帕金森病细胞模型的构建及氨基酸表达观察
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摘要
目的建立帕金森病(PD)人神经母细胞瘤细胞模型,并筛选PD细胞中的氨基酸标志物。方法将人神经母细胞瘤细胞SH-SY5Y分为正常组、溶媒组、鱼藤酮组。鱼藤酮组分别加入0.15、0.25μmol/L的鱼藤酮,溶媒组加入二甲基亚砜(DMSO),正常组不加药物。分别于给药24、48 h后采用MTT法测算细胞存活率,倒置显微镜观察细胞形态,选择对细胞存活、形态影响较小的鱼藤酮作用浓度及作用时间(0.15μmol/L、24 h),采用Western blotting法检测SH-SY5Y细胞中的α-突触核蛋白以验证PD模型。采用高效液相色谱法检测PD细胞中的谷氨酸(Glu)、丝氨酸(Ser)、谷氨酰胺(Gln)、组氨酸(His)、甘氨酸(Gly)、丙氨酸(Ala)、脯氨酸(Pro)、色氨酸(Trp)、苯丙氨酸(Phe)、异亮氨酸(Ile)、亮氨酸(Leu)、赖氨酸(Lys),对鱼藤酮组和溶媒组差异有统计学意义的氨基酸进入偏最小二乘判别分析,选择P<0.05且变量重要性投影值(VIP)>1的氨基酸为PD的标志物。结果鱼藤酮组细胞中α-突触核蛋白相对表达量高于溶媒组(P均<0.05),表明SH-SY5Y细胞PD模型制作成功。给药24 h时,鱼藤酮组细胞中Glu、Ser、Gln、His、Gly、Pro、Phe、Ile、Leu、Lys水平低于溶媒组(P均<0.05);给药48 h时,鱼藤酮组除Gln外,其余11种氨基酸水平均低于溶媒组(P均<0.05)。筛选氨基酸标志物为Glu、Ser、Gly、Pro(给药24 h时VIP值分别为1.016、1.016、1.016、1.009,给药48 h时VIP值均为1.001)。结论利用鱼藤酮诱导成功建立了PD细胞模型,筛选出Glu、Ser、Gly、Pro可能为PD的潜在标志物。
Objective To establish the human neuroblastoma cell model with Parkinson's disease( PD),and screen out the amino acid biomarkers in PD cells. Methods Human neuroblastoma SH-SY5 Y cells were divided into the normal group,solvent group and rotenone group. Cells in the rotenone group were given 0. 15,0. 25 μmol/L rotenone,cells in the solvent group were given dimethyl sulfoxide( DMSO),and the normal group was not added with any drug. Cell viability was assessed by MTT assay at 24,48 h after medication,and inverted microscope was used to observe the cell morphology.Then we determined the concentration( 0. 15 μmol/L) and treatment time( 24 h) of rotenone which had the little effect according to the changes of cell viability and cell morphology. The expression of α-synuclein was detected by Western blot to validate the success of PD model. And 12 kinds of amino acids,including glutamic acid( Glu),serine( Ser),glutamine( Gln),histidine( His),glycine( Gly),alanine( Ala),proline( Pro),tryptophan( Trp),phenylalanine( Phe),isoleucine( Ile),leucine( Leu),and lysine( Lys),in PD cells were determined by high performance liquid chromatography,and we analyzed the significant changed amino acids between solvent group and rotenone group with partial least squares discriminant analysis( PLS-DA). Amino acid biomarkers in the PD model were determined with a variable importance in the projection( VIP) value greater than 1 and a P value less than 0. 05. Results The expression of α-synuclein in the cells of the rotenone group was significantly higher than that of the solvent group( P < 0. 05),indicating the successful construction of SH-SY5 Y cell model with PD. At 24 h,the Glu,Ser,Gln,His,Gly,Pro,Phe,Ile,Leu,Lys levels in the rotenone group were lower than those in the solvent group( all P < 0. 05). At 48 h,the levels of 11 kinds of amino acid except Gln in the rotenone group were lower than those in the solvent group( all P < 0. 05). Glu,Ser,Gly,Pro were screened out as the amino acid biomarkers( At 24 h,VIP values were 1. 016,1. 016,1. 016,1. 009; at 48 h,all VIP values were 1. 001). Conclusion The PD cell model is successfully established by rotenone induction and Glu,Ser,Gly,and Pro may be the potential diagnostic biomarkers for PD.
