自噬在不同年龄大鼠终板软骨中的变化

Change of autophagy in endplate chondrocytes of rats during aging process

目的 观察不同月龄SD大鼠终板软骨中自噬的变化及其意义.方法 取3、6、12月龄SD大鼠的原代终板软骨细胞,分为自然培养组和雷帕霉素组.茜素红染色观察细胞形态,实时聚合酶链反应和免疫印迹检测软骨标志基因Ⅱ型胶原、SOX-9、蛋白多糖转录因子和基质分解酶相关基因MMP-13以及自噬相关基因Beclin-1和LC3基因的表达,单丹磺酰戊二胺（MDC）染色观察细胞的自噬率,四甲基偶氮唑盐（MTT）法检测细胞存活率.结果 茜素红染色显示细胞呈梭形改变,蛋白多糖转录因子、Sox-9和Ⅱ型胶原基因表达呈年龄依赖性降低趋势（P〈0.05）,提示随年龄增长,终板软骨细胞出现退变;自噬相关基因Beclin-1和LC3在各组终板软骨细胞中均有表达,且呈年龄依赖性降低趋势[（1.0±0.1）比（0.6±0.1）,（1.0±0.1）比（0.3±0.2）;（1.0±0.2）比（0.4±0.1）,（1.0±0.2）比（0.1±0.1）]（P〈0.05）,自噬发生率也呈现明显降低（P〈0.05）.自然培养组中,细胞存活率呈逐渐降低趋势（P〈0.05）;与自然培养组相比,雷帕霉素组中各组细胞存活率明显增加（P〈0.05）.结论 自然增龄大鼠中,终板软骨细胞逐渐出现退变,且存活率降低;上调其自噬水平,可提高细胞存活率,提示呈年龄依赖性降低的自噬活性,可能与椎间盘的自然退变有关.

Objective To explore the expression and significance of autophagy in endplate cartilage of rats during aging process. Methods The end-plate chondrocytes were isolated from 3, 6 and 12-month SD rats respectively. And the natural culture and rapamyein groups were assigned. Alizarin red staining was used to observe the morphological changes of cells. And RT-PCR was employed to detect the expressions of type Ⅲ collagen, proteoglycan, SOX-9 and matrix metalloproteinase （MMP-13）. The expressions of Beclin- I and LC3 were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The rate of autophagy was observed by monodansylcadaverine （ MDC ） staining and methyl thiazolyl tetrazolium （MTT） for cell survival rate. Results Alizarin red staining showed that cells might reflect the process of intervertebral disc degeneration. The expressions of polysaecharide, Sox-9, type Ⅱ collagen, Beclin-1 and LC3 in endplate chondrocytes significantly decreased with advancing age （ P 〈 0. 05 ）. The incidence of autophagy significantly decreased （ P 〈 0. 05 ）. The cell viability of each group significantly decreased （ P 〈 0. 05 ） . Compared with control group, the cell viability of rapamycin group significantly increased （ P 〈 0.05 ）. Conclusion During aging process, the expressions of autophagy related-gene LC3 and Beclin-1 significantly decrease with the reduced activity of end-plate ehondroeyte. And autophagy activity may be correlated with the development and degeneration of intervertebral disc.