Establishment of the EMT model of CoCl2 -induced NRK-52E cells
10.19405/j.cnki.issn1000-1492.2025.10.014
- VernacularTitle:CoCl2 诱导 NRK⁃52E 细胞 EMT 模型的建立
- Author:
Lei Ni
;
Qingqing Sun
;
Jiangrui Cheng
;
Wei Wei
;
Chun Wang
- Publication Type:Journal Article
- Keywords:
NRK-52E cells;
Cobalt(Ⅱ) dichloride;
epithelial-mesenchymal transition;
hypoxia-inducible factor-1α;
α-smooth muscle actin
- From:
Acta Universitatis Medicinalis Anhui
2025;60(10):1879-1886
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish an in vitro cell model of Cobalt dichloride(CoCl2)-induced epithelial-mesenchymal transition(EMT) in rat renal tubular epithelial cells(NRK-52E).
Methods:NRK-52E cells were cultured in vitro and randomly divided into a blank control group(NC group) and a CoCl2 treatment group. The CoCl2 treatment group underwent 1, 2, 3, and 4 cycles of treatment, with each cycle consisting of CoCl2 treatment for 9 h followed by recovery in CoCl_2-free medium for 3 h. The optimal concentration and time of CoCl_2-induced EMT were screened using the CCK-8 assay. Morphological changes in cells were observed using light microscopy and phalloidin staining. The expression levels of EMT marker proteins were detected by Western blot and immunofluorescence.
Results:Compared with the NC group , stimulation by 100 μmol/L CoCl2 for 48 h significantly induced apoptosis (P < 0. 01) , meeting the requirements for subsequent experiments. Western blot results showed that the expression of hypoxia-inducible factor-1 α (HIF-1α ) and α-smooth muscle actin ( α-SMA) significantly increased in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h ( P < 0. 001) , indicating the most pronounced fibrotic response. Observations under light microscopy and rhodamine-labeled phalloidin staining revealed that the morphological changes and cytoskeletal rearrangement of NRK-52E cells were most significant in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h , demonstrating the best model stability. Immunofluorescence results showed that compared with the NC group , the fluorescence intensity of the fibrous matrix protein Collagen I significantly increased in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h ( P < 0. 001) .
Conclusion:The protocol involves treating NRK-52E cells with 100 μmol/L CoCl2 for 9 h , following 3 h of recovery in CoCl2 -free medium. Repeating this cycle three times can establish an in vitro EMT model.
