摘要
目的 双同源盒蛋白4基因(DUX4)及其靶基因的激活是面肩肱型肌营养不良症(FSHD)的主要发病因素。通过单细胞核转录组测序分析FSHD患者肌肉微环境中各细胞谱系的组成及转录异质性。方法 选择2例FSHD患者,男性1例,年龄65岁,病程43年;女性1例,年龄46岁,病程32年;采集双侧不对称病变的口轮匝肌(轻度组:2例患者肌肉损伤相对正常侧;重度组:2例患者肌肉损伤较重侧)。2例健康志愿者,男性1例,年龄42岁;女性1例,年龄38岁;采集男性左侧和女性右侧正常口轮匝肌(对照组)。对6个样本进行单细胞核转录组测序分析。过滤掉低质量细胞后,对所有样本的44 303个细胞进行聚类分群和细胞类型鉴定,寻找各细胞类型中疾病组和对照组之间的差异表达基因,并对这些基因进行通路富集分析,分析其参与的生物学过程。通过基因集特征评分探索不同样本中特定细胞类型的功能状态差异,利用拟时序分析构建特定细胞类型的单细胞发育轨迹。结果 从6个样本的肌肉组织中共鉴定出10种细胞类型,即肌细胞、卫星细胞、纤维/脂肪祖细胞(FAPs)、巨噬细胞、T淋巴细胞、肥大细胞、内皮细胞、血管平滑肌细胞、脂肪细胞、施旺细胞。其中免疫细胞和FAPs介导的微环境失调在FSHD疾病进展中起重要作用。重度组M1型巨噬细胞的比例增加,促进了肌肉炎症和脂肪替代。疾病组FAPs处于分化紊乱的异常状态,并激活了促炎症、纤维化及脂肪化的相关基因,与FSHD的双侧肌肉不对称改变有关。最后,在重度组的巨噬细胞和FAPs中鉴定了一组关键基因:脂肪酸转位酶基因(CD36),过氧化物酶体增殖物激活受体γ基因(PPARG),半乳糖凝集素1基因(LGALS1),骨形态发生蛋白5基因(BMP5),核受体亚家族4A组成员1基因(NR4A1),磷酸二酯酶基因10A(PDE10A),可作为FSHD的潜在治疗靶点。结论实验研究揭示了FSHD双侧肌肉样本的细胞谱系组成和转录异质性,探索了免疫细胞和FAPs在FSHD肌肉微环境中的作用,有助于FSHD的治疗和预后。
Objective To analyze the composition and transcriptional heterogeneity of each cell lineage in muscle microenvi ronment of facioscapulohumeral muscular dystrophy(FSHD) by single-cell nuclear transcriptome sequencing, because the activation of double homeobox 4 gene(DUX4) and its target genes is the key mechanism in pathogenesis of FSHD. Methods Two patients with FSHD were enrolled, which included 1 male(aged 65-year-old, duration disease 43 years) and 1 female(aged46-year-old, duration disease 32 years). The bilateral asymmetric lesions of orbicularis oris muscle were collected(mild group: 2 patients with relatively normal muscle side;severe group: 2 patients with more severe muscle damage side). Two healthy volunteers were selected,which inclued 1 male(aged 42-year-old) and 1 female(aged 38-year-old), the normal orbicularis oris muscle was collected from the left side of male and the right side of female(control group). The single-cell nuclear transcriptome sequencing analysis was performed on 6 samples. After the low-quality cells were filtered out, all samples of 44 303 cells were clustered and cell types were identified, the differentially expressed genes between disease group and control group in each cell type were searched, and pathway enrichment analysis was performed to analyze the biological processes involved. The functional status differences of specific cell types in different samples were explored by gene set feature scoring, and the single cell development trajectory of specific cell types was constructed by pseudo time series analysis.Results A total of 10 cell types were identified from muscle tissue of 6 samples: myocyte, satellite cells, fibrous/adipose progenitor cells(FAPs), macrophages, lymphoid T cells, mastocyte, endothelial cells, vascular smooth muscle cells, adipocytes,and Schwann cells. Among them, immune cells and FAP-mediated microenvironmental dysregulation play an important role in FSHD disease progression. The increased proportion of M1-type macrophages in severe group promoted muscle inflammation and fat replacement. FAPs in disease group were in an abnormal state of disturbed differentiation and activated genes promoting muscle inflammation, fibrosis and fat infiltration, associated with bilateral muscle asymmetric changes in FSHD. Finally, a set of key genes were identified in macrophages and FAPs in severe group: Fatty acid translocase gene(CD36), peroxisome proliferator activated receptor gamma gene(PPARG), galectin 1 gene(LGALS1), bone morphogenetic protein 5 gene(BMP5), nuclear receptor subfamily 4 group A member 1 gene(NR4A1), phosphodiesterase 10A gene(PDE10A), which were potential therapeutic targets for FSHD. Conclusion It is demonstrated that the study reveals the cell lineage composition and transcriptional heterogeneity of FSHD bilateral muscle samples and explores the role of immune cells and FAPs in FSHD muscle microenvironment,contributing to the treatment and prognosis of FSHD.
作者
刘思宇
宋涛
尹宁北
LIU Si-yu;SONG Tao;YIN Ning-bei(Department of Cleft Lip and Palate,Plastic Surgery Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100144,China)
出处
《生物医学工程与临床》
CAS
2023年第4期494-502,共9页
Biomedical Engineering and Clinical Medicine
关键词
单细胞转录组测序
面肩肱型肌营养不良症
肌肉微环境
免疫失调
纤维/脂肪祖细胞(FAPs)
single-cell transcriptome sequencing
facioscapulohumeral muscular dystrophy
muscle microenvironment
immune dysregulation
fibro/adipose progenitor cells(FAPs)
