Rat Hepatic Stellate Cells Oxidative Stress Model
Figure 1. A simplified overview of primary drivers of non-alcoholic steatohepatitis (NASH)-induced hepatic fibrosis. (Zisser et al., 2021)
Hepatic stellate cell activation is the central link of the occurrence and development of hepatic fibrosis. At present, one of the ideas of international research and development of hepatic fibrosis drugs is to find targets from the mechanism of hepatic fibrosis, that is, the activation mechanism of hepatic stellate cells. Oxidative stress is an important factor in the activation of hepatic stellate cells in the occurrence and development of liver fibrosis. Therefore, it is very necessary to construct a scientific hepatic stellate cell oxidative stress model in vitro.
Liver is the main place of iron metabolism and the main target organ of iron overload. Iron overload is closely related to the occurrence and development of liver fibrosis. Creative Bioarray induced oxidative stress model of primary rat hepatic stellate cells (rHSC) by iron overload.
Creative Bioarray can establish a variety of cellular oxidative stress and other stress models according to the specific needs of customers. Colleagues can provide administration experiments, pharmacopharmacology, efficacy evaluation and pharmacokinetic analysis, and assist customers in all-round scientific research projects.
Model Information
Model application
Iron overload cell oxidative stress injury model can be used as an important tool to test the effectiveness of drugs in the treatment of liver cancer, speech and liver fibrosis in vitro.
Modeling method
The cells were isolated and cultured according to standard procedures. All cells were 1 × 105 / ml was inoculated on 96 well plates, in which rHSC cells were cultured for 5 days after separation. After overnight culture, a certain concentration of Fe-NTA was added.
If you need, we can also induce the oxidative stress model of human hepatic stellate cell line lx-2 by iron overload.
Modeling Effect Guarantee
Isolation and identification of rHSC
Trypan blue was used to identify the survival rate of freshly isolated rHSC, which should be > 95%. Desmin staining and purity of isolated rHSC detected by SMA staining was more than 95%, which met the experimental requirements.
Cell proliferation was detected by crystal violet
The cell proliferation was detected by crystal violet at 24, 48 and 72 hours respectively. Cells were washed with formaldehyde twice and fixed with PBS. Add crystal violet dye solution for incubation, rinse with PBS, then add acetic acid aqueous solution for dissolution, and read at 570nm.
Intracellular superoxide detection
After the cells were treated accordingly, the superoxide anion fluorescent probe working solution was added, incubated at 37 ℃ for 60min, washed twice with s buffer, and observed by green excitation under fluorescence microscope.
MTT assay (applicable to LX-2 cell line)
After reaching the set time, continue to culture MTT reagent in the sample for 4h, terminate the culture, suck and discard the culture medium, add DMSO, shake to fully dissolve the crystal, measure the OD value at 492 nm of the microplate reader, and use 630 nm as the reference wavelength.
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Reference
- Zisser A, Ipsen DH, Tveden-Nyborg P. Hepatic Stellate Cell Activation and Inactivation in NASH-Fibrosis-Roles as Putative Treatment Targets? Biomedicines. 2021 Mar 31;9(4):365.
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