敲低小鼠中脑神经元多巴胺D2受体使其生脂基因表达上凋
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摘要
目的研究小鼠中脑神经细胞多巴胺D2受体(DRD2)对神经元生脂基因的调节作用。方法构建针对DRD2短发夹RNA(shRNA)的慢病毒载体,特异性下调小鼠原代中脑神经元的DRD2表达;神经元分为阴性病毒对照组和DRD2-shRNA病毒感染组,进行高通量转录组测序,分析两组差异基因并以共表达网络筛选核心基因;实时荧光定量PCR及Western blot法验证该差异基因表达并检测神经元脂肪酸含量变化。结果成功培养小鼠中脑神经元并下调DRD2;高通量转录组测序发现中脑神经细胞DRD2下调对多个生脂基因有调节作用,主要包括δ(14)-固醇还原酶、乙酰辅酶A合成酶、胰岛素诱导基因1蛋白、硬脂酰辅酶A去饱和酶等,其中硬脂酰辅酶A去饱和酶1(SCD1)上调最显著(上调4倍),并经实时定量PCR和Western blot结果得到验证。同时脂肪酸含量检测发现与SCD1关系密切的游离脂肪酸在DRD2下调组显著增加。结论下调小鼠中脑神经元DRD2可以显著上调SCD1的表达,SCD1上调可以加速饱和脂肪酸向单不饱和脂肪酸转化,防止脂毒性对细胞的损伤。
Objective To study the effects of dopamine receptor D2(DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing(HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group.Real-time quantitative PCR(qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes.Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation,mainly including delta(14)-sterol reductase,acetyl-coenzyme A synthetase,insulin-induced gene 1 protein and especial y stearoyl-coenzyme A desaturase 1(SCD1,4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2. 6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover,the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.
Objective To study the effects of dopamine receptor D2(DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing(HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group.Real-time quantitative PCR(qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes.Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation,mainly including delta(14)-sterol reductase,acetyl-coenzyme A synthetase,insulin-induced gene 1 protein and especial y stearoyl-coenzyme A desaturase 1(SCD1,4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2. 6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover,the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.
引文
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[2]Allen Reish H E,Standaert D G.Role ofα-synuclein in inducing innate and adaptive immunity in Parkinson disease[J].J Parkinsons Dis,2015,5(1):1-19.
[3]Van der Perren A,Macchi F,Toelen J,et al.FK506 reduces neuroinflammation and dopaminergic neurodegeneration in anα-synuclein-based rat model for Parkinson’s disease[J].Neurobiol Aging,2015,36(3):1559-1568.
[4]Foucault-Fruchard L,Antier D.Therapeutic potential ofα7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases[J].Neural Regen Res,2017,12(9):1418-1421.
[5]Agim Z S,Cannon J R.Dietary factors in the etiology of Parkinson’s disease[J/OL].Biomed Res Int,2015,2015:672838.DOI:10.1155/2015/672838.Epub 2015 Jan 20.
[6]Fessler M B.Regulation of adaptive immunity in health and disease by cholesterol metabolism[J/OL].Curr Allergy Asthma Rep,2015,15(8):48.DOI:10.1007/s11882-015-0548-7.
[7]陈杰,王圆圆,叶海霞.帕金森病及帕金森综合征患者血脂水平的临床分析[J].中西医结合心脑血管病杂志,2017,15(9):1119-1121.
[8]Schulte E C,Altmaier E,Berger H S,et al.Alterations in lipid and inositol metabolisms in two dopaminergic disorders[J/OL].PLo S One,2016,11(1):e0147129.DOI:10.1371/journal.pone.0147129.e Collection 2016.
[9]Madore C,Nadjar A,Delpech J C,et al.Nutritional n-3 PUFAs deficiency during perinatal periods alters brain innate immune system and neuronal plasticity-associated genes[J].Brain Behav Immun,2014,41:22-31.
[10]Delpech J C,Thomazeau A,Madore C,et al.Dietary n-3 PUFAs deficiency increases vulnerability to inflammation-induced spatial memory impairment[J].Neuropsychopharmacology,2015,40(12):2774-2787.
