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  • 邵瑾良,施佳玉,刘佶平,等.牙本质基质蛋白1突变小鼠血脑屏障通透性变化的分子机制[J].同济大学学报(医学版),2021,42(4):445-452.    [点击复制]
  • SHAO Jin-liang,SHI Jia-yu,LIU Ji-ping,et al.Molecular mechanism of changes in blood brain barrier permeability in DMP1 mutant mice[J].同济大学学报(医学版),2021,42(4):445-452.   [点击复制]
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牙本质基质蛋白1突变小鼠血脑屏障通透性变化的分子机制
邵瑾良,施佳玉,刘佶平,孙毅
0
(同济大学医学院,上海200092; 同济大学附属同济医院干细胞临床转化中心,上海200065)
摘要:
目的通过单细胞转录组数据,探究牙本质基质蛋白1(dentin matrix protein 1, DMP1)突变小鼠血脑屏障通透性改变的分子机制。方法通过同源重组的方法,在C57BL/6J背景的小鼠体内敲入一段突变序列,将DMP1糖基化位点的丝氨酸错义突变为分子量更小的甘氨酸,构建S89G-DMP1小鼠模型。前序研究发现DMP1糖基化位点无义突变的S89G-DMP1小鼠会导致血脑屏障(blood brain barrier, BBB)功能障碍。分离小鼠脑组织后通过酶消化制备单细胞悬液,上机测序得到24167个细胞的转录组数据。在R语言中利用Seurat包进行细胞分群,寻找各亚群的差异基因后进行细胞类型定义。结合血脑屏障损伤对神经炎症和神经功能的影响,分析对照组(野生组,简称WT组)与S89G-DMP1组的神经元、小胶质细胞、少突胶质细胞、星形胶质细胞等细胞群体的变化和细胞类型特异性基因表达差异,找到影响血脑屏障完整性的关键细胞和因子。结果在S89G-DMP1小鼠大脑中,少突细胞比例明显上升,神经细胞比例下降。S89G-DMP1组与WT组神经细胞的基因表达较为接近,S89G-DMP1组与WT组星形胶质细胞的基因表达差异较大,共筛选出10个上调基因,85个下调基因,通过GO分析,发现下调基因显著富集在细胞黏附、离子跨膜转运等生理功能上。除此之外,在小胶质细胞、少突胶质细胞中也发现与细胞黏附生理功能的基因在S89G-DMP1小鼠大脑中显著下调。结论S89G-DMP1小鼠大脑神经细胞的比例降低,可能与BBB功能障碍互为因果,但基因表达谱无明显改变,主要通过下调星形胶质细胞、少突胶质细胞和小胶质细胞中与细胞黏附功能相关的基因表达来增加小鼠BBB的通透性,导致BBB功能障碍。
关键词:  牙本质基质蛋白1  血脑屏障  神经系统  单细胞转录组测序  GEO数据库
DOI:10.12289/j.issn.1008-0392.21099
投稿时间:2021-03-18
基金项目:国家自然科学基金(81601975);科技部重点研发计划课题(2016YFA0100801)
Molecular mechanism of changes in blood brain barrier permeability in DMP1 mutant mice
SHAO Jin-liang,SHI Jia-yu,LIU Ji-ping,SUN Yi
(School of Medicine, Tongji University, Shanghai 200092, China; Stem Cell Translational Research Center, Tongji Hospital, School of Medicine, Tongji University, Shanghai 200065, China)
Abstract:
ObjectiveTo investigate the molecular mechanism of altered blood brain barrier(BBB) permeability in DMP1 mutant mice by single-cell transcriptome data. MethodsThe S89G-DMP1 mouse model was constructed by homologous recombination in C57BL/6J background mice by knocking in a mutant sequence that missense mutates the serine at the DMP1 glycosylation site to a smaller molecular weight glycine. The S89G-DMP1 mice with nonsense mutations in the DMP1 glycosylation site were found to cause BBB dysfunction. Single cell suspensions were prepared by enzymatic digestion after isolation of mouse brain tissue, and the transcriptome data of 24167 cells were sequenced. Cell typing was defined in R language using the Seurat package for cell subgrouping and after searching for differential genes in each subgroup. The effects of combined bloodbrain barrier injury on neuroinflammation and neurological function were analyzed for changes in cell populations of neurons, astrocytes, oligodendrocytes and microglia and differences in cell type-specific gene expression in control and S89G-DMP1 mice to find the key cells and factors affecting BBB integrity. ResultsIn the brain of S89G-DMP1 mice, the proportion of oligodendrocytes increased significantly and the proportion of nerve cells decreased.The gene expression of S89G-DMP1 group was similar to that of WT group.The gene expression of astrocytes in S89G-DMP1 group and WT group was significantly different, and 10 up-regulated genes and 85 down-regulated genes were screened out. By GO analysis, it was found that down-regulated genes were significantly enriched in physiological functions such as cell adhesion and ion transmembrane transport.In addition, it was also found in microglia and oligodendrocytes that the genes related to the physiological function of cell adhesion were significantly downregulated in the brain of S89G-DMP1 mice. ConclusionThe gene expression profile of neuronal cells in the brain of S89G-DMP1 mice was not significantly altered. The increased permeability of the BBB in mice is mainly induced by down-regulating the expression of genes related to cell adhesion function in astrocytes, oligodendrocytes and microglia.
Key words:  dentin matrix protein 1  blood brain barrier  nervous system  single cell transcriptome sequencing  GEO database

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