The analysis base study on mechanical double enzyme technique for isolating and culturing primary chondrocytes
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PROVIDER: S-EPMC10630635 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:ObjectiveTo study the effect of co-culturing chondrocytes with osteoblasts on hypoxia-inducible factor (HIF)-1 pathway of chondrocytes and its mechanism.MethodsChondrocytes and osteoblasts were separately extracted from the knee joint and skull of newborn mice by trypsin digestion. The co-culturing system of osteoblasts and chondrocytes was constructed by using Transwell inserts to culture the osteoblasts and 6-well plate to culture the chondrocytes. We used qRT-PCR to examine changes in the mRNA expression of HIFs and its target gene pyruvate dehydrogenase kinase 1 ( PDK1) in chondrocytes co-cultured for 24 h. Western blot was used to analyze changes in the protein expression of HIFs and PDK1 and the changes in the activation of mitogen activated protein kinase (MAPK) signaling pathway after the cells were co-cultured for 48 h. Reactive oxygen species (ROS) staining was done to show the changes of ROS production in chondrocytes co-cultured for 48 h.ResultsThe results of qRT-PCR and Western blot showed upregulated levels of HIF-1α gene and protein expression ( P<0.05) in the chondrocytes after they were co-cultured with osteoblasts. The gene and protein expression levels of PDK1 , the target gene of HIF-1, were also upregulated ( P<0.05). ROS staining showed that co-culturing of chondrocytes with osteoblasts decreased ROS production in chondrocytes. Western blot revealed that extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling of co-cultured chondrocytes were enhanced ( P<0.05).ConclusionCo-culturing with osteoblasts enhanced the ERK1/2 and p38 signaling of chondrocytes and upregulated the HIF-1 pathway of chondrocytes.
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