摘要
目的通过大鼠终板软骨细胞体外自然传代的退变模型,探讨大鼠终板软骨细胞体外自然退变过程中Asporin基因的表达及意义。方法取大鼠腰椎终板软骨细胞,酶消化法及自然传代法分离培养大鼠终板软骨细胞,选取P1代及P5代,体外培养6d,在倒置显微镜下观察细胞形态,利用HE染色及甲苯胺蓝染色对软骨细胞进行观察及鉴定。用RT-PCR及Westernblot检测Ⅱ型胶原、蛋白多糖及Asporin基因变化。结果随着细胞传至P5代,细胞形态上有梭形变趋势,Ⅱ型胶原(P5/P1=0.111767,P=0.009842)及蛋白多糖表达(P5/P1=0.328965,P=0.002371)明显下调,Asporin蛋白表达明显上调。结论终板软骨细胞传至P5代,细胞发生自然退变,Asporin基因表达明显上调,提示Asporin基因表达与终板软骨的退变具有重要联系。
Objective To investigate Asporin gene expression and significance in rat endplate chondrocytes through an in vitro degeneration model by propagating rat endplate chondrocytes during a natural degeneration process.Methods Endplate chondrocytes were extracted from rat lumbar vertebrae,isolated by enzyme digestion and P1 and P5 generations selected for a 6-day in vitro culture.The specimens were photographed microscopically to observe the cellular differences by HE staining and toluidine blue staining.Type Ⅱ collagen,proteoglycan and Asporin are detected by RT-PCR and Western blot.Results Compared with P1 cells,P5 cells tended to assume a spindle-shaped morphology.Type Ⅱ collagen（P5/P1=0.111767,P=0.009842）and proteoglycan（P5/P1=0.328965,P=0.002371）were significantly reduced.Asporin protein expression was significantly increased.Conclusion P5 endplate chondrocytes undergo natural degeneration in vitro with the rising passage number.Asporin gene expression was significantly increased.It has an important relationship between endplate degeneration and expression of Asporin gene.
出处
《中国骨与关节外科》
2011年第6期479-483,共5页
Chinese Journal of Bone and Joint Surgery
基金
国家自然科学基金(30973025)
安徽省教育厅自然科学基金(KJ2010A320)
弋矶山医院人才基金(YR2009013)
