TOM7在糖尿病大血管内皮损伤中的作用和机制研究

Endothelial cell damage is the initiative process of diabetic cardiovascular complications which is the most important factor relating to the death of diabetic patients. Translocase of the out membrane (Tom) which is the vital protein regulating the import of mitochondrial protein encoding by nuclear genome plays an important role in the regulation of mitochondrial function, oxidative damage and cell-aging. Tom7 which is one of the sub-units of Tom take a part in assembling of Tom sub-units and maintaining it stability. Our research-group has detected that Tom especially the Tom7 were reducing by the stimulation of high concentration of glucose (HG) when we researched in the relationship of HG and oxidative damage in vascular-endothelial cell. As the fold change of Tom7 is the most significantly one，we propose that Tom7 may be the key protein in the process of protectiing endothelial cell from damage induced by HG. In order to clarifying the function of Tom and Tom7 in vascular-endothelial cell damage induced by diabetes, in this project we plan to research in the following four parts including the relationship of Tom7 and glucose, the interaction of TOM7 and Bcl/Bax, the effect of NOX4 on TOM7 and the change of Tom7 and other sub-units of Tom under high concentration of glucose. These may provide new therapeutic targets and the points of penetration for the prevention and cure of diabetic vascular diseases.

糖尿病大血管并发症是糖尿病患者的首要致死原因，内皮细胞损伤是血管并发症的始动环节。线粒体外膜易位酶（Tom）是核基因编码的线粒体蛋白进入线粒体的关键蛋白，Tom在细胞的线粒体功能调节、氧化应激损伤、细胞衰老等过程发挥了重要作用。Tom7是Tom的亚基之一，主要负责Tom各亚基装配及结构稳定性调节。课题组在前期探究大血管内皮细胞中高糖与氧化损伤的关系的研究中发现，Tom亚基在高糖刺激的内皮细胞中显著下调，其中Tom7是表达差异最大的亚基，因此提出假设：上调Tom7水平可能对高糖导致的血管内皮损伤发挥保护作用。本项目计划通过探究Tom7和高糖导致的内皮损伤的关系、Tom7和凋亡相关Bcl-2/Bax蛋白的相互作用、NOX4对Tom7的影响以及Tom7与Tom其余亚基在高糖作用中的变化等四部分内容，以期阐明Tom7及Tom在糖尿病血管病变中的作用，为糖尿病大血管病变的防治研究提供新的靶点。

糖尿病大血管并发症是糖尿病患者的首要致死原因，内皮细胞损伤是血管并发症的始动环节。线粒体外膜易位酶（Tom）是核基因编码的线粒体蛋白进入线粒体的关键蛋白，Tom在细胞的线粒体功能调节、氧化应激损伤、细胞衰老等过程发挥了重要作用。Tom22是Tom的中心受体，负责核编码线粒体蛋白进入线粒体的易位和输入，对维持正常的线粒体功能至关重要。课题组在前期探究大血管内皮细胞中高糖与氧化损伤的关系的研究中发现，Tom亚基在高糖刺激的内皮细胞中显著下调，其中Tom22是表达差异最大的亚基，因此提出假设：Tom22水平上调可能对高糖导致的血管内皮损伤发挥保护作用。.本项目通过胶原酶消化分离人脐静脉内皮细胞，用不同浓度的葡萄糖溶液和孵育时间处理细胞，发现Tom22的mRNA和蛋白水平均显著下降，线粒体融合蛋白Mfn1显著减少，且线粒体裂解增加，氧化磷酸化相关蛋白表达也减少，ATP水平和膜电位显著降低，凋亡增加。慢病毒转染构建Tom22过表达内皮细胞发现Tom22能逆转高糖引起的线粒体裂解增加，Mfn1和氧化磷酸化蛋白表达增加，ATP水平和膜电位升高，凋亡减少。CO-IP显示Tom22与Mfn1蛋白直接作用，敲低内皮细胞Mfn1表达的影响类似于下调Tom22表达，而过表达Mfn1可以逆转高糖引起的变化，改善ATP水平和膜电位，减少凋亡。.我们得出结论：高糖通过减少Tom22的表达引起线粒体融合和氧化磷酸化系统受损，导致内皮细胞功能障碍和凋亡增加，其机制可能通过减少下游效应因子Mfn1介导。本项目通过探究Tom22和高糖导致的内皮损伤的关系、Tom22在线粒体动力学和氧化磷酸化方面的机制，阐明了Tom22及Tom在糖尿病血管病变中的作用，为糖尿病大血管病变的防治研究提供新的靶点。