【作者】 陈键；

【导师】 王金福； 李莹辉； 戴钟铨；

【作者基本信息】 浙江大学 ， 细胞生物学， 2016， 博士

【摘要】 长期空间飞行过程中,由于重力的消失迫使机体的多种组织和器官去适应新的微重力环境,从而产生一系列的适应性变化,称之为“空间适应综合症”。这些航天医学问题已成为人类长期太空探索的重要障碍之一,主要包括骨丢失、认知功能损伤、肌肉萎缩、心血管功能紊乱等。航天微重力环境对航天员的生理健康和任务执行造成了严重影响,如何对这些影响进行监测和防护并揭示其机制,是航天医学研究的首要任务。开发简单有效的检测指标对于航天员的生理健康监测具有重要意义。同时,以系统生物学的思维来探索微重力对机体动态的、全身性的影响,将是航天医学问题研究的有效手段。本论文以尾吊大鼠作为主要的动物模型,通过对血清miRNAs表达谱的研究来探索模拟微重力对机体的影响,获得以下主要结果：首先对血清miRNAs的内参基因进行了筛选和验证。通过miRNAs芯片筛选,并结合多批次、大样本的qPCR及2个专业内参筛选软件(geNorm和Normfinder)的运算分析,确定了miR-25-3p是最稳定的内参基因。进一步分析表明,miR-25-3p是一个在不同物种间高度保守的基因。它在大鼠尾吊、猕猴卧床等模拟微重力模型,以及大鼠OVX、绝经后骨质疏松等模型中的表达均有很好的稳定性,从而最终确定miR-25-3p可作为这些模型中稳定的血清miRNAs内参基因。这些结果为后续开展的的血清miRNAs研究奠定了基础。随后我们对失重性骨丢失和绝经后骨质疏松的血清miRNAs标志物进行了筛选。利用大鼠OVX血清miRNAs芯片技术高通量筛选差异表达的miRNAs,并经qPCR验证得到15个下调的miRNA。进一步结合临床样本检测后发现,其中miR-30b-5p在骨质减少和骨质疏松病人血清中都显著下调,miR-103-3p、 miR-142-3p和miR-328-3p在骨质疏松病人血清中显著下调。偏相关分析发现它们与H-BMD之间均存在显著的正相关,而且miR-30b-5p和miR-142-3p还同时与]N-BMD显著相关。这4个niRNAs可作为绝经后骨质疏松的生物标志物。利用猕猴卧床实验对模拟失重性骨丢失的标志物进行了鉴定,结果发现miR-30b-5p、miR-103-3p和miR-142-3p在卧床后猕猴血清中的表达显著下降,ROC曲线分析表明它们有很好的诊断价值,但miR-328-3p未发生显著变化。从而确定miR-30b-5p、miR-103-3p和miR-142-3p可作为模拟失重性骨丢失的标志物。miRNA表达谱和代谢组学数据都包含了生理变化的重要信息,为了对数据进行深度挖掘,我们进行了二者的联合分析。血清miRNAs芯片和qPCR检测确定了13个在尾吊4周和5周后大鼠血清中均上调的miRNAs,pather和DAVID软件的分析表明它们与神经系统,尤其是海马高度相关。连续尾吊5周后,血清代谢组发生显著性变化,与变化miRNAs的预测靶基因进行联合分析发现,模拟失重对机体的神经系统尤其是海马区有显著影响。根据生物信息学分析结果,我们检测了尾吊大鼠海马组织miRNA、mRNA和蛋白的表达,发现miR-383-5p的表达显著上调,同时其预测靶基因AQP4的mRNA和蛋白水平均显著下调,推测它可能直接受miR-383-5p的靶向调控。通过体外双荧光素酶报告基因实验,确认了AQP4是miR-383-5p的靶基因。AQP4是调节脑组织水平衡的重要蛋白,也与海马的学习记忆和认知功能密切相关。因此,推测它的变化是微重力影响海马功能的潜在机制之一,并可能与微重力下的血液头向分布相关。综上所述,我们通过对血清miRNAs的研究发现：miR-25-3p是模拟微重力模型和骨质疏松模型稳定的内参基因；并筛选得到一组血清miRNAs可作为模拟失重性骨丢失和绝经后骨质疏松的潜在标志物；最后对模拟微重力导致的认知功能损伤进行了初步的机制探索。更多还原

