摘要目的 探讨6-羟多巴胺(6-OHDA)对大鼠嗜铬细胞瘤(PC12)细胞单胺类5-羟色胺(5-HT)、去甲肾上腺素(NE)、多巴胺递质及其合成限速酶基因色氨酸羟化酶(TpH) mRNA、多巴胺β羟化酶(DβH)mRNA和酪氨酸羟化酶(TH)mRNA的影响,及囊泡单胺类转运体(VMAT)功能抑制对上述物质的作用.方法 分别用不同浓度6-OHDA(25、50、100、200 μmol/L),不同浓度的VMAT功能抑制剂利血平(50、100、400、1600 nmol/L)与6-OHDA(100 μmol/L)作用于PC12细胞,于不同时间点(0、12、24、36、48 h)用四甲基偶氮唑盐(MTT)法检测细胞活性,酶联免疫(ELISA)法检测细胞内5-HT、NE、多巴胺递质,并用荧光实时定量PCR(RT-PCR)法检测TpHmRNA、DβHmRNA及THmRNA相对表达量.结果 (1)加入不同浓度的6-OHDA时,PC12细胞中细胞活性随6-OHDA浓度增加而下降,并随作用时间的延长浓度降低更加明显.而在100 μmol/L 6-OHDA组中,随着利血平浓度的增加,细胞活性显著下降,差异有统计学意义.PC12细胞中5-HT浓度随6-OHDA浓度增加下降不明显,但在同一浓度组中随作用时间的延长浓度降低明显,尤其在36 h时间点浓度最低.NE随6-OHDA浓度增加显著下降,并随作用时间的延长表达量降低更加明显.多巴胺浓度随6-OHDA浓度增加显著降低,但随作用时间的延长浓度变化不明显.100μmol/L 6-OHDA组中,随着利血平浓度的增加,5-HT、NE及多巴胺浓度显著下降,差异有统计学意义.(2)与对照组相比,加入不同浓度的6-OHDA时PC12细胞TpHmRNA、DβHmRNA及THmRNA表达随6-OHDA浓度增加显著下降,并随作用时间的延长表达量降低更加明显,而在100μmol/L 6-OHDA组中,随着利血平浓度的增加(50、100、400、1600 nmol/L),TpHmRNA(0.006±0.001、0.003±0.000、0.003±0.000、0.002±0.000),DβHmRNA(0.005 ±0.002、0.003±0.001、0.002±0.001、0.001 ±0.000)及THmRNA (0.005±0.002,0.003 ±0.001,0.002 ±0.001,0.001±0.000)表达显著下降,差异有统计学意义(F值分别为13.336、9.000、9.393,均P=0.000).结论 6-OHDA能诱导PC12细胞活性降低,诱导5-HT、NE、多巴胺递质浓度及TpHmRNA、DβHmRNA、THmRNA表达的降低,并呈剂量及时间依赖性,VMAT功能抑制加重上述变化.

abstractsObjective To study the effects of 6-hydroxydopamine (6-OHDA),and inhibitors for vesicular monoamine transporter (VMAT) on 5-hydroxytryptamine (5-HT),norepinephrine (NE) and dopamine (DA) and the expressions of tryptophan hydroxylase (TpH) mRNA,dopamine-beta-hydroxylase (DβH) mRNA and tyrosine hydroxylase (TH) mRNA in PC12 cells.Methods The cell viability was determined using MTT assay, the density of 5-HT, NE and DA was detected using enzyme-linked immunosorbent assay,and the expressions of TpHmRNA,DβHmRNA and THmRNA were detected using RT-PCR in PC12 cells at different time points (0,12,24,36,48 h ) after exposure to different concentrations of 6-OHDA(25,50,100,200 μmol/L),and VMAT inhibitors,reserpine (50,100,400,1600 nmol/L),which combined with 6-OHDA( 100 μmol/L).Results (1)The cell viability declined with the increasing concentration of 6-OHDA which showed time dependence.The cell viability in PC12 cell which treated with reserpine decreased significantly in the responding group.The density of 5-HT in PC12 cell did not decrease with the increasing concentration of 6-OHDA,but the change had the time dependence,and the density of 5-HT was lowest at 36 h.The density of NE decreased with the increasing concentration of 6-OHDA which showed time dependence. The density of DA in PC12 cell decreased with the increasing concentration of 6-OHDA,but the change did not have the time dependence.The density of 5-HT,NE and DA in PC12 cell which treated with reserpine decreased significantly in the responding group. (2) The expressions of TpHmRNA, DβHmRNA and THmRNA in PC12 cell decreased with the increasing concentration of 6-OHDA which showed time dependence.The expressions of TpHmRNA(0.006 ± 0.001,0.003 ± 0.000,0.003 ± 0.000,0.002 ± 0.000) ; DβHmRNA (0.005 ± 0.002,0.003 ± 0.001,0.002 ±0.001,0.001 ± 0.000) and THm RNA (0.005 ± 0.002,0.003 ± 0.001,0.002 ± 0.001,0.001 ± 0.000) in PC12 cell which treated with reserpine decreased significantly in the responding group(F =13.336,9.000,9.393,all P =0.000).Conclusions 6-OHDA can decrease the cell viability in PC12 cell,reduce the density of 5-HT,NE and DA and decrease the expressions of TpHmRNA,DβHmRNA and THmRNA,and the effects have dose and time dependence.Reserpine can aggravate this damage.