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FPR1 channel function-based chemical carcinogenicity in-vitro detection method

An in vitro detection and chemical technology, applied in biochemical equipment and methods, microbial determination/inspection, material analysis by optical means, etc., can solve the problems of complex and cumbersome direct detection methods and high requirements for experimental environment, and achieve results. Objectively quantifiable, reducing interference factors and shortening detection time

Pending Publication Date: 2021-06-25
NANJING INST OF PROD QUALITY INSPECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FPR1 is a GPCR coupled to Gαi, the direct detection method is complex and cumbersome, and requires high experimental environment

Method used

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  • FPR1 channel function-based chemical carcinogenicity in-vitro detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 An in vitro detection method for chemical carcinogenicity based on the FPR1 channel, optimization of high-throughput real-time fluorescence detection conditions;

[0042] (1) Cell seed plate

[0043] FPR1-CHO cells in the logarithmic growth phase were divided into 5×10 3 pcs / hole, 1×10 4 pcs / hole, 2×10 4 pcs / hole, 4×10 4 The density of cells / well was planted in 96-well black transparent flat-bottomed cell plates. The next day, the cell confluence was observed under an inverted microscope.

[0044] (2) Dye Incubation

[0045] Add calcium ion dye Fluo-4 AM at a volume of 60 μL per well, and incubate at 37°C for 45min-60min. After incubation, wash 3-4 times with HBSS buffer to remove dye that has not entered the cells. After washing, add HBSS buffer to a final volume of 175 μL.

[0046] (3) Treatment of chemicals to be tested

[0047] The FPR1 agonist fMLP was dissolved in DMSO into a 100 mM sample solution. Dilute with HBSS buffer at a ratio of 1:125, ...

Embodiment 2

[0056] An in vitro assay of chemical carcinogenicity based on the FPR1 channel, evaluating the carcinogenicity of ochratoxin A (OTA);

[0057] (1) Cell seed plate

[0058] Take FPR1-CHO cells in the logarithmic growth phase in 2×10 4 The density of cells / well was planted in 96-well black transparent flat-bottomed cell plates. On the next day, the degree of cell fusion was observed under a microscope, and the dye incubation could be carried out when it reached more than 70%.

[0059] (2) Dye Incubation

[0060] Add calcium ion dye Fluo-4 AM at a volume of 60 μL per well, and incubate at 37°C for 45min-60min. After incubation, wash 3-4 times with HBSS buffer to remove dye that has not entered the cells. After washing, add HBSS buffer to a final volume of 175 μL.

[0061] (3) Treatment of chemicals to be tested

[0062] OTA was first dissolved in DMSO into 20mM, 10mM, 5mM sample solutions. Dilute them with HBSS buffer at a ratio of 1:125, add 60 μL per well to a 96-well tr...

Embodiment 3

[0069] A chemical carcinogenicity detection method based on FPR1 channel in vitro to evaluate the carcinogenicity of patulin;

[0070] (1) Cell seed plate

[0071] The steps of seeding the cells are the same as in Example 2.

[0072] (2) Dye Incubation

[0073] Dye incubation steps are the same as in Example 2.

[0074] (3) Treatment of chemicals to be tested

[0075] Dissolve patulin in DMSO into sample solutions with concentrations of 20mM, 10mM, and 5mM. Dilute with HBSS buffer at a ratio of 1:125, and add to a 96-well transparent conical-bottom microtiter plate at a volume of 60 μL per well.

[0076] (4) High-throughput real-time fluorescence detection

[0077] The high-throughput real-time fluorescence detection is the same as that in Example 2.

[0078] (5) Results

[0079] Such as image 3 As shown, compared with the negative control, after patulin at the final concentration of 5 μM, 10 μM, and 20 μM acted on FPR1-CHO cells, the intracellular calcium ion concent...

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Abstract

The invention provides an FPR1 channel function-based chemical carcinogenicity in-vitro detection method, which comprises the following steps of culturing FPR1-CHO cells until the fusion degree of the cells reaches more than 70%, adding a dye for incubation, exposing the incubated cells to a chemical-containing test solution, carrying out high-flux real-time fluorescence detection, and if the FPR1 channel of the cells is activated, determining that the chemical is carcinogenic. FPR1 target function detection and chemical carcinogenicity screening are combined, and the abnormal activation of FPR1 and the change of a downstream signal transduction pathway can significantly promote the malignant progression of tumors, so that the sensitivity of a chemical carcinogenicity prediction method can be greatly improved, the detection range is expanded, the test period is shortened, interference factors are reduced, and the result is more stable and reliable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an in vitro detection method of chemical carcinogenicity based on FPR1 channel function. Background technique [0002] Cancer is the largest disease burden in most countries around the world. 80% to 90% of human tumors are related to environmental factors, mainly chemical factors. Heavy metals and biological toxins are the main environmental chemical carcinogens, which enter the human body through the atmosphere, water, soil, food, etc., and cause serious harm to health after accumulation and metabolism. [0003] The carcinogenic process of chemicals is very long, and the growth period of tumors usually lasts for decades. There are no obvious clinical manifestations on the body in the early stage of exposure to carcinogenic chemicals. Once clinical symptoms or imaging manifestations appear, it indicates that the tumor has entered the growth and development stage, and the cells have i...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/02
CPCG01N21/6428G01N33/5005
Inventor 杨淼张驰强雨薇吴肖肖纪晗旭
Owner NANJING INST OF PROD QUALITY INSPECTION
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