Objective: This study aimes to investigate whether the senescence and apoptosis of rat endplate chondrocytes during in vitro monolayer culture can imitate that in vivo and whether the insulin-like growth factor-I(IGF-I)can slow down that process.Methods: Primary cells,in this experiment,were isolated from the lumbar spine endplate cartilage of young rats.Passage 2 cells passing from the Primary cells were selected to investigate the following tests about the mechanism of phenotypic preservation of chondrocytes.The alteration of relative expression of m RNA,as the object of observation,was used to determine the dose-and time-dependent assay.The endplate chondrocytes cultured in IGF-I(20ng/ml)were observed for the change of phenotype and signal pathway,finally using LY294002 and U0126 to block the PI3K/Akt,MAPK/ERK signal pathway respectively and observing the change of phenotype.Then we divided rats into three groups: young rats,adult rats and older rats.Histological staining was performed to examine the disc tissue morphology.Primary chondrocytes were isolated from the lumbar spine endplate cartilage of young rats.Then we treated the passage 2(P2)chondrocytes with IGF-I for 24 h,IGF-I enhances COL-2A m RNA expression in chondrocytes in a dose-dependent manner.From P2,the chondrocytes were passaged every third day,finally reaching P6,no matter whether IGF-I was used.Three cell staining methods were performed to examine the cell morphology and extracellular matrix.The main phenotypic protein and gene of chondrocytes were respectively examined by western blotting and RT-q PCR.And finally various strategies were used to examine the activation of AKT and ERK signaling pathways after the application of IGF-I in vitro.Results: Time-and dose-dependent assay indicated that the COL-2A expression of chondrocytes,cultured in IGF-I(20ng/ml)for 24 hours,could be increased as approximately 2.5 times as those not being cultured in IGF-I.After chondrocytes were cultured in IGF-I(20ng/ml)for 24 hours,we found that MMP-13 protein expression significantly reduced and Sox9 protein expression dramatically increased.What is more,phosphorylation of Akt protein and phosphorylation of ERK1/2 were strongest after chondrocytes being cultured in IGF-I for 15 min,then decreasing to bottom out after chondrocytes being cultured in IGF-I for 60 min and 30 min respectively.Furthermore,the increase in phosphorylated ERK protein was located in nucleus and cytoplasm,while phosphorylated Akt protein was mainly located in nucleus.The results indicated that cartilaginous end plates underwent age-related degenerative changes.20 ng/ml of IGF-I was the optimal concentration for rat's endplate chondrocytes to enhance the expression of COL-2A.With the passage increasing,monolayer culture chondrocytes showed significant phenotypic and morphological changes depending on the culture period.In accordance with that,IGF-I could maintain chondrocyte phenotype and activate the ERK and AKT signaling pathways during in vitro monolayer culture.Above all,the increased quantity of P-ERK and P-AKT was mainly located in the nucleus.Conclusion: The expression of COL-2A is stimulated by IGF-I via PI3K/AKT signal pathway,while Sox9 and MMP13 expression are stimulated by IGF-I both via MAPK/ERK signal pathway.To some extent,the senescence and apoptosis of chondrocytes cultured in vitro can be used to imitate those,caused by age,of chondrocytes in vivo,and IGF-I delays the process probably mediated by the ERK and AKT signaling pathways. |