线粒体ROS通过NAD+调控的线粒体分裂/融合在衰老细胞增殖抑制中的作用及机制研究

Reactive oxygen species(ROS) is closely related to aging,in which ROS-induced injury plays an essential role.ROS can stimulate cell proliferation.However, the ROS level is greatly increased while the cell proliferation and apoptosis are inhibited in the aged cell. Mitochondira is the main source of endogenous ROS.It has been found that the size of mitochodria in aged cell is significantly enlarged, and our previous study also show that the protein mitofusin 2(Mfn2) level is greatly increased,suggesting the imbalance of mitochondrial fission and fusion in the aged cell.Studies show that Mfn2 can inhibit the cell proliferation and apoptosis, as well as the increasd ROS.Based on the literatures and our previous studies, we hypothesize that mitochondrial fission/fusion regualted by mitochondrial ROS through NAD+ pathway plays a key role in the proliferation inhibition of aged cell. In the present study,we will employ the natural aging models both in vivo and in vitro.The specific gene knockout mice and molecular techniques will be used to regulate the targeted proteins to demonstrate our hypothesis and the detail molecular pathways.This study will benefit to understand the molecular mechanism of aging.

活性氧（ROS）与衰老密切相关。ROS损伤是促进衰老的重要机制，同时ROS也能刺激细胞增殖。但在衰老细胞中，ROS水平显著升高，而细胞增殖却显著降低，并且出现凋亡抵抗的矛盾现象。线粒体是正常细胞内源性ROS的最主要来源。研究发现，衰老细胞线粒体形态普遍不规则增大，而我们课题组前期工作也发现，线粒体融合蛋白（Mfn2）表达显著升高，提示衰老细胞线粒体分裂/融合的动态平衡出现了偏移。而研究表明，Mfn2可以通过多途径抑制细胞增殖和凋亡，并且能够反馈抑制ROS水平。根据线粒体分裂/融合调控机制等最新进展并结合课题组前期工作，我们提出了"线粒体ROS通过NAD+调控线粒体分裂/融合平衡偏移抑制衰老细胞增殖"的课题新设想。本项目拟采用离体和在体自然衰老模型，利用基因敲除小鼠和多种分子生物学干预手段，系统证明该设想的正确性并阐明其中的具体分子信号通路，为揭示衰老分子机制提供新思路和实验室数据。

活性氧（ROS）与衰老密切相关。ROS损伤是促进衰老的重要机制，但具体分子机制并不清楚。线粒体是正常细胞内源性ROS的最主要来源。研究发现，衰老细胞线粒体形态普遍不规则增大，提示衰老细胞线粒体分裂/融合的动态平衡出现了偏移。结合文献和课题组的研究结果，我们认为线粒体来源的ROS通过调控线粒体分裂/融合平衡偏移而发挥调控衰老的作用。我们通过细胞和动物实验，证实了课题的基本设想。发现在衰老过程中，线粒体分裂/融合蛋白特别是线粒体融合蛋白2（Mfn2）发生明显的升高，引起线粒体融合增加。此外NAD/NADH比例降低，衰老相关指标变化明显。通过RNA干扰等技术发现Sirt1可以影响PGC-1β、Mfn2的水平，而线粒体来源的ROS是影响衰老的最主要因素。我们通过联合表达靶向线粒体的抗氧化酶SOD2和M-CAT，有效降低线粒体ROS和细胞整体ROS水平，同时联合过表达靶向线粒体抗氧化酶可以显著抑制升高的Mfn2的表达，提高NAD/NADH的比例，改善衰老相关指标，从而发挥抗衰老作用。我们的研究结果表明，线粒体来源的ROS通过影响NAD+水平，影响Sirt1，进一步调节PGC-1β和Mfn2，从而调节线粒体分裂/融合偏移，调控衰老。这些结果为阐明衰老分子机制提供了新的实验室数据，也为将来的可能的抗衰老研究提供了新的干预靶点。