线粒体氧化应激信号通路参与糖基化终末产物诱导椎间盘细胞凋亡的机制研究

Diabetes mellitus (DM) has been clinically proved as a risk factor of intervertebral disc (IVD) degeneration. Elevated glucose concentration and the accumulation of intracellular advanced glycation end products （AGEs）could not only directly affect the activity of disc cell, but also indirectly induce increased mitochondrial oxidative stress levels significantly in the cells. Abnormal high mitochondria oxidative stress level was the important pathway of the IVD cell apoptosis by external factors. Furthermore, excessive apoptosis is an important mechanism of IVD degeneration. So we deduced that mitochondrial oxidative stress pathways likely plays an important role in disc cells apoptosis induced by AGEs. But there have not been reported systematically about the influence of AGEs on the oxidative stress level in IVD cells and the correlation with IVD cells apoptosis, even the IVD degeneration. Nucleus pulposus cells and annulus fibrosus cells play a main role in nutrition, development and repair of IVD. The topic of this study proposed to build the rat hyperglycemia model of intervertebral disc cells in vivo and vitro respectively, so as to implore the role of mitochondrial oxidative stress pathways in IVD cells apoptosis induced by AGEs and to investigate the mechanism of mitochondrial oxidative stress level and cellular apoptosis influened by hyperglycemia in disc cells. It will provide new thought and theory basis for further clarifying the mechanism of intervertebral disc degeneration and prevention and treatment of intervertebral disc degeneration.

糖尿病已被临床证实为椎间盘退变的重要诱因。体内高血糖水平和细胞内糖基化终末产物的积聚不仅能直接影响椎间盘细胞活性，且能间接诱发细胞内线粒体氧化应激水平明显升高。细胞内线粒体氧化应激水平的异常升高是外界因素诱发椎间盘内细胞凋亡的重要通路。而过度凋亡是椎间盘退变的重要机制。由此我们推论，线粒体氧化应激通路很可能在糖基化终末产物诱发的椎间盘细胞凋亡中扮演着重要角色。但糖基化终末产物对椎间盘内细胞线粒体氧化应激水平的影响及其与椎间盘细胞凋亡乃至椎间盘退变的相关性未见系统报道。髓核细胞及纤维环细胞是对椎间盘营养、发育及修复起主要作用的细胞。本课题拟分别构建鼠椎间盘细胞的体内外高糖模型，考察线粒体氧化应激信号通路在糖基化终末产物诱导的椎间盘内细胞凋亡中的作用；进而探讨高糖环境影响椎间盘细胞氧化应激水平及诱导细胞凋亡的机制，将为进一步阐明椎间盘退变机制及防治椎间盘退变的发生提供新的思路及理论依据。

目的：最近研究表明糖基化终末产物（AGEs)的大量聚集被证实为是糖尿病椎间盘退变的重要因素。本研究的目的是评估AGEs诱导椎间盘细胞后细胞凋亡及线粒体氧化应激通路变化情况，进一步探讨线粒体氧化应激通路在AGEs诱导的椎间盘细胞凋亡中的作用机制。.方法：椎间盘细胞经过3天的AGEs处理后，用CCK-8及Edu标记实验检测细胞增殖情况；细胞凋亡用Annexin V/PI 凋亡检测试剂盒及 Hoechst 33342进行检测； Western blotting检测凋亡相关蛋白表达（Bax, Bcl-2, cytochrome c，caspase-3 and caspase-9）； RT-PCR检测Bax及Bcl-2基因表达变化。线粒体膜电位及细胞内活性氧分别使用JC-1及DCFH-DA荧光探针标记后检测。应用抗氧化剂干预后观察线粒体膜电位及细胞凋亡率。.结果：研究结果显示AGEs可明显抑制椎间盘细胞增殖且能诱导椎间盘细胞凋亡；分子生物学检测结果提示AGEs明显上调Bax基因表达及抑制Bcl-2基因表达。此外，AGEs显著增加细胞色素C的释放及增强Caspase-9及Caspase-3的激活。而且经过AGEs干预后椎间盘细胞线粒体膜电位明显降低、细胞内活性氧大量聚集。抗氧化剂N-acetyl-L-cysteine能明显逆转AGEs诱导的线粒体膜电位及降低椎间盘细胞凋亡。.结论：这些研究结果提示AGEs能诱导椎间盘细胞凋亡，且线粒体氧化应激通路可能参与了AGEs介导的椎间盘细胞凋亡中，这将为研究糖尿病椎间盘退变提供了一定的理论基础。