糖皮质激素个体化用药系统平台的建立与评价  

Establishment and evaluation of glucocorticoid individualized medication system platform

作　　者：王昌富[1,2] 彭长华[1,2] 李琳芸[1,2] 梅冰[1,2] 

机构地区：[1]华中科技大学同济医学院附属荆州医院检验医学部,湖北荆州434020 [2]长江大学医学院免疫研究室,湖北荆州434020

出　　处：《分子诊断与治疗杂志》2015年第2期103-106,共4页Journal of Molecular Diagnostics and Therapy

基　　金：湖北省科技厅攻关计划引导项目(2007AA301C54)

摘　　要：目的建立可靠的糖皮质激素（GC）实验诊断系统平台。方法采用超高效液相色谱串联质谱法检测人工合成GC药物（泼尼松、甲基泼尼松和地塞米松）血药浓度，流式细胞分析法检测糖皮质激素受体（GR）-α蛋白，逆转录实时荧光基因扩增定量分析法检测GR-α mRNA，并进行了方法学分析。结果质谱法检测PNS、DPNS、DX血药浓度分别在1～15ng／mL、1～100ng／mL、20～400ng／mL范围内线性关系良好。标本置26℃6h、置-20℃30d、加甘油三酯13．21mmol／L、加总胆红素162．8μmol／L，比较各组血药浓度未发现统计学差异。PNS、DPNS、DX血药浓度日内CV分别小于10．81％、7．98％、5．47％。日间CV分别小于10．72％、6．29％、12．36％。回收率试验显示PNS、DPNS、DX在20ng／mL、20ng／mL、400ng／mL浓度的平均回收率分别是99．91％、102．49％、109．89％。流式细胞术分析GR-α蛋白，标本立即处理检测与放置6h、24h后处理检测结果比较无统计学差异．标本处理后5h检测结果无显著性变化，3种抗体反应条件（室温孵育30min、37℃孵育30min、4℃孵育6h）检测结果无统计学差异，百分率及荧光强度CV值分别为3．4％与9．8％。逆转录实时荧光基因扩增定量分析法检测GR—α mRNA检测灵敏度达到10^1copies／μL，批内和批间Ct值变异系数均小于2．0％。结论初步证实本体系设计科学、方法先进、系统可靠。Objective To establish reliable glucocorticoid hormone experimental diagnosis system platform. Methods Blood drug concentrations of synthetic glucocorticoid （GC）, including prednisone （PNS）, methyl prednisone （DPNS） and dexamethasone （DX）, were measured through ultra performance liquid chromatography tandem mass spectrometry. Expression levels of Glucocorticoid receptor-alpha （GR-α） protein were detected through flow cytometry, mRNA levels of GR-α were measured by using reverse transcription real-time fluorescent quantitative PCR. Methodological index were also analyzed, including sensitivity, precision, interference and recovery ratio, etc. Results Detections of PNS, DPNS and DX blood concentration using mass spectrometry showed good linear relationship in 1-15 ng/mL, 1-100 ng/mL and 20 - 400 ng/mL, respectively. Preservation of specimens in 26 ℃ for 6h, -20 ℃ 30 days, addition of 13.21 mmol/L triglyceride or 162.8 μmol/L total bilirubin had no significant effect on the results. The intra-day CVs of PNS, DPNS and DX blood concentration were less than 10.81%, 7.98% and 5.47%, and inter-day CV less than 10.72%, 6.29% and 12.36%, respectively. The average recovery ratio of PNS, DPNS and DX （concentration at 20 ng/mL, 20 ng/mL or 400 ng/mL, respectively） were 99.91%, 102.49% and 109.89%. No significant difference existed between the levels of GR-α protein detected immediately and after preserving samples for 6h or 24 h using flow cytometry. The results detected at 5h time point after sample processing also showed no significant discrepancy. The results of 3 antibody reaction conditions （incubation for 30 min at room temperature, 30 min at 37 ℃ or 6 h at 4 ℃） were no statistically different. The precision test showed CVs of percentage and fluorescence intensity were 3.4% and 9.8%. The sensitivity of GR-α mRNA detection with reverse transcription real-time fluorescent quantitative PCR was up to 101 copies/L. The precision test showed that the intra- and inter- assay coefficient

关 键 词：糖皮质激素 个体化用药 质谱 

分 类 号：R927.2[医药卫生—药学] O657.63[理学—分析化学]