Chicken Hepatocyte Oxidative Stress Model
Oxidative stress refers to the excessive production of highly active molecules such as reactive oxygen free radicals (ROS) in the body when the body is subjected to various stimuli, which exceeds the scavenging capacity of the body, resulting in the imbalance of oxidation and antioxidant status and oxidative damage.
As an important metabolic organ of chicken, liver has vigorous lipid metabolism, which is prone to metabolic oxidative damage, fatty liver and other diseases. H2O2 is one of the important ROS, which can cause cell oxidative damage. Creative Bioarray isolated and cultured laying chicken hepatocytes by in-situ two-step perfusion method. With H2O2 as inducer, an in vitro oxidative stress cell model was constructed through time-dependent and dose-response tests, which can provide a technical platform and theoretical reference for the subsequent research and development of antioxidant additives applied to laying hens.
Modeling Application
The establishment of chicken hepatocyte oxidative stress model is of great significance for revealing the mechanism of oxidative stress, researching and developing new antioxidant feed and reducing laying chicken liver injury.
Modeling Principle
Isolation and culture of chicken primary hepatocytes
The liver was obtained by in situ two-step perfusion. After passing 100 mesh, 200 mesh and 400 mesh cell sieves respectively, count the blood cell count plate, resuspend with adherent culture medium, and inoculate the cell culture plate for adherent culture. The growth medium was changed after 6 hours, and then every 24 hours.
Treatment of chicken hepatocytes with H2O2
Different hydrogen peroxide concentrations were used to treat multiple groups of cells for different times in order to find out the best modeling concentration and time.
Modeling Quality Assurance
Determination of cell viability
The survival rate of hepatocytes was detected by thiazole blue (MTT) method. Hepatocytes were inoculated into 96 well plates. MTT was added to each well. After shaking, the absorbance value of each well at 490nm wavelength was measured by enzyme labeling instrument.
Determination of reactive oxygen species (ROS)
The production of ROS was measured by NBT (nitrotetrazolium blue chloride) method. The hepatocytes were inoculated into 96 well plates and NBT reaction solution was added. After subsequent fixation and cleaning, the absorbance value of each well was measured at 620 nm by enzyme-linked immunosorbent assay.
Determination of MDA, SOD, cat and other indicators
After H2O2 treatment, break and centrifuge with cell crusher, collect the supernatant as the sample to be tested, and use the kit to determine the indexes such as MDA, SOD and cat.
Services and Deliverable
We can also provide administration experiment, pharmacology, pharmacodynamics evaluation and pharmacokinetic analysis based on the establishment of model according to the specific needs of customers, so as to help customers study the mechanism of liver injury and find feed and food antioxidant additives to prevent and treat liver injury.
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