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Method for analyzing action of drugs on monoamine transporter based on liquid chromatography-mass spectrometry

A technology of mass spectrometry and liquid chromatography, which is applied in the field of liquid chromatography-mass spectrometry analysis of the action of drugs on monoamine transporters, can solve the problems of expensive substrates, hidden radioactive safety hazards, etc., and achieve high operability and safety performance, high sensitivity, and a wide range of applications

Pending Publication Date: 2021-05-21
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the HEK293 cells specifically and highly express monoamine transporters through transfection technology, the traditional research method is to use [ 3 The substrate of the H]-labeled transporter is detected by a liquid scintillation counter (liquid scintillation counter), but this radioactive isotope detection method, except [ 3 In addition to the expensive price of H]-labeled substrates, there are also potential radioactive safety hazards, which require specific personnel with strict training to operate, and involve the samples used and the remaining [ 3 Subsequent processing of H]-labeled substrates, etc.

Method used

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  • Method for analyzing action of drugs on monoamine transporter based on liquid chromatography-mass spectrometry
  • Method for analyzing action of drugs on monoamine transporter based on liquid chromatography-mass spectrometry
  • Method for analyzing action of drugs on monoamine transporter based on liquid chromatography-mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Construction of DAT and SERT overexpression cell lines

[0022] 1.1 Cell culture

[0023] HEK293 cells were cultured in DMEM medium containing 10% standard fetal bovine serum, 60 U / mL-1 penicillin, and 100 μmol / L streptomycin at 37°C and 5% CO 2 , The humidity is saturated, and the culture medium is replaced every 72 hours. After the HEK293 cells reached more than 80% confluence, they were passaged at a ratio of 1:4.

[0024] 1.2 DAT and SERT plasmids

[0025] Plasmid pcDNA3.1-DAT and pcDNA3.1-SERT plasmid maps are as follows figure 1 As shown, the full-length cDNAs of the genes DAT and SERT were inserted into the pcDNA3.1 vector between the HindIII and Xhol restriction sites, respectively.

[0026] 1.3 Transfection of DAT and SERT overexpression HEK293 cells

[0027] HEK293 cells one day in advance according to 0.25-1x10 6 The density of the cells was spread in 6-well plates and cultured overnight to keep the cell density at 70%-90% during transfection.

[0...

Embodiment 2

[0046] Example 2 Establishment of LC-MS / MS analysis technology for detecting DA and 5-HT in HEK293 cells

[0047] 2.1 Sample pretreatment

[0048] Wash HEK293 cells transfected to express DAT and SERT gently twice with 500 μL KHB buffer solution, add 200 μL 0.2M HOAc solution, overnight at 4°C, collect the lysate the next day and transfer to a centrifuge tube, centrifuge at 12,000 rpm at 4°C Take the supernatant, combine the lysates of DAT and SERT at the same drug concentration, take 350 μL of the sample to be tested, 50 μL of IS working solution, 50 μL of 7% ammonia solution and 250 μL of 0.2% PBA buffer solution (pH 8.5) and mix well , slowly load the mixed solution to pre-activated with 200μL MeOH and 200μL 0.2M NH 4 Cl solution (pH 8.5) equilibrated on the OasisHLB 96-well μElution microextraction plate, and then 200 μL 0.2M NH 4 After washing with Cl solution three times, it was vacuum filtered until completely dry, and then eluted with 100 μL of 5% MeOH in 0.2M HOAc s...

Embodiment 3

[0066] Example 3 The reuptake inhibitory effect test of drugs on DAT and SERT

[0067] Follow the steps below:

[0068] 24 hours before the experiment, the HEK293 cell lines expressing DAT and SERT were respectively transfected and plated for culture. The cells were gently washed twice with 500 μL of 37°C preheated KHB buffer solution, then 250 μL of KHB buffer solution containing serial concentrations of drugs were added to incubate at 37°C for 20 minutes, and then DA (to examine DAT) and 5-HT (to examine SERT) were added. 50 μL of KHB buffer solution, so that the final concentration of DA is 1 μM, and the final concentration of 5-HT is 1 μM. Incubate at 37°C for 15 minutes, discard the reaction solution to terminate the reaction, and operate according to "2.1 Sample pretreatment". For non-specific binding, 250 μL of KHB buffer solution containing 10 μM mazindol (to examine DAT) and 10 μM fluoxetine (to examine SERT) were added respectively, and no drug was added to the bla...

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Abstract

The invention belongs to the technical field of biological analysis, and relates to a method for analyzing the action of drugs on monoamine transporters, in particular to a method for analyzing the action of drugs on monoamine transporters based on liquid chromatography-mass spectrometry. The method can be used for researching the action mechanism of drugs on dopamine transporters and 5-hydroxytryptamine transporters. The method is high in sensitivity, strong in specificity and high in safety, and can be used for replacing a traditional radioisotope detection method.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and relates to an analysis method for the effect of drugs on monoamine transporters, in particular to a method for analyzing the effect of drugs on monoamine transporters based on liquid chromatography-mass spectrometry. The method can be used to study the effect of drugs on monoamine transporters. Mechanism of action of the dopamine and serotonin transporters. The method has high sensitivity, strong specificity and high safety, and can be used to replace the traditional radioisotope detection method. Background technique [0002] The prior art discloses that monoamine transporters in the nervous system are the main targets of action of several drugs. Monoamine transporters are a class of SLC6 solute transporters, including dopamine transporter (dopamine transporter, DAT), serotonin transporter (serotonin transporter, SERT) and so on. Studies have shown that the role of drugs and mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/88
CPCG01N30/02G01N30/06G01N30/7266G01N30/88G01N2030/027G01N2030/8818
Inventor 饶渝兰林泽彬刘晨阳王昊樊恩杉
Owner FUDAN UNIV
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