AMP活化蛋白激酶在终板软骨细胞体外传代培养中的表达及意义

Expression of AMP-activated protein kinase in subcultured rat endplate chondrocytes

背景:终板软骨功能障碍是椎间盘退变的始动因素,AMPK在软骨的形成和降解过程中也具有相应的调节作用。目的:观察AMP活化蛋白激酶(AMPK)在终板软骨细胞体外自然传代退变模型中的作用。方法:分离大鼠腰椎终板软骨细胞,体外自然传代培养,取原代细胞(P0)、第2代细胞(P2)及第5代细胞(P5),倒置显微镜及细胞骨架染色观察终板软骨细胞形态的变化,甲苯胺蓝染色观察终板软骨细胞表型的改变;MTT法检测3组细胞的增殖能力;通过Real time-PCR评价3组终板软骨细胞中软骨标志基因(Ⅱ型胶原、蛋白多糖、SOX-9)以及基质金属蛋白酶3和13的表达变化,Western blot检测终板软骨细胞中AMPK蛋白磷酸化的改变。结果与结论:(1)随着传代次数的增加终板软骨细胞形态发生改变,增殖能力降低,软骨细胞表型逐渐丢失;(2)P5代终板软骨细胞中Ⅱ型胶原、蛋白聚糖及SOX-9基因表达明显下调(P<0.05或0.01),而基质金属蛋白酶3,基质金属蛋白酶13基因表达较P0代及P2代均明显增高(P<0.01);(3)Western blot结果显示P5代终板软骨细胞中AMPK蛋白磷酸化水平明显降低。提示:AMPK磷酸化的水平与终板软骨细胞体外自然传代过程中软骨细胞的退变密切相关,调控AMPK活性有可能减轻或阻止终板软骨及椎间盘的退变。

BACKGROUND：Endplate cartilage degeneration initiates intervertebral disc degeneration. AMP-activated protein kinase （AMPK） regulates the formation and degradation of cartilage. OBJECTIVE：To explore the role of AMPK in an in vitro natural degeneration model of chondrocytes derived from endplate of rat intervertebral discs. METHODS：Morphology of in vitro subcultured endplate chondrocytes of rat intervertebral discs at passages 0, 2, and 5 were observed under an inverted microscope fol owing cytoskeleton staining. Chondrocyte phenotype, proliferation, and the cartilage marker genes （type II col agen, proteoglycan, SOX-9, matrix metal oproteinase-3 and-13）, and AMPK phosphorylation were determined by toluidine blue staining, MTT assay, real-time PCR analysis, and western blot assay, respectively. RESULTS AND CONCLUSION：The altered morphology, decreased proliferation ability, and phenotype loss were observed in chondrocytes with increased passage number. Gene expression of type II col agen, proteoglycan, SOX-9 was significantly decreased;while gene expression of matrix metal oproteinase-3 and-13 was significantly increased in endplate chondrocytes at passage 5 compared with those at passages 0 and 2. AMPK phosphorylation in endplate chondrocytes at passage 5 was significantly decreased. These findings indicate that AMPK phosphorylation is involved in in vitro natural degeneration of chondrocytes derived from the endplate of rat intervertebral discs, and the degeneration of endplate chondrocytes and intervertebral discs can be inhibited through the regulation of AMPK activity.