Objective To establish the human neuroblastoma cell model with Parkinson's disease( PD),and screen out the amino acid biomarkers in PD cells. Methods Human neuroblastoma SH-SY5 Y cells were divided into the normal group,solvent group and rotenone group. Cells in the rotenone group were given 0. 15,0. 25 μmol/L rotenone,cells in the solvent group were given dimethyl sulfoxide( DMSO),and the normal group was not added with any drug. Cell viability was assessed by MTT assay at 24,48 h after medication,and inverted microscope was used to observe the cell morphology.Then we determined the concentration( 0. 15 μmol/L) and treatment time( 24 h) of rotenone which had the little effect according to the changes of cell viability and cell morphology. The expression of α-synuclein was detected by Western blot to validate the success of PD model. And 12 kinds of amino acids,including glutamic acid( Glu),serine( Ser),glutamine( Gln),histidine( His),glycine( Gly),alanine( Ala),proline( Pro),tryptophan( Trp),phenylalanine( Phe),isoleucine( Ile),leucine( Leu),and lysine( Lys),in PD cells were determined by high performance liquid chromatography,and we analyzed the significant changed amino acids between solvent group and rotenone group with partial least squares discriminant analysis( PLS-DA). Amino acid biomarkers in the PD model were determined with a variable importance in the projection( VIP) value greater than 1 and a P value less than 0. 05. Results The expression of α-synuclein in the cells of the rotenone group was significantly higher than that of the solvent group( P < 0. 05),indicating the successful construction of SH-SY5 Y cell model with PD. At 24 h,the Glu,Ser,Gln,His,Gly,Pro,Phe,Ile,Leu,Lys levels in the rotenone group were lower than those in the solvent group( all P < 0. 05). At 48 h,the levels of 11 kinds of amino acid except Gln in the rotenone group were lower than those in the solvent group( all P < 0. 05). Glu,Ser,Gly,Pro were screened out as the amino acid biomarkers( At 24 h,VIP values were 1. 016,1. 016,1. 016,1. 009; at 48 h,all VIP values were 1. 001). Conclusion The PD cell model is successfully established by rotenone induction and Glu,Ser,Gly,and Pro may be the potential diagnostic biomarkers for PD.
引文
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[13]王军玲,杨阳,高健,等.基于氨基酸代谢的抗脑缺血益气解毒配伍中药协同作用研究[J].中国药理学通报,2014,30(5):725-731.
[14]Xia T,Zhang Q,Xiao Y,et al.CREB/TRH pathway in the central nervous system regulates energy expenditure in response to deprivation of an essential amino acid[J].Int J Obes(Lond),2015,39(1):105-113.
[15]杨晓运,李智,秦绿叶,等.星形胶质细胞和神经元之间谷氨酸-谷氨酰胺的代谢偶联[J].生理科学进展,2003,34(4):350-352.
[16]Phang JM,Liu W,Hancock CN,et al.Proline metabolism and cancer:emerginglinks to glutamine and collagen[J].Curr Opin Clin Nutr Metab Care,2015,18(1):71-77.
[17]Wolfson RL,Chantranupong L,Saxton RA,et al.Sestrin2 is a leucine sensor for the m TORC1 pathway[J].Science,2016,351(6268):43-48.
[18]李双,王小琴,郭春燕,等.低浓度DMSO对SH-SY5Y细胞存活率和氨基酸代谢的影响[J].神经药理学报,2015,5(4):9-13.
