polymerase chain reaction(RT-PCR). Results Cell morphology comparison between the G93 A plasmid group and normal NSC-34 cell group,cell bodies became rounded,axons shortened and the processes decreased. There were no significant changes among the empty plasmid group,the normal SOD1 protein expression plasmid group and the normal NSC-34 cell group. QRT-PCR result showed that SOD1 was positive-expression in the normal SOD1 protein expression plasmid group and the G93 A plasmid group,and SOD1 was negative-expression in the empty plasmid group and the normal NSC-34 cell group,indicating that ALS cell model was successfully constructed. Compared with the normal SOD1 protein expression plasmid group,the expression of mRAGE was significantly increased in the G93 A plasmid group(P < 0. 05),and the expression of esRAGE was significantly decreased(P < 0. 05). Conclusion Aberrant splicing regulation of RAGE in glial cells changes abnormally in the course of ALS pathology,it cause mRAGE subtype increased and esRAGE subtype decreased.
Objective To explore alternative splicing changes in mRAGE and esRAGE two subtypes of advanced glycation end products receptor(RAGE) in amyotrophic lateral sclerosis(ALS) cell model. Methods NSC-34 cell of rat motor like cell line was selected as the study objects,and SH-SY5 Y cell c DNA was used as template,and copper/zinc superoxide dismutase(SOD1) mutant vector was structured. NSC-34 cells were divided into empty plasmid group,normal SOD1 protein expression plasmid group and SOD1 mutant protein expression plasmid(G93 A plasmid group). The empty plasmid,the normal SOD1 protein expression plasmid,and the G93 A plasmid were transfected into the cells,respectively. What's more,the normal NSC-34 cell group without transfection was used as a control. 48 hours after transfection,the morphological changes of each group were observed,and the expression of SOD1,mRAGE and esRAGE was detected by real-time polymerase chain reaction(RT-PCR). Results Cell morphology comparison between the G93 A plasmid group and normal NSC-34 cell group,cell bodies became rounded,axons shortened and the processes decreased. There were no significant changes among the empty plasmid group,the normal SOD1 protein expression plasmid group and the normal NSC-34 cell group. QRT-PCR result showed that SOD1 was positive-expression in the normal SOD1 protein expression plasmid group and the G93 A plasmid group,and SOD1 was negative-expression in the empty plasmid group and the normal NSC-34 cell group,indicating that ALS cell model was successfully constructed. Compared with the normal SOD1 protein expression plasmid group,the expression of mRAGE was significantly increased in the G93 A plasmid group(P < 0. 05),and the expression of esRAGE was significantly decreased(P < 0. 05). Conclusion Aberrant splicing regulation of RAGE in glial cells changes abnormally in the course of ALS pathology,it cause mRAGE subtype increased and esRAGE subtype decreased.
引文
[1]陈虹,万欢英,李庆云.以呼吸困难为首诊症状的肌萎缩侧索硬化症一例并文献复习[J].临床内科杂志,2012,29(6):398-400.
[2]崔博,崔丽英.肌萎缩侧索硬化流行病学研究现状[J].中华神经科杂志,2015,48(6):542-544.
[3]Armon C,Graves MC,Moses D,et al.Linear estimates of disease progression predict survival in patients with amyotrophic lateral sclerosis[J].Muscle Nerve,2015,23(6):874-882.
[4]吕翠.晚期糖基化终末产物受体及其抑制剂的研究进展[J].中国药理学通报,2013,29(4):452-456.
[5]陈嬿,董漪,卢家红,等.中国华东地区家族性肌萎缩侧索硬化症患者SOD1基因突变检测及临床特点分析[J].中国临床神经科学,2016,24(2):146-152.
[6]邓娜,周瑾瑕,廖迪,等.铜/锌超氧化物岐化物1突变致肌萎缩侧索硬化一家系分析并文献复习[J].国际神经病学神经外科学杂志,2017,44(1):49-53.
[7]Evans MC,Couch Y,Sibson N,et al.Inflammation and neurovascular changes in amyotrophic lateral sclerosis[J].Mol Cell Neuroscience,2013,53(3):34-41.
[8]徐陶.小胶质细胞极化在神经系统疾病中的研究进展[J].重庆医学,2017,46(27):3866-3869.
[9]邱惠,李虹伟.糖基化终末产物及其受体在动脉粥样硬化中的作用及意义[J].临床和实验医学杂志,2018,17(4):443-446.
[10]孙筱璇,薛莉,周畅,等.晚期糖基化终末产物受体对帕金森病模型小鼠脑内酪氨酸羟化酶表达的影响[J].中国临床神经科学,2017,25(1):1-7.
[11]Saha A,Kim SJ,Zhang Z,et al.RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis[J].Febs Letters,2008,582(27):3823-3831.
[12]岳丽霞.OECs过表达esRAGE对AGE诱导RMECs炎症反应的影响作用[D].南昌:南昌大学,2014.
[13]Wang P,Huang R,Lu S,et al.RAGE and AGEs in mild cognitive impairment of diabetic patients:a cross-sectional study[J].Plos One,2016,11(1):e0145521.
[14]Sternberg Z,Chiotti A,Tario J,et al.Reduced expression of membrane-bound(m)RAGE is a biomarker of multiple sclerosis disease progression[J].Immunobiol,2016,221(2):193-198.
[2]崔博,崔丽英.肌萎缩侧索硬化流行病学研究现状[J].中华神经科杂志,2015,48(6):542-544.
[3]Armon C,Graves MC,Moses D,et al.Linear estimates of disease progression predict survival in patients with amyotrophic lateral sclerosis[J].Muscle Nerve,2015,23(6):874-882.
[4]吕翠.晚期糖基化终末产物受体及其抑制剂的研究进展[J].中国药理学通报,2013,29(4):452-456.
[5]陈嬿,董漪,卢家红,等.中国华东地区家族性肌萎缩侧索硬化症患者SOD1基因突变检测及临床特点分析[J].中国临床神经科学,2016,24(2):146-152.
[6]邓娜,周瑾瑕,廖迪,等.铜/锌超氧化物岐化物1突变致肌萎缩侧索硬化一家系分析并文献复习[J].国际神经病学神经外科学杂志,2017,44(1):49-53.
[7]Evans MC,Couch Y,Sibson N,et al.Inflammation and neurovascular changes in amyotrophic lateral sclerosis[J].Mol Cell Neuroscience,2013,53(3):34-41.
[8]徐陶.小胶质细胞极化在神经系统疾病中的研究进展[J].重庆医学,2017,46(27):3866-3869.
[9]邱惠,李虹伟.糖基化终末产物及其受体在动脉粥样硬化中的作用及意义[J].临床和实验医学杂志,2018,17(4):443-446.
[10]孙筱璇,薛莉,周畅,等.晚期糖基化终末产物受体对帕金森病模型小鼠脑内酪氨酸羟化酶表达的影响[J].中国临床神经科学,2017,25(1):1-7.
[11]Saha A,Kim SJ,Zhang Z,et al.RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis[J].Febs Letters,2008,582(27):3823-3831.
[12]岳丽霞.OECs过表达esRAGE对AGE诱导RMECs炎症反应的影响作用[D].南昌:南昌大学,2014.
[13]Wang P,Huang R,Lu S,et al.RAGE and AGEs in mild cognitive impairment of diabetic patients:a cross-sectional study[J].Plos One,2016,11(1):e0145521.
[14]Sternberg Z,Chiotti A,Tario J,et al.Reduced expression of membrane-bound(m)RAGE is a biomarker of multiple sclerosis disease progression[J].Immunobiol,2016,221(2):193-198.