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In-vitro model construction method for predicting toxicity of compound upon fatty liver diseases

A fatty liver disease and construction method technology, which is applied in the field of in vitro model construction to predict the virality of compounds on fatty liver, can solve the problems of animal ethics and complicated operation, and achieve the effect of significant susceptibility

Inactive Publication Date: 2019-02-15
NANJING DRUM TOWER HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

While obese mice and rats may be useful, there are numerous differences in hepatic drug metabolism between rodents and humans
In addition, research at the animal level is operationally complex and raises animal ethical issues

Method used

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  • In-vitro model construction method for predicting toxicity of compound upon fatty liver diseases
  • In-vitro model construction method for predicting toxicity of compound upon fatty liver diseases
  • In-vitro model construction method for predicting toxicity of compound upon fatty liver diseases

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Embodiment Construction

[0034] The present invention will be further described below in conjunction with specific embodiments.

[0035] 1. Two-dimensional or three-dimensional culture of hepatocytes;

[0036] The primary hepatocytes can be obtained from rodent liver or human liver by Seglen two-step collagenase perfusion method. The principle is based on Ca 2+ It is a key molecule for maintaining cell adhesion, so it does not contain Ca first 2+ And contains Ca 2+ The chelating agent solution (such as citrate or a solution containing EDTA or EGTA) is perfused for the first step, which then destroys the calcium-dependent adhesion factors between cells and breaks the desmosomes. Use containing Ca 2+ The type IV collagenase solution digests the extracellular matrix as the second step of perfusion to make the hepatocytes fall off from the extracellular matrix, and then through a series of steps such as centrifugation and washing to obtain higher purity liver cells.

[0037] The obtained primary hepatocytes wer...

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Abstract

The invention relates to an in-vitro model construction method for predicting toxicity of a compound upon fatty liver diseases. The method comprises the following steps: culturing different liver cells under a two-dimensional or three-dimensional condition, and culturing a fat converted liver cell in-vitro culture model by using a culture medium with a free fatty acid component. By adopting the in-vitro culture system, a growth micro environment in a liver cell can be simulated, the morphology and the function of the liver cell can be maintained for a long time, a cytochrome enzyme level is approximate to that in vivo, and the model has typical characteristics of fat converted liver cells, is capable of replacing fatty liver animal models, and achieving toxicology metabolism detection or pharmacodynamics study on a compound in long-term action on fat converted liver tissue in vitro.

Description

Technical field [0001] The invention belongs to the technical field of toxicology detection, and specifically relates to an in vitro model construction method for predicting the virality of a compound to fatty liver. Background technique [0002] Non-alcoholic fatty liver disease is defined as fatty degeneration of more than 5% of liver cells and little or no alcohol consumption. It is a metabolic stress liver disease closely related to insulin resistance and genetic susceptibility. Its disease progression includes non-alcoholic simple liver steatosis, non-alcoholic steatohepatitis, liver cirrhosis and hepatocellular carcinoma. Existing studies have shown that non-alcoholic fatty liver has an effect on the activity of drug metabolism enzymes and changes the levels of protective substances in cells such as reduced glutathione. For example, the activity of cytochrome enzyme CYP3A4 is down-regulated in steatohepatitis, while the activity of CYP2E1 and efflux transporter MRP3 is up-...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/071C12Q1/02
CPCC12N5/0653C12N5/067C12N2500/32C12N2500/36C12N2501/11C12N2501/33C12N2501/39C12N2533/54G01N33/5014G01N2500/10
Inventor 巫国谊施晓雷丁义涛
Owner NANJING DRUM TOWER HOSPITAL
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