A detection method of 5-fluorocytosine and 5-fluorouracil in cells based on capillary electrophoresis

A technology of capillary electrophoresis and flucytosine, which is applied in the field of cell detection, can solve the problem that the separation of 5-fluorocytosine and 5-fluorouracil has not been carried out, and achieves the effects of less sample amount, short separation time and high separation efficiency.

Inactive Publication Date: 2019-06-04
DONGHUA UNIV
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Problems solved by technology

[0005] At present, the separation of extracellular bases, especially the study of purine bases, has not been carried out on the separation of 5-fluorocytosine and 5-fluorouracil from intracellular extracts.

Method used

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  • A detection method of 5-fluorocytosine and 5-fluorouracil in cells based on capillary electrophoresis
  • A detection method of 5-fluorocytosine and 5-fluorouracil in cells based on capillary electrophoresis
  • A detection method of 5-fluorocytosine and 5-fluorouracil in cells based on capillary electrophoresis

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Experimental program
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Embodiment 1

[0038] instrument

[0039]PA800Plus capillary electrophoresis instrument (Beckman Coulter Instruments, Fullerton, CA, USA), PDA detector, controlled by 32Krarat Station software. The effective length of the quartz capillary is 44 cm, the inner diameter of the capillary is 50 μm, and the 0.22 μm polyethersulfone membrane filter (Shanghai Anpu Experimental Technology Co., Ltd.).

[0040] 2. Cell culture

[0041] The human kidney epithelial cell line 293T was preserved and cultivated in the laboratory; DMEM dry powder, fetal calf serum, and trypsin (containing EDTA) were purchased from Gibco; the transfection reagent Lipofectamine2000 was purchased from Invitrogen; the cells were cultured in 10% serum, 1% bis Anti-(penicillin, streptomycin) DMEM medium. The cells were cultured in a 5% CO2, 37°C incubator, passaged every 2 to 3 days, and the cells in the logarithmic growth phase were used for experiments.

[0042] 3. Cell transfection

[0043] 3×10 by cell concentration 5 293...

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Abstract

The invention relates to a method for detecting intracellular 5-flucytosine and 5-fluorouracil based on capillary electrophoresis. The method comprises the following steps: transfecting cells with a plasmid carrying a suicide gene FCY1, adding 5-flucytosine and / or 5-fluorouracil into a medium, and carrying out culture; then collecting cell metabolite extract; and carrying out qualitative and quantitative detection of 5-flucytosine and / or 5-fluorouracil in the cell metabolite extract by using capillary electrophoresis. The method provided by the invention has the characteristics of high separation efficiency, low sample usage amount, short separation time, low analysis cost and the like, and realizes qualitative and quantitative detection of intracellular 5-flucytosine and 5-fluorouracil.

Description

technical field [0001] The invention belongs to the field of cell detection, in particular to a method for detecting 5-fluorocytosine and 5-fluorouracil in cells based on capillary electrophoresis. Background technique [0002] 5-Fluorouracil (5-FU) is a congener of uracil, a component of ribonucleotides. 5-Fluorouracil acts as an anti-metabolite. After being converted into effective fluorouracil nucleotides in cells, it is converted into thymidylate by blocking deoxyribouridine acid by intracellular thymidylate synthase, while It interferes with the synthesis of DNA and also has a certain inhibitory effect on the synthesis of RNA. After it is converted into nucleotides, it is preferentially taken up by actively dividing tissues and tumors. It is clinically used for the effective treatment of various tumors, such as digestive tract tumors, breast cancer, ovarian cancer, choriocarcinoma, cervical cancer, bladder cancer, liver cancer, skin cancer, etc., especially for digest...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N21/33G01N15/10
CPCG01N15/1031G01N21/33G01N27/447G01N2015/1006
Inventor 黄志伟史萍刘亚敬王桥
Owner DONGHUA UNIV
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