[19]Maynard TM,Manzini MC.Balancing Act:Maintaining Amino Acid Levels in the Autistic Brain[J].Neuron,2017,93(3):476-479.
[20]李鞠,唐凤英.原发性肾病综合征患者血清异常代谢通路的代谢组学分析[J].山东医药,2016,56(30):1-4.
[21]王风萍,郭春燕.代谢组学及其研究新进展[J].神经药理学报,2012,2(6):49-55.
[22]Zheng P,Chen JJ,Zhou CJ,et al.Identification of sex-specific urinary biomarkers for major depressive disorder by combined application of NMR-and GC-MS-based metabonomics[J].Transl Psychiatry,2016,6(11):e955.
[23]吴宏伟,高健,李韶菁,等.基于液相色谱-串联质谱的氨基酸代谢组学方法研究黄芪注射液治疗脑缺血[J].分析化学,2013,41(3):344-348.
[24]Da Silva LB,Poulsen JN,Arendt-nielsen L,et al.Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells[J].J Cell Mol Med,2015,19(8):1900-1909.
[25]吴帅,安书成,陈慧彬,等.慢性应激性抑郁发生中大鼠眶额叶多巴胺D1受体对谷氨酸及其NMDA受体的调节[J].心理学报,2014,46(1):69-78.
[26]吴倩,汪宁,王艳,等.藁本内酯对谷氨酸诱导的PC12细胞凋亡的保护作用[J].药学学报,2015,50(2):162-168.
[27]Fossat P,Turpin FR,Sacchi S,et al.Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex[J].Cereb Cortex,2012,22(3):595-606.
[28]何文娟,阮怀珍.D-丝氨酸在神经元-胶质细胞间通讯的新进展[J].生理科学进展,2009,40(4):303-307.
[29]Maguire EP,Mitchell EA,Greig SJ,et al.Extrasynaptic Glycine Receptors of Rodent Dorsal Raphe Serotonergic Neurons:A Sensitive Target for Ethanol[J].Neuropsychopharmacology,2014,39(5):1232-1244.
[30]Lu Y,Dong H,Gao Y,et al.A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia[J].J Clin Invest,2013,123(9):4050-4062.
[31]Wyse AT,Netto CA.Behavioral and neurochemical effects of proline[J].Metab Brain Dis,2011,26(3):159-172.
[2]胡雅琼,罗理勇,曾亮.茶叶提取物对帕金森病的防治作用研究进展[J].中草药,2014,45(9):1342-1348.
[3]Chorfa A,Temps D,Morignat E,et al.Specific pesticide-dependent increases inα-synuclein levels in human neuroblastoma(SHSY5Y)and melanoma(SK-MEL-2)cell lines[J].Toxicol Sci,2013,133(2):289-297.
[4]Klingelhoefer L,Reichmann H.Pathogenesis of Parkinson disease--the gut-brainaxis and environmental factors[J].Nat Rev Neurol,2015,11(11):625-636.
[5]Sequra-Aquilar J,Kostrzewa RM.Neurotoxin mechanisms and processes relevantto Parkinson's disease:an update[J].Neurotox Res,2015,27(3):328-354.
[6]白莹,徐斌.利用诱导性多能干细胞技术研究环境致病因素诱导帕金森病发病的进展[J].环境与职业医学,2015,32(2):181-185.
[7]柴星星,鲍波,叶成,等.鱼藤酮诱导制备SD大鼠帕金森病模型的可行性[J].山东医药,2015,55(1):9-14.
[8]CorbilléAG,Letournel F,Kordower JH,et al.Evaluation of alpha-synuclein Immuno-histochemical methods for the detection of Lewy-type synucleinopathy in gastrointestinal biopsies[J].Acta Neuropathol Commun,2016,4(1):35-41.
[9]Wenzel S,Mollenhauer B,Trenkwalder C.Diagnosis and clinical therapy for Parkinson's disease[J].Nervenarzt,2006,77(12):1439-1443.
[10]Meredith GE,Totterdell S,Beales M,et al.Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease[J].Exp Neuro,2009,219(1):334-340.
