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用毛细管电泳-激光诱发荧光技术检测清醒吗啡戒断大鼠导水管周围灰质微透析液中谷氨酸和精氨酸的含量

曹君利, 张亚海, 顾钧, 周文华, 杨国栋, 曾因明

浙江省宁波戒毒研究中心. 浙江, 宁波 315010；江苏省徐州医学院附属医院麻醉科. 江苏,徐州 221002；江苏省麻醉学重点实验室. 江苏, 徐州 221002

摘要

清醒动物脑微透析技术能够用于动态观察某一特定核团中生物活性物质变化及其与行为的关系,结合高效毛细管电泳-激光诱发荧光对衍生后的透析样品进行检测,使这一技术更趋完善.本实验用荧光素异硫氰酸酯(FITC)与痕量氨基酸样品进行衍生,通过适当增加衍生温度,能明显缩短衍生时间,衍生效果与传统的室温下衍生16 h无明显差别;进一步确定30℃水浴反应5 h是较理想的FITC与痕量氨基酸的衍生条件.在此优化衍生条件下,成功检测了清醒吗啡戒断大鼠脑导水管周围灰质(periaqueductal gray matter,PAG)微透析液中谷氨酸(glutamate, Glu)和精氨酸(arginine,Arg)含量变化.结果表明,非吗啡依赖和吗啡依赖大鼠脑PAG中L-精氨酸(L-arginine,L-Arg)和L-谷氨酸(L-glutamate,L-Glu)含量无明显差别,纳洛酮催促戒断后的第一个10 min内PAG中的L-Arg和L-Glu含量显著增加,分别比戒断前增加了63%和105%, 10 min后含量逐渐下降,这种变化趋势与同时观察的吗啡戒断评分变化相一致。

关键词： 毛细管电泳; 微透析; 谷氨酸; 精氨酸; 导水管周围灰质

Application of combination of capillary electrophoresis with laser-induced fluorescence:measurement of glutamate and arginine in PAG microdialytes of conscions morphine-withdrawal rats

Cao Junli, Zhang Yahai, Gu Jun, Zhou Wenhua, Yang Guodong, Zeng Yinming

Department of Anesthesiology，Affiliated Hospital of Xuzhou Medical College，Jiangsu Province Key Laboratory of Anesthesiology. Xuzhou 221002, China；Ningbo Addiction Research and Treatment Center. Ningbo 315010,China

Abstract

Microdialysis technique in free-moving animals can be used to monitor continuously the chan-ges of many extracellular neurotransmitters in certain brain areas and study the relationship between neuro-transmitter and functions. Using detection of capillary electrophoresis combined with laser-induced fluores-cence (CE-LIF) further improves the above-mentioned technique. In the present study, fluorescein isothio-cyanate (FITC ) was used to derivatizate amino acid in very low concentration. We found that increasing de-rivatization temperature could shorten derivatization time and that the derivatizative efficiency was not differ-ent from that when experiment was performed under the traditional derivatization condition (room tempera-ture for 16 h). We also got an optimized condition of amino acid derivatization with FITC at 30℃ waterbath for 5 h. Using the optimized condition of amino acid derivatization, we investigated the changes in L- arginine (L-Arg) and L-glutamate (L-Glu) concentration in periaqueductal gradually. These changes were in correspondence with the scores of morphine withdrawal symptom.

Key words: Capillary electrophoresis;Microdialysis glutamate (Glu);Arginine (Arg); Periaqueductal gray matter ;

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引用本文：

曹君利, 张亚海, 顾钧, 周文华, 杨国栋, 曾因明. 用毛细管电泳-激光诱发荧光技术检测清醒吗啡戒断大鼠导水管周围灰质微透析液中谷氨酸和精氨酸的含量[J]. 生理学报 2003; 55 (5): .

Cao Junli, Zhang Yahai, Gu Jun, Zhou Wenhua, Yang Guodong, Zeng Yinming. Application of combination of capillary electrophoresis with laser-induced fluorescence:measurement of glutamate and arginine in PAG microdialytes of conscions morphine-withdrawal rats. Acta Physiol Sin 2003; 55 (5): (in Chinese with English abstract).