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Hepatoxic substance sieving and evaluating method based on fluorescence labeling

An evaluation method, fluorescent labeling technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial measurement/inspection, etc., can solve the problem of high price and achieve reliable results

Inactive Publication Date: 2010-07-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, commercial HCS equipment is often very expensive, and only large pharmaceutical companies have the ability to use it abroad; at present, only a few scientific research institutes in China have related equipment

Method used

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  • Hepatoxic substance sieving and evaluating method based on fluorescence labeling
  • Hepatoxic substance sieving and evaluating method based on fluorescence labeling
  • Hepatoxic substance sieving and evaluating method based on fluorescence labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Screening system construction and application to liver toxicity screening

[0053] 1. Hardware and software system

[0054] The purpose of the present invention is to provide a screening and evaluation method for hepatotoxic substances based on the state of fluorescently labeled cells, which is a screening and evaluation method combining hardware / software systems. The screening and evaluation hardware system consists of a German Leica DMI 6000B Composition of fluorescent inverted microscope 1 and computer 2 (see figure 1 ). Fluorescence inverted microscope 1 includes fluorescent inverted microscope main body A, high-precision controllable electric platform B, platform controller C, charge coupler D (1392×1040 pixels, model Leica DFC 310FX, Leica Company of Germany), mercury lamp E, mercury Lamp controller F, coarse adjustment screw G and manual joystick H, platform controller C is connected to microscope body A through wiring, charge coupler D is connected t...

Embodiment 2

[0071] Embodiment 2 Fluorescent image recognition processing

[0072] The fluorescence image recognition system is mainly to process the fluorescence images obtained by the fluorescence microscope, so that the cell biological information is converted into data and the results are visualized. The specific processing steps are background signal subtraction of fluorescent images, binarization processing, and data statistics to generate reports. Through the processing of these three steps, the fluorescent signals that cannot be distinguished by the human eye can be visualized, the recognition sensitivity is high, the detection limit is low, and the fluorescent images are processed automatically in batches, and the results are faster and more stable, and are multi-parameter ( Provide number, area, and fluorescence intensity). For the identification diagram, see image 3 , where panel A. FDA-stained HepG2 cells in whole wells of a 96-well plate; panel B. after image processing.

Embodiment 3

[0073] Example 3 Based on this hardware and software system, the FDA staining and labeling of living cells is used in the dose-effect test of the toxicity of two compounds of acetaminophen and chlorpromazine hydrochloride to liver cells

[0074] HepG2 cells and L-O2 cells were seeded in 96-well plates at a density of 3000 cells per well, and the medium was changed the next day. Compounds acetaminophen and chlorpromazine hydrochloride were selected as positive damage controls, and concentration gradient experiments were performed. Paracetamol was diluted to 9 concentrations starting from 13.65 mM, and chlorpromazine hydrochloride was diluted to 9 concentrations starting from 163.5 μM, and each concentration was replicated in triplicate. Compounds were incubated with cells in a cell culture incubator for 48 hours. In a dark room, discard the culture medium in each well, add 100 μL / well PBS containing 2.5 μg / mL FDA, and incubate at room temperature (25°C) for 15 minutes, and then ...

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Abstract

The invention provides a hepatoxic substance sieving and evaluating method based on cell states labeled by fluorescence, which is a sieving and evaluating method combining a hardware / software system. The images are acquired by a fluorescence inverted microscope and a computer in real time, the biological information is processed and visualized, the liner dynamic range width of the living cell is measured by combining FDA dyeing, and then the relevant parameters for evaluating the hepatoxic substances are provided. The method is quick, sensitive and economical, and is used for quickly detecting the influence of the sieved sample on cell quantity, forms, migration, apoptasis and the like under the condition of maintaining cell structure and function integrity, accurately carrying out quantitative analysis on the activity of the cell labeled by the fluorescence specialty and detecting the activity change of the cell under the function of the hepatoxic substances. The method has reasonable design. The provided sieving and evaluating system has complete structure, and can be used for sieving and evaluating the hepatoxic substances.

Description

technical field [0001] The invention belongs to the field of toxicological screening and evaluation methods of substances, and relates to the application of fluorescent probes to specifically mark cells, automatic acquisition technology of cell microscopic images, fluorescent image recognition and data generation, and to obtain hepatotoxic substance-related indicators by analyzing image information and use them in Hepatotoxicity screening and evaluation of exogenous substances, and this method can be extended to the screening and evaluation of other cytotoxic substances (such as nephrotoxicity, cardiotoxicity, anti-tumor, etc.). Background technique [0002] The liver is the most important place for the metabolism of drugs and exogenous substances in the body. While many drugs play a role in disease prevention and treatment in the body, they will inevitably affect the structure and function of the liver, leading to drug-induced liver injury of different mechanisms. According...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/02
Inventor 程翼宇范骁辉王毅瞿海斌
Owner ZHEJIANG UNIV
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