【Abstract】 During long-term space flight, the organism is forced to adapt to the new microgravity environment for the absence of gravity. These adaptive changes of physiology that result from microgravity, including bone loss, cognitive impairment, muscle atrophy and so on, was named space adaptation syndrome and has been a major obstacle of human long-term space exploration.Microgravity seriously impacts the health and mission performance of astronauts. To monitor and counteract these negative effects and reveal the mechanism are the priority of space medical research. Developing simple and effective biomarkers is of great significance for the health monitoring of astronauts. Meanwhile, using a systems biology approach to investigate the dynamic and systematic effect of microgravity will be an effective method in space medical research. In this study, we used rat hindlimb unloading as main animal model to explore the effects of simulated microgravity by serum miRNAome. The main results are summarized as following:Firstly, we performed a systematic study on serum miRNAs to identify suitable reference gene for normalization. miR-25-3p was identified as the suitable reference gene through a large-scale screening using miRNA microarray, qPCR validation and statistical algorithms (geNorm and Normfinder). Further analysis showed that miR-25-3p was highly conserved among species, and its expression showed fairly stable in simulated microgravity model (hindlimb unloading rat and bedrest rhesus monkey) and osteoporosis model (OVX rat and postmenopausal woman). These results confirmed that miR-25-3p was a stable reference gene and provided foundation for the following serum miRNAs research.Then, we screened potential serum miRNAs biomarkers for the postmenopausal osteoporosis and the simulated microgravity-induced osteoporosis. miRNA microarray and qPCR were performed in OVX rats and 15 serum miRNAs were identified differentially expressed. We further detected that miR-30b-5p was significantly downregulated in serum of postmenopausal women with bone loss (osteopenia or osteoporosis), and miR-103-3p, miR-142-3p, miR-328-3p were significantly downregulated in osteoporosis. Multiple correlation analysis showed that these miRNAs were significantly positive correlate with H-BMD, meanwhile, miR-30b-5p and miR-142-3p were significantly associated with FN-BMD. These miRNAs were potential biomarker for postmenopausal osteoporosis. To investigate the potential biomarkers for simulated microgravity induced osteoporosis, we detected these miRNAs in the serum of rhesus monkeys with or without long-duration bedrest. miR-30b-5p, miR-103-3p and miR-142-3p were significantly downregulated after bedrest, while miR-328-3p had no significant change. These three miRNAs were potential biomarkers for simulated microgravity induced osteoporosis and showed considerable diagnostic value by ROC analysis.To gain better insight into the impacts of microgravity on organism, an association analysis was performed on serum miRNAome and metabonomics data to provide clue and direction for the following research. miRNAs microarray and qPCR identified that 13 miRNAs were upregulated in the serum of rat with hindlimb unloading for 4w or 5w and highly correlated with nervous system especially hippocampus by the analysis of Panther and DAVID. Moreover, the serum metabolites showed the significant changes after hindlimb unloading for 5w. The association analysis of serum miRNAome and metabonomics showed that the simulated microgravity had important effect on nervous system especially hippocampus.According to the bioinformatics analysis, we detected the expression of miRNAs, mRNAs and proteins in hippocampus. miR-383-5p was significant upregulated after hindlimb unloading, while its predicted target gene AQP4 showed significant downregulated in both mRNA and protein level. We then confirmed that AQP4 is a target gene of miR-383-5p by dual luciferase reporter experiment in vitro. AQP4 plays important role in water balance of brain and is closely related to the cognitive function of hippocampus. We hypothesized that the downregulated of AQP4 is an underlying mechanism of microgravity impacting cognitive function of hippocampus and may associate with the head hyperemia under microgravity.Taken together, we identified that miR-25-3p is a suitable reference gene for simulated microgravity and osteoporosis model; A group of miRNAs screened in the serum could serve as non-invasive biomarkers in detection of postmenopausal osteoporosis and simulated microgravity-induced osteoporosis; Meanwhile, we explored that the underlying mechanism of microgravity impacts cognitive function of hippocampus.更多还原