摘要
该实验观察白藜芦醇(Resveratrol,Resv)对兔关节软骨细胞去分化现象的作用并探讨其可能的机制。首先,在无菌条件下取4周龄兔关节软骨细胞,行软骨细胞鉴定后将细胞随机分成5组:空白组、0μmol/L Resv组、2.5μmol/L Resv组、5μmol/L Resv组和10μmol/L Resv,体外单层培养传代至第5代。分别取P1、P3、P5代细胞在倒置相差显微镜下观察细胞形态变化,倒置相差显微镜下观察到各组P1代软骨细胞形态基本一致,P3、P5代2.5μmol/L Resv组细胞形态变化不大,其余各组细胞出现胞体变大,胞浆变淡,镜下胞核不明显等衰老的形态变化;Alcian Blue染色检测蛋白聚糖(Aggrecan)含量和β-半乳糖苷酶染色检测衰老软骨细胞,各组P1代细胞Alcian Blue染色和β-半乳糖苷酶染色无明显差别,P3、P5代2.5μmol/L白藜芦醇组较其他组明显深染、衰老软骨细胞数明显减少;RT-PCR检测蛋白多糖(Aggrecan),沉默信息调节因子1(SIRT1),抑癌基因p53、p21以及P16的表达,2.5μmol/L Resv组的Aggrecan和SIRT1基因表达明显上调,P53、P21及P16基因表达显著下调。综上所述:低浓度白藜芦醇(2.5μmol/L)可能通过上调SIRT1,下调P53、P21、P16基因的表达抑制软骨细胞去分化的现象。
In this study,the role of resveratrol on dedifferentiation of rat chondrocyte and its possible mechanism were investigated.Cartilage cells was harvested under sterile conditions from normal rabbit′s joint of 4 weeks old rabbit and was then identified.The experiment were randomly divided into five groups：control group,0 μmol/L Resv group,2.5 μmol/L Resv group,5 μmol/L Resv group and 10 μmol/L group.The rabbit articular chondrocytes were subcultured in vitro to the 5th generation （named P1-P5）.Inverted microscope was used to observe cell morphology at 1,3 and 5 generation respectively.The results indicated that the morphology at passage 1 chondrocytes were similar.The morphology of 2.5 μmol/L Resv group at passage 3 and 5 were nearly the same with passage 1,but other groups became enlarged,cytoplasmic stained light and the nucleus were not obvious;Alcian Blue staining was used to observe the changes of Proteoglycans.The results of Alcian Blue staining showed that the poly polysaccharide protein expression of 2.5 μmol/L Resv group were higher than other groups; β-galactosidase staining were used to identify the ageing cells.The results showed that the number of aging cells of 2.5 μmol/L Resv group was lower than other groups.RT-PCR were adopted to detect the RNA expression level of Aggrecan gene,SIRT1 gene,p53 gene,p21gene and P16 gene.RT-PCR analysis showed that SIRT1 gene of2.5 μmol/L Resv was significantly upregulated,and P53,P21 and P16 gene was downregulated.In conclusion,this experiment showed that 2.5 μmol/L Resv can inhibit the dedifferentiation of chondrocytes via upregulating expression of SIRT1 gene and downregulating expression of P53,P21 and P16.
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2017年第1期7-13,21,共8页
Natural Product Research and Development
基金
国家自然科学基金(81160226)
