基于单细胞转录组测序分析人肋来源软骨细胞的异质性

Heterogeneity of chondrocytes derived from human ribs based on single-cell transcriptome sequencing

背景:肋软骨是人体组织中一类重要的软骨来源,常作为软骨自体移植和组织工程构建的种子细胞来源。单细胞测序是分析细胞异质性的强大工具,可针对肋软骨细胞进行细胞异质性的深入研究。目的:通过单细胞转录组测序分析人肋软骨组织细胞分群情况,以及每个细胞亚群参与的生物学过程。方法:临床获取1例31岁小耳重建手术后废弃的肋软骨组织制成原代细胞悬液,经10X Genomics平台进行单细胞分离,使用Gel Bead Kit V3构建单细胞RNA-seq文库,Illumina Novaseq6000测序仪对文库进行测序,并利用主成分分析和T分布随机邻域嵌入进行降维,获得4个亚群细胞,进而获得不同亚群细胞的标记基因,再对每个细胞亚群的标记基因进行GO和KEGG分析,分析这些基因可能参与的生物学过程。结果与结论:测序共获取6634个细胞,符合质控标准。将肋软骨细胞划分为4个细胞亚群,分别为以COL10A1、S100A2为标记基因的肥大软骨细胞群;以BMP2、COL2A1为标记基因的软骨细胞群;以FOS、JUN为标记基因的增殖性细胞群;以MYLK、CD146为标记基因的干细胞群。将肋软骨细胞进一步划分细胞亚群,有利于深入了解肋软骨细胞的异质性,对其作为种子细胞应用及疾病认识都有积极意义。

BACKGROUND:Costal cartilage is an important source of cartilage in human tissues.It is often used as a source of seed cells for cartilage autograft and tissue engineering.As a powerful tool for analyzing cell heterogeneity,single-cell sequencing can conduct in-depth research on costal chondrocyte heterogeneity.OBJECTIVE:Single-cell transcriptome sequencing is used to analyze the cell grouping of human costal cartilage tissue and the biological processes involved in each cell subgroup.METHODS:A 31-year-old case of costal cartilage tissue discarded after a microtia reconstruction operation was obtained clinically and made into a primary cell suspension.Single-cell isolation was performed on the 10 X Genomics platform.A single-cell RNA-seq library was constructed using Gel Bead Kit V3,and the library was analyzed by Illumina Novaseq6000 Sequencing and dimensionality reduction using Principal Component Analysis and t-Distributed Stochastic Neighbor Embedding were used to obtain four subgroups of cells,and then to obtain marker genes for different subgroups of cells.GO and KEGG analyses were performed on the marker genes of each cell subgroup to analyze the biological processes that these genes may participate in.RESULTS AND CONCLUSION:A total of 6634 cells were obtained by sequencing,which met the quality control standards.The costal chondrocytes are divided into four cell subgroups,namely,hypertrophic chondrocyte population with COL10 A1 and S100 A2 as marker genes;chondrocyte population with BMP2 and COL2 A1 as marker genes;and proliferative cells with FOS and JUN as marker genes;stem cell population with MYLK and CD146 as marker genes.The further division of costal chondrocytes into cell subgroups is helpful for in-depth understanding of the heterogeneity of cost chondrocytes,and has positive significance for its application as seed cells and understanding of diseases.