硒超营养致胰岛素抵抗的分子网络构建及关键基因解析

Recent studies, including our previous work, have found that long-term overnutrition of selenium induced insulin resistance through the regulation of some gene expressions, but the exact mechanism has not been fully elucidated. These findings have complicated recommendations for safe and reasonable selenium supplementation. The present application intends to establish selenium-induced insulin resistance models in inbred rats fed with a supranutritional level of selenium for two generations. RNA sequencing technique will be applied to screen the differentially expressed genes in the whole genome. A gene interaction and regulatory network of the differentially expressed genes will be built up by using bioinformatics approaches. The expression differences of key selenoprotein genes and node genes in the network will be confirmed by real-time quantitative PCR, and protein abundances of these key genes will be assayed with Western blot, ELISA, and enzymatic ways. Among the candidate genes whose mRNA and protein abundances are significantly different, two or three genes ranking the highest difference, but having unclear relationship with selenium overnutrition or insulin resistance, will be subjected to functional analyses with RNA interference technique in cell culture experiments. These results, from the perspective of systematic biology and nutrigenomics, are expected to help reveal the mechanisms of supranutritional selenium-induced insulin resistance, to advance new directions for further research, to develop a preliminary understanding of the functions of key genes involved in that pathology, and to broaden the knowledge of the control and prevention of insulin resistance-related diseases.

包括我们前期工作在内的新近研究发现：长期硒超营养可改变某些基因表达而致胰岛素抵抗，但详细机制未完全阐明，这给人们安全合理的补硒带来极大困扰。本项目拟以近交系大鼠为模型，以常规硒饲养为对照，超营养剂量硒饲养两代动物诱发胰岛素抵抗，以RNA测序技术从4种最相关组织筛选全基因组的差异表达基因，用生物信息学手段构建这些基因的互作与调控网络；结合实时荧光定量PCR技术确认网络中关键的硒蛋白基因和节点基因的表达差异，并以免疫印迹、酶联免疫、酶活性等蛋白质检测技术分析它们的蛋白质丰度。接着，从mRNA和蛋白质均呈显著差异表达的关键候选基因中，选取2-3个差异最为显著而又与待考察问题关系不明的基因进行体外细胞RNA干扰的功能研究。项目结果将有助于人们从系统生物学和营养基因组学的角度解开硒超营养致胰岛素抵抗的谜团，发现后续研究的新线索，初步认识部分关键基因在这其中的功能，拓展胰岛素抵抗相关疾病的防治思路。

经过1年紧张工作，用3种硒（Se）浓度饲料完成了第1代BALB/c雌鼠、第2代雄鼠和雌鼠动物实验，评价了胰岛素抵抗。在两代雌鼠妊前、妊末和授乳末及2代雄鼠成年时，进行了多阶段胰岛素耐受和葡萄糖耐量试验。雌鼠授乳结束、雄鼠成年后屠杀，检测了空腹血糖、胰岛素（FPI）和谷胱甘肽过氧化物酶活性。结果发现0.15、1.2和3.0 mg Se/kg饲料对非妊期小鼠体重无影响；3.0 mg Se/ke饲料致第1代和第2代雌鼠妊末出现外源性胰岛素清除障碍和糖耐量升高（P < 0.05），且使2代雌鼠妊前和授乳末出现外源性胰岛素抵抗；实验结束时，1.2和/或3.0 mg Se/kg饲养小鼠出现FPI和/或HOMA-IR增高趋势（0.05 < P < 0.1）。由此判定：Se超营养摄入是导致小鼠发生胰岛素抵抗的危险因素。.在花费较多时间完成配套经费筹集、公开招标等程序后，与华大基因公司签定检测服务合同，小鼠样本转录组测序与分子网络构建待春节后开展。在此之前，利用另一项研究中不同Se水平饲养大鼠的组织样品，用基因芯片对肝、骨骼肌和内脏脂肪组织（VAT）进行了全基因组表达谱分析，对差异基因进行了筛选、注释和分子互作网络分析，这些数据将与小鼠转录组测序分析结果相呼应，提高候选靶基因筛选的可靠性。该实验4组动物分别饲以0.0、0.25、3.0和5.0 mg Se/kg饲料，发现的差异表达基因中，肝中Sepp1、Selt、Selh、Selk、Gpx3、Gpx1和Dio1，骨骼肌中Txnrd1、Txnrd2、Sepw1、Selt和Selh，以及VAT中Sepn1和Gpx1为硒蛋白基因。分子互作网络显示：肝中氧化还原相关基因、骨骼肌和VAT中核糖体相关基因受影响最多。.综上，本课题完成了1年期研究内容，找到线索支持Se超营养摄入是胰岛素抵抗危险因素这一观点。大鼠基因芯片检测结果发现肝和骨骼肌这两个重要的糖代谢组织中存在多个硒蛋白基因的差异表达，并涉及相互作用的其它基因；同时，主持人与外国专家合作，就Se与糖代谢领域完成了1篇1.3万字的外文图书章节。