[11]Assous M,Had-Aissouni L,Gubellini P,et al.Progressive Parkinsonism by acute dysfunction of excitatory amino acid transporters in the rat substantia nigra[J].Neurobio Dis,2014,65(1):69-81.
[12]罗瑞静,何建成.兴奋性氨基酸与帕金森病研究进展[J].医学研究杂志,2010,39(10):13-15.
[13]王军玲,杨阳,高健,等.基于氨基酸代谢的抗脑缺血益气解毒配伍中药协同作用研究[J].中国药理学通报,2014,30(5):725-731.
[14]Xia T,Zhang Q,Xiao Y,et al.CREB/TRH pathway in the central nervous system regulates energy expenditure in response to deprivation of an essential amino acid[J].Int J Obes(Lond),2015,39(1):105-113.
[15]杨晓运,李智,秦绿叶,等.星形胶质细胞和神经元之间谷氨酸-谷氨酰胺的代谢偶联[J].生理科学进展,2003,34(4):350-352.
[16]Phang JM,Liu W,Hancock CN,et al.Proline metabolism and cancer:emerginglinks to glutamine and collagen[J].Curr Opin Clin Nutr Metab Care,2015,18(1):71-77.
[17]Wolfson RL,Chantranupong L,Saxton RA,et al.Sestrin2 is a leucine sensor for the m TORC1 pathway[J].Science,2016,351(6268):43-48.
[18]李双,王小琴,郭春燕,等.低浓度DMSO对SH-SY5Y细胞存活率和氨基酸代谢的影响[J].神经药理学报,2015,5(4):9-13.
[19]Maynard TM,Manzini MC.Balancing Act:Maintaining Amino Acid Levels in the Autistic Brain[J].Neuron,2017,93(3):476-479.
[20]李鞠,唐凤英.原发性肾病综合征患者血清异常代谢通路的代谢组学分析[J].山东医药,2016,56(30):1-4.
[21]王风萍,郭春燕.代谢组学及其研究新进展[J].神经药理学报,2012,2(6):49-55.
[22]Zheng P,Chen JJ,Zhou CJ,et al.Identification of sex-specific urinary biomarkers for major depressive disorder by combined application of NMR-and GC-MS-based metabonomics[J].Transl Psychiatry,2016,6(11):e955.
[23]吴宏伟,高健,李韶菁,等.基于液相色谱-串联质谱的氨基酸代谢组学方法研究黄芪注射液治疗脑缺血[J].分析化学,2013,41(3):344-348.
[24]Da Silva LB,Poulsen JN,Arendt-nielsen L,et al.Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells[J].J Cell Mol Med,2015,19(8):1900-1909.
[25]吴帅,安书成,陈慧彬,等.慢性应激性抑郁发生中大鼠眶额叶多巴胺D1受体对谷氨酸及其NMDA受体的调节[J].心理学报,2014,46(1):69-78.
[26]吴倩,汪宁,王艳,等.藁本内酯对谷氨酸诱导的PC12细胞凋亡的保护作用[J].药学学报,2015,50(2):162-168.
[27]Fossat P,Turpin FR,Sacchi S,et al.Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex[J].Cereb Cortex,2012,22(3):595-606.
[28]何文娟,阮怀珍.D-丝氨酸在神经元-胶质细胞间通讯的新进展[J].生理科学进展,2009,40(4):303-307.
[29]Maguire EP,Mitchell EA,Greig SJ,et al.Extrasynaptic Glycine Receptors of Rodent Dorsal Raphe Serotonergic Neurons:A Sensitive Target for Ethanol[J].Neuropsychopharmacology,2014,39(5):1232-1244.
[30]Lu Y,Dong H,Gao Y,et al.A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia[J].J Clin Invest,2013,123(9):4050-4062.
[31]Wyse AT,Netto CA.Behavioral and neurochemical effects of proline[J].Metab Brain Dis,2011,26(3):159-172.
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