[11]Tang K S.Protective effect of arachidonic acid and linoleic acid on1-methyl-4-phenylpyridinium-induced toxicity in PC12 cells[J/OL].Lipids Health Dis,2014,13(1):197.DOI:10.1186/1476-511X-13-197.
[12]Kubo S I.Membrane lipids as therapeutic targets for Parkinson’s disease:a possible link between Lewy pathology and membrane lipids[J].Expert Opin Ther Targets,2016,20(11):1301-1310.
[13]Romaní-Pérez M,Lépinay A L,Alonso L,et al.Impact of perinatal exposure to high-fat diet and stress on responses to nutritional challenges,food-motivated behaviour and mesolimbic dopamine function[J].Int J Obes(Lond),2017,41(4):502-509.
[14]Begieneman M P,Ter Horst E N,Rijvers L,et al.Dopamine induces lipid accumulation,NADPH oxidase-related oxidative stress,and a proinflammatory status of the plasma membrane in H9c2 cells[J/OL].Am J Physiol Heart Circ Physiol,2016,311(5):H1097-H1107.DOI:10.1152/ajpheart.00633.2015.Epub 2016 Aug 12.
[15]Freyberg Z,Aslanoglou D,Shah R,et al.Intrinsic and antipsychotic drug-induced metabolic dysfunction in schizophrenia[J/OL].Front Neurosci,2017,11:432.DOI:10.3389/fnins.2017.00432.e Collection 2017.
[16]Matuskey D,Worhunksy P,Correa E,et al.Age-related changes in binding of the D2/3 receptor radioligand[11C](+)PHNO in healthy volunteers[J].Neuroimage,2016,130:241-247.
[17]李伟,孙茂民,林雪霞,等.多巴胺受体D2基因Taq I位点多态性与帕金森病的关系[J].苏州大学学报(医学版),2009,29(2):303-306.
[18]Baik J H,Picetti R,Saiardi A,et al.Parkinsonian-like locomotor impairment in mice lacking dopamine D2 receptors[J].Nature,1995,377(6548):424-428.
[19]李凡,舒斯云,包新民.多巴胺受体的结构和功能[J].中国神经科学杂志,2003,19(6):405-410.
[20]Bains M,Roberts J L.Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway[J/OL].Neurosci Lett,2016,610:79-85.DOI:10.1016/j.neulet.2015.10.054.Epub 2015 Oct 28.
[21]Morin N,Morissette M,Grégoire L,et al.m Glu5,dopamine D2 and adenosine A2A receptors in L-DOPA-induced dyskinesias[J/OL].Curr Neuropharmacol,2016,14(5):481-493.
[22]FernJ,Skrede S,Vik-Mo A O,et al.Drug-induced activation of SREBP-controlled lipogenic gene expression in CNS-related cell lines:marked differences between various antipsychotic drugs[J/OL].BMC Neurosci,2006,7:69.DOI:10.1186/1471-2202-7-69.
[23]Iwai T,Kume S,Chin-Kanasaki M,et al.Stearoyl-Co A desaturase-1protects cells against lipotoxicity-mediated apoptosis in proximal tubular cells[J/OL].Int J Mol Sci,2016,17(11):E1868.DOI:10.3390/ijms17111868.
[24]Tan L C,Methawasin K,Tan E K,et al.Dietary cholesterol,fats and risk of Parkinson’s disease in the Singapore Chinese Health Study[J].J Neurol Neurosurg Psychiatry,2016,87(1):86-92.
[25]Kamel F,Goldman S M,Umbach D M,et al.Dietary fat intake,pesticide use,and Parkinson’s disease[J].Parkinsonism Relat Disord,2014,20(1):82-87.
[26]Fabelo N,Martin V,Santpere G,et al.Severe alterations in lipid composition of frontal cortex lipid rafts from Parkinson’s disease and incidental Parkinson’s disease[J].Mol Med,2011,17(9/10):1107-1118.
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