The Research Of Resveratrol In Inhibiting Dedifferentiation Of Rabbit’s Cartilage Cell In Vitro

ObjectiveTo study the effect of resveratrol on dedifferentiation of rabbit articular cartilage cells when monolayer cultured in vitro and to explore the mechanism of resveratrol in inhibting dedifferentiation of rabbit articular cartilage cells monolayer cultured in vitro.MethodsTrypsin and type II collagenase were used to isolate primary rabbit articular chondrocyte separated from normal rabbit’s hip joint and then cultured.Alcian Blue staining was used to identify chondrocyte.The experiments were randomly divided into five groups: control group,0μM Resv group,2.5μM Resv group,5μM Resv group and 10μM Resv group,total RNA and protein were extracted when cell reached80% fusion at 1,3 and 5 generation respectively.CCK-8 assay was used to detect the absorbance of chondrocytes in 450 nm of each group in each generation and the growth curve were drawed.β-galactosidase(senescence-associated β-galactosidase,SA-β-gal)staining were used to identify dedifferentiated cells,inverted microscope was used to observe cell morphology everyday,RT-PCR and western blot were adopted to detect the expression level of protein polysaccharide Aggrecan gene,Sirt-1gene,p53 gene,p21 gene and P16 gene.ResultsThe cell cultured were identified as rabbit articular chondrocytes by Alcian Blue staining and morphological observations.The results of morphology,SA-β-gal and Alcian Blue staining showed that rabbit articular chondrocytes were prone to dedifferentiating when monolayer cultured continuously in vitro.whereas,resveratrol can inhibit this process under certain ranges of concentrations and the trend became more obvious after more passages.The results of Alcian Blue staining showed that the poly polysaccharide protein expression levels of 2.5μmol/L Resv group were significantly increased than that in other groups,and so that of β-galactosidase staining in aging cells.RT-PCR analysis showed that Resv significantly upregulatedSirt1 gene expression level,and reduced P53,P21 and P16 gene expression level,inhibiting cell dedifferentiation promoting cell proliferation under low concentration of Resv.ConclusionRabbit articular chondrocytes were prone to dedifferentiating when monolayer cultured consecutively in vitro,and this phenomenon was associated with senescence and could be inhibited by resveratrol via promoting the synthesis of extracellular matrix,inhibiting the dedifferentiation of chondrocytes,upregulatingexpression level of Sirt1 gene.