硒蛋白S介导蛋白激酶CβⅡ苏素化修饰在糖尿病血管内皮损伤中的作用及分子机制

Endothelial dysfunction is the initiating key step for diabetic macrovascular complications, and it is closely associated with vascular endothelial selenoprotein S (SelS) expression. Studies related to SelS role in vascular endothelial function currently mainly stay at in vitro cytological level, the evidence from in vivo studies remains insufficient. In our previous research, we found that overexpression of SelS could increase human umbilical vein endothelial cells (HUVECs) resistance to high glucose and correlated with protein kinase CβⅡ (PKCβⅡ) phosphorylation. Thus, on the basis of previous results, we intend to construct vascular endothelial specific SelS knockout mice model through utilizing Cre/loxP conditional knockout technology as foundation. Furthermore, the antiapoptotic role and mechanism of SelS-mediated PKCβⅡSUMOylation, recognized as another post-translational modification, in diabetic vascular endothelial dysfunction will be explored. Our results will provide a reliable and effective animal model for investigation on endothelial SelS function. Moreover, this study will not only elucidate the pathological mechanism of diabetic macrovascular complications but also offer solid experimental evidence for accelerating the development of SelS as a novel targeting antiapoptotic drug to treat diabetic macrovascular complications.

内皮功能紊乱是糖尿病大血管并发症的始动环节，与血管内皮硒蛋白S（SelS）的表达密切相关。目前针对SelS与血管内皮功能的研究主要停留在与机体内环境差别较大的体外细胞水平，尚欠缺体内研究证据。我们前期研究发现上调血管内皮SelS表达可抗凋亡保护人脐静脉内皮细胞（HUVECs）抵抗高糖损伤并与介导蛋白激酶CβⅡ（PKCβⅡ）磷酸化有关。因此，本课题在此基础上，以Cre/loxP条件性基因敲除技术为支撑，首次构建血管内皮特异性SelS基因敲除小鼠，并利用上述在体动物模型探讨SelS抗凋亡保护血管内皮的作用及可能涉及的调控PKCβⅡ苏素化（另一种蛋白质翻译后修饰形式）的分子机制。成果有助于为在在体动物水平研究血管内皮SelS的生物学功能提供一种可靠、有效的动物模型，深入阐释糖尿病大血管并发症发病机制，并为SelS作为靶向抗凋亡防治糖尿病大血管并发症的新药研发提供可靠的实验依据。

内皮功能紊乱是糖尿病大血管并发症的始动环节，与血管内皮硒蛋白S（SelS）的表达密切相关。我们前期研究发现上调血管内皮SelS表达可抗凋亡保护人脐静脉内皮细胞（HUVECs）抵抗高糖损伤并与介导蛋白激酶CβⅡ（PKCβⅡ）磷酸化有关。因此，本课题在此基础上，构建糖尿病小鼠模型，初步探讨SelS与糖尿病（DM）小鼠血管内皮功能紊乱及凋亡的关系；并应用DNA重组和RNA干扰技术构建SelS与小泛素相关修饰物蛋白1（SUMO1）过表达载体和小干扰RNA（siRNAs），探讨SelS抗凋亡保护血管内皮的作用及可能涉及的调控蛋白苏素化（另一种蛋白质翻译后修饰形式）的分子机制。结果显示，DM小鼠主动脉内膜粗糙、内皮脱落，内皮剪切型caspase3表达增加，同时伴有血管内皮内源性SelS表达增加。进一步，应用RNA干扰技术下调人主动脉内皮细胞（HAECs）SelS表达，发现SelS敲低后HAECs凋亡增多，剪切型caspase3（Cleaved caspase3）和B细胞淋巴瘤/白血病-2（Bcl-2）相关x蛋白（Bax）表达增加、Bcl-2表达减低；内皮功能紊乱相关指标细胞间黏附分子-1（ICAM-1）和血管细胞粘附分子-1（VCAM-1）表达升高、内皮型一氧化氮合酶（eNOS）表达降低；并证实上述作用在过表达SUMO1后减弱，而在沉默SUMO1表达后结果则与之相反。提示SelS抗凋亡保护血管内皮抵抗高糖损伤的作用与介导蛋白苏素化修饰水平有关，成果将深入阐释糖尿病大血管并发症的发病机制，并为SelS作为靶向抗凋亡防治DM大血管并发症的新药研发提供可靠的实验依据。

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