6-羟基多巴模型大鼠salsolinol氮甲基转移酶活性检测

摘要

目的 应用6-羟基多巴(6-hydroxydopamine,6-OHDA)制作帕金森病(Parkinson''s disease,PD)大鼠模型,并对大鼠外周血淋巴细胞中salsolinol氮甲基转移酶(SNMT)的活性进行测定.方法 利用单侧双点注射6-OHDA损毁大鼠纹状体,构建PD模型并对模型进行行为学评价;从模型大鼠外周血淋巴细胞中提取SNMT粗酶液,建立酶反应产物N-methyl-salsolinol(NMSal)的高效液相色谱串联三重四极杆质谱检测方法,以NMSal的生成量表征SNMT的活性.结果 18只经过6-OHDA注射的大鼠共有7只经阿朴吗啡诱导后表现为恒定向未损毁侧旋转(>7 r/min),建模成功率为38.9%.建立了基于多反应监测技术的SNMT活性的高选择性、高灵敏度、高重复性的表征方法.该方法中NMSal的检出限和定量限分别达到49 pmol/L和98 pmol/L,日内和日间精密度均在6.0%以下.检测结果显示PD组大鼠外周血淋巴细胞中SNMT的活性与假手术组、正常组相比差异有显著性(P<0.01,n=5),而正常组和假手术组之间差异无显著性(P>0.05,n=5).结论 外周血淋巴细胞中SNMT活性可能反映出PD发病状况,有望成为诊断的指标之一.%Objective The aim of this study was to establish a rat model of Parkinson''s disease ( PD) by using 6-hydroxydopamine (6-OHDA) and detect the salsolinol N-methyltransferase ( SNMT) activity in peripheral lymphocytes of PD rats for the development of a biomarker for early diagnosis of PD. Methods Rat model of PD was established by unilateral double-pointed injection of 6-OHDA into the striatum and was verified by behavior observation. An analytical method was developed based on multiple reaction monitoring with HPLC-ESI-QQQ to determine the SNMT activity in peripheral lymphocytes. Results Seven of 18 rats injected with 6-OHDA showed steadily apomorphine-induced rotation ( >7 r/min) . The success rate was 38. 9%. A sensitive and stable quantitative method with internal standard added was created, based on multiple reaction monitoring mode to analyze SNMT activity. The limit of detection ( LOD) and limit of quantitation ( LQD) of N-methyl-salsolinol, which is the product of Salsolinol catalyzed by SNMT, were 49 pmol/L and 98 pmol/L, respectively. The precisions of intra-day and inter-day assays both were below 6. 0%. SNMT activity of peripheral lymphocytes in the 6-OHDA-lesioned rats was significantly increased [43. 37 ±9. 49 pmol/(h·mg)NMSal] in comparison with that in the normal group [2. 16 ±5. 82 pmol/(h·mg)NMSal] and the sham-operated group [0. 58 ±2. 32 pmol/(h· mg)NMSal](P< 0. 01, n=5). There was no significant difference between the normal group and sham-operated group (P< 0. 05, n =5). Conclusions Our results indicate that SNMT activity may reflect the changes in the course of Parkison''s disease and may become a potential clinical biomarker in diagnosis of this disease.