羟多巴胺%PC12细胞%利血平%囊泡单胺转运蛋白质类%多巴胺β羟化酶
羥多巴胺%PC12細胞%利血平%囊泡單胺轉運蛋白質類%多巴胺β羥化酶
간다파알%PC12세포%리혈평%낭포단알전운단백질류%다파알β간화매
Oxidopamine% PC12 cells% Reserpine% Vesicular monoamine transporter proteins%Dopamine beta-hydroxylase
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.
目的 探討6-羥多巴胺(6-OHDA)對大鼠嗜鉻細胞瘤(PC12)細胞單胺類5-羥色胺(5-HT)、去甲腎上腺素(NE)、多巴胺遞質及其閤成限速酶基因色氨痠羥化酶(TpH) mRNA、多巴胺β羥化酶(DβH)mRNA和酪氨痠羥化酶(TH)mRNA的影響,及囊泡單胺類轉運體(VMAT)功能抑製對上述物質的作用.方法 分彆用不同濃度6-OHDA(25、50、100、200 μmol/L),不同濃度的VMAT功能抑製劑利血平(50、100、400、1600 nmol/L)與6-OHDA(100 μmol/L)作用于PC12細胞,于不同時間點(0、12、24、36、48 h)用四甲基偶氮唑鹽(MTT)法檢測細胞活性,酶聯免疫(ELISA)法檢測細胞內5-HT、NE、多巴胺遞質,併用熒光實時定量PCR(RT-PCR)法檢測TpHmRNA、DβHmRNA及THmRNA相對錶達量.結果 (1)加入不同濃度的6-OHDA時,PC12細胞中細胞活性隨6-OHDA濃度增加而下降,併隨作用時間的延長濃度降低更加明顯.而在100 μmol/L 6-OHDA組中,隨著利血平濃度的增加,細胞活性顯著下降,差異有統計學意義.PC12細胞中5-HT濃度隨6-OHDA濃度增加下降不明顯,但在同一濃度組中隨作用時間的延長濃度降低明顯,尤其在36 h時間點濃度最低.NE隨6-OHDA濃度增加顯著下降,併隨作用時間的延長錶達量降低更加明顯.多巴胺濃度隨6-OHDA濃度增加顯著降低,但隨作用時間的延長濃度變化不明顯.100μmol/L 6-OHDA組中,隨著利血平濃度的增加,5-HT、NE及多巴胺濃度顯著下降,差異有統計學意義.(2)與對照組相比,加入不同濃度的6-OHDA時PC12細胞TpHmRNA、DβHmRNA及THmRNA錶達隨6-OHDA濃度增加顯著下降,併隨作用時間的延長錶達量降低更加明顯,而在100μmol/L 6-OHDA組中,隨著利血平濃度的增加(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)錶達顯著下降,差異有統計學意義(F值分彆為13.336、9.000、9.393,均P=0.000).結論 6-OHDA能誘導PC12細胞活性降低,誘導5-HT、NE、多巴胺遞質濃度及TpHmRNA、DβHmRNA、THmRNA錶達的降低,併呈劑量及時間依賴性,VMAT功能抑製加重上述變化.
목적 탐토6-간다파알(6-OHDA)대대서기락세포류(PC12)세포단알류5-간색알(5-HT)、거갑신상선소(NE)、다파알체질급기합성한속매기인색안산간화매(TpH) mRNA、다파알β간화매(DβH)mRNA화락안산간화매(TH)mRNA적영향,급낭포단알류전운체(VMAT)공능억제대상술물질적작용.방법 분별용불동농도6-OHDA(25、50、100、200 μmol/L),불동농도적VMAT공능억제제리혈평(50、100、400、1600 nmol/L)여6-OHDA(100 μmol/L)작용우PC12세포,우불동시간점(0、12、24、36、48 h)용사갑기우담서염(MTT)법검측세포활성,매련면역(ELISA)법검측세포내5-HT、NE、다파알체질,병용형광실시정량PCR(RT-PCR)법검측TpHmRNA、DβHmRNA급THmRNA상대표체량.결과 (1)가입불동농도적6-OHDA시,PC12세포중세포활성수6-OHDA농도증가이하강,병수작용시간적연장농도강저경가명현.이재100 μmol/L 6-OHDA조중,수착리혈평농도적증가,세포활성현저하강,차이유통계학의의.PC12세포중5-HT농도수6-OHDA농도증가하강불명현,단재동일농도조중수작용시간적연장농도강저명현,우기재36 h시간점농도최저.NE수6-OHDA농도증가현저하강,병수작용시간적연장표체량강저경가명현.다파알농도수6-OHDA농도증가현저강저,단수작용시간적연장농도변화불명현.100μmol/L 6-OHDA조중,수착리혈평농도적증가,5-HT、NE급다파알농도현저하강,차이유통계학의의.(2)여대조조상비,가입불동농도적6-OHDA시PC12세포TpHmRNA、DβHmRNA급THmRNA표체수6-OHDA농도증가현저하강,병수작용시간적연장표체량강저경가명현,이재100μmol/L 6-OHDA조중,수착리혈평농도적증가(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)급THmRNA (0.005±0.002,0.003 ±0.001,0.002 ±0.001,0.001±0.000)표체현저하강,차이유통계학의의(F치분별위13.336、9.000、9.393,균P=0.000).결론 6-OHDA능유도PC12세포활성강저,유도5-HT、NE、다파알체질농도급TpHmRNA、DβHmRNA、THmRNA표체적강저,병정제량급시간의뢰성,VMAT공능억제가중상술변화.
Objective 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.
