帕金森病细胞模型的构建及氨基酸表达观察

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、0.25 μmol/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(0.15 μmol/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-SY5Y 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-SY5Y 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.