目的·基于单细胞转录组测序（single-cell RNA sequencing，scRNA-seq）探究单次和重复七氟烷麻醉对新生小鼠前额叶皮层（prefrontal cortex，PFC）神经元发育的损伤机制。方法·将新生小鼠分为重复麻醉暴露（Sev3）组、单次麻醉暴露（Sev1）组和对照组，每组3只。Sev3组于出生后第6、第7、第8日接受3%七氟烷和60%氧气的麻醉，Sev1组仅在出生后第6日接受麻醉，出生后第9日获取3组小鼠的PFC进行scRNA-seq。通过UMAP（uniform manifold approximation and projection）聚类、RNA速度分析、转录因子分析（SCENIC）获得七氟烷麻醉后新生小鼠PFC细胞图谱和神经元细分亚群图谱，并进行差异表达基因分析；通过基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）数据库分析研究差异基因富集的生物学过程和通路；利用QuSAGE分析描述细胞周期及Hippo信号通路基因集的激活情况。通过CytoTRACE评分判断七氟烷麻醉后新生小鼠PFC神经元谱系细胞转录本丰富度和干性；利用拟时序分析（pseudo-time analysis）确定神经元分化轨迹，通过分支表达分析模型（BEAM）对发育节点进行解析，以确定决定细胞不同命运的关键基因。结果·3组新生小鼠PFC经scRNA-seq共获得40 061个细胞，10种细胞类型。单次七氟烷麻醉后PFC细胞下调的基因富集在细胞分化、前脑神经元分化、去甲肾上腺素能神经元分化和大脑皮层γ-氨基丁酸能中间神经元分化。重复七氟烷麻醉后下调基因富集在细胞分化的正向调节。单次七氟烷麻醉后下调的差异基因富集的KEGG通路有转化生长因子β信号通路。重复七氟烷麻醉后下调的差异基因富集的KEGG通路有Notch信号通路。SCENIC显示，早期生长反应因子1（early growth response 1，Egr1）、SRY-box转录因子7（SRY-box transcription factor 7，Sox7）在单次和重复七氟烷麻醉后均上调（均P<0.01），HES家族bHLH转录因子6（HES family bHLH transcription factor 6，Hes6）、NKX同源框-1基因（NK2 homeobox 1，Nkx2-1）仅在单次七氟烷麻醉后下调（均P<0.01）。七氟烷麻醉后放射状胶质细胞和神经元与细胞周期相关的基因集激活度增加，并且重复麻醉暴露后激活度增加更明显。重复七氟烷麻醉后神经元与Hippo信号通路相关的基因集由抑制变为激活。将8 224个神经元单独进行亚群分析获得8种神经元谱系细胞，CytoTRACE评分表明七氟烷麻醉后神经元干性增加，神经元发育迟滞。PFC神经元经拟时序分析区分为3个发育阶段，重复七氟烷麻醉使PFC神经元分化拟时间后退（P=0.000）。单次七氟烷麻醉暴露后新生小鼠PFC神经元下调的基因富集在细胞周期蛋白依赖性蛋白丝氨酸/苏氨酸激酶活性的调节、有丝分裂细胞周期相变、细胞分化、长期记忆、有丝分裂细胞周期G₁/S转换。重复七氟烷麻醉暴露后PFC神经元下调基因富集在细胞群增殖的负向调控、细胞分化的正向调节、前脑神经元的分化、典型Wnt信号通路的正向调控和细胞分化。结论·单次和重复七氟烷麻醉均促进新生小鼠PFC神经祖细胞增殖和神经元迁移，重复七氟烷麻醉抑制PFC的神经祖细胞向神经元分化，其潜在机制可能与细胞周期转换有关。

Objective ·To investigate the mechanism of sevoflurane damaging neuron development in the prefrontal cortex (PFC) of the neonatal mice after single or multiple sevoflurane anesthesia based on the single-cell RNA sequencing (scRNA-seq). Methods ·The neonatal mice were divided into multiple anesthesia exposure (Sev3) group, single anesthesia exposure (Sev1) group, and control group with 3 mice each. The Sev3 group received anesthesia with 3% sevoflurane and 60% O₂ on postnatal day 6, 7, and 8, and the Sev1 group received anesthesia only on postnatal day 6. The PFC from mice in the 3 groups was harvested on postnatal day 9 for scRNA-seq. PFC cell profiles and neuronal subpopulation profiles of newborn mice after sevoflurane anesthesia were obtained by UMAP (uniform manifold approximation and projection) clustering, RNA velocity analysis, and transcription factor analysis (SCENIC). Differential expression gene analysis was performed. The biological processes and pathways of the differential genes were investigated through Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database; QuSAGE analysis was used to describe the activation of the cell cycle and Hippo signaling pathway gene sets. Transcript enrichment and stemness of PFC neuronal lineage cells of neonatal mice after sevoflurane anesthesia was determined by CytoTRACE score. The differentiation trajectory of PFC neurons was determined by using pseudo-time analysis, and the developmental nodes were resolved by BEAM analysis to identify key genes that determine different cell fates. Results ·A total of 40 061 cells with 10 cell types were obtained from the PFC of newborn mice in the 3 groups by scRNA-seq. The down-regulated genes in the PFC cells after single sevoflurane anesthesia were enriched in cell differentiation, forebrain neuron differentiation, noradrenergic neuron differentiation, and cerebral cortex GABAergic interneuron differentiation. The down-regulated genes after multiple sevoflurane anesthesia were enriched in positive regulation of cell differentiation. KEGG analysis showed that the down-regulated genes after single sevoflurane anesthesia were enriched in transforming growth factor-β signaling pathway, and the down-regulated genes after multiple sevoflurane anesthesia were enriched in the Notch signaling pathway. SCENIC analysis showed that early growth response 1 (Egr1) and SRY-box transcription factor 7 (Sox7) were up-regulated after both single and multiple sevoflurane anesthesia (both P<0.01), and HES family bHLH transcription factor 6 (Hes6) and NK2 homeobox 1 (Nkx2-1) were down-regulated only after single sevoflurane anesthesia (P<0.01). Activation of the gene set of the cell cycle in radial glial cells and neurons increased after sevoflurane anesthesia, and the increase in activation was more pronounced after multiple sevoflurane anesthesia. The gene set of the Hippo signaling pathway in neurons changed from inhibition to activation after multiple sevoflurane anesthesia. Subpopulation analysis of 8 224 neurons identified 8 neuronal lineage cells, and CytoTRACE scores indicated increased neuron stemness and delayed neuron development after sevoflurane anesthesia. The PFC neurons were divided into 3 developmental stages by pseudo-time analysis, and multiple sevoflurane anesthesia receded the differentiation of PFC neurons in pseudo-time (P=0.000). The down-regulated genes in PFC neurons of newborn mice after single sevoflurane anesthesia were enriched in the regulation of cyclin-dependent protein serine/ threonine kinase activity, mitotic cell cycle phase transition, cell differentiation, long-term memory, and G₁/S transition of the mitotic cell cycle. The down-regulated genes in PFC neurons after multiple sevoflurane anesthesia were enriched in the negative regulation of cell population proliferation, positive regulation of cell differentiation, forebrain neuron differentiation, positive regulation of canonical Wnt signaling pathway, and cell differentiation. Conclusion ·Both single and multiple sevoflurane anesthesia promote PFC neuron proliferation and migration, and multiple sevoflurane anesthesia inhibits the differentiation of neural progenitor cell into neuron in PFC. The underlying mechanism might be related to cell cycle transitions.