Sox9基因在终板软骨细胞退变模型中的表达变化及意义

目的 建立大鼠腰椎终板软骨细胞体外自然退变模型,并探讨Sox9基因在终板软骨细胞自然退变中的变化及作用.方法 取大鼠腰椎终板软骨细胞,酶消化及自然传代法分离培养大鼠终板软骨细胞,建立体外终板软骨细胞培养模型.采取绘制细胞生长曲线、HE、甲苯胺蓝染色及免疫组织化学染色等方法,对该模型进行鉴定.RT-PCR检测各代细胞中Sox9、Ⅱ型胶原mRNA的表达变化.结果 大鼠终板软骨细胞表达特征性Ⅱ型胶原,其生长情况及细胞表型类似于关节软骨,细胞传至4、5代后表现出细胞形态呈梭形、细胞增殖速度减慢等退变的特征.与原代相比,Sex9基因mRNA在4、5代终板软骨细胞的表达明显下降(P<0.05),受其调控的Ⅱ型胶原mRNA的表达也相应降低,两者表达变化呈正相关(r=0.912,P<0.05).结论 终板软骨细胞自然退变模型成功建立,为椎间盘退变机制研究提供了较好的细胞学基础.Sox9基因与终板软骨细胞自然退变存在明显关联,Sox9基因的表达下降可抑制软骨终板细胞的增殖及基质合成,从而促进或诱发椎间盘的退变.

Establishment of an in vitro model of natural degeneration of lumbar endplate chondrocytes and expression of Sox9 therein

Objective To establish an in vitro model of natural degeneration of lumbar endplatechondrocytes and explore the role and the expression change of Sox9 gene, an important gene in thedifferentiation and maturation of chondrocyte, in the process of natural degeneration of endplatechondrocytes. Methods The lumbar vertebrae of 35 SD rats were taken out to obtain the endplates.Endplate chondrocytes were isolated by enzyme digestion and cultured so as to establish an in vitro naturaldegeneration model of chondrocytes. The morphological appearances and biological characteristics of thechondrocytes of different generations were observed by HE staining, immunocytochemical staining andtoluidine blue et cetera; RT-PCR was used to detect the mRNA expression of Sex9 gene and type Ⅱ collagenin differential generations. Results The lumbar cartilaginous endplate chondrocytes of rat expressedcollagen Ⅱ, and it's phenotype and biological characteristics were similar to those of articular cartilage cells.When the cells were passaged to the forth or fifth generations they were fusiform and their proliferative speeddecreased. Compared with the primary generation chondrocytes, the expression of Sox9 mRNA in the forthand fifth generation chondrocytes was markedly decreased (P < 0.05). And the mRNA expression level oftype Ⅱ collagen, regulated by Sox9 gene, decreased too (P < 0.05). The mRNA expression of Sox9 waspositively correlated with the mRNA expression of type Ⅱ collagen (r = 0.912, P < 0.05). Conclusion Amodel of natural degeneration of lumbar endplate chondrocytes has been established successfully, thusproviding a good cytological basis for the study of degeneration of lumbar endplate. Sox9 gene may play a rolein the process of natural degeneration of endplate chondrocytes.