基于单细胞组学的松果体细胞亚群分析及其在脑损伤节律紊乱中的作用与机制研究

新生儿缺氧缺血性脑病(HIE)是常见的围产窒息后严重的并发症，至今仍缺乏特异性诊疗手段。申请人前期研究发现：伴随松果体损伤的HIE患儿，常有昼夜节律紊乱，预后不良。但机制不明。由于松果体组织中细胞量极少，异质性高，基于基因芯片或转录组测序结果差异较大，因此，我们利用单细胞测序分析了新生大鼠缺氧缺血性脑损伤（HIBD）松果体各细胞亚群基因图谱，发现：HIBD后24h和72h，松果体细胞主体由β亚群转变为α亚群；并筛选出差异表达基因Upf3b。本项目拟采用单细胞测序技术结合时序研究明确松果体中主要细胞亚群对HIBD损伤的动态响应及调控机制，尤其是松果体细胞中β和α亚群转变在松果体功能损伤和修复中的作用和机制，并就潜在标志基因Upf3b病理生理作用进行鉴定。本项目的实施对认识HIE后松果体损伤修复及生物节律调控机制具有重要意义。

Hypoxic-ischemic encephalopathy (HIE), a severe syndrome occurred during laboring, lacks specific diagnosis markers and effective treatments. Our previous clinical studies indicated that circadian disorders accompanying pineal gland injuries widely occur in patients with HIE. However, due to the high heterogeneity and low cell numbers of the pineal gland, previously performed analysis by microarray and/or RNA-Seq often led to highly variable results. To our knowledge, no systematic analysis of HIE evoked transcriptional changes in the pineal gland has been reported, so we sought out to apply single cell RNA sequencing to explore transcriptional changes of multiple cell types in the pineal gland of neonatal rat with HIBD. Our preliminary data showed a subtype conversion between alpha and beta pinealocytes at 24h and 72h post HIBD. In this proposal, we will first characterize transcriptional changes of different populations of pineal cells post HIBD, primarily focusing on the role of subtype conversion between alpha and beta pinealocytes in the pathophysiology and circadian functional recovery post HIBD. Next, we will investigate the pathophysiological roles of Upf3b in pinealocytes. Our study will therefore provide new insights into the pathophysiological mechanisms of circadian disorders and potential therapeutic target after HIE.