猪耳成纤维细胞转录组异质性及对核移植胚胎发育的潜在影响

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation

同一来源的供体细胞之间存在异质性。许多研究已经表明体细胞核移植(somatic cell nuclear transfer,SCNT)效率与供体细胞有关。然而,鲜有在单细胞水平分析供体细胞异质性对核移植效率的潜在影响。本研究利用单细胞转录组测序技术对同一来源且随机挑选的52个猪耳组织成纤维细胞进行测序分析。结果表明有48个单细胞的基因表达模式相似,4个单细胞(编号为D11_1、D12_1、DW61_2和DW99_2)的基因表达模式与其他单细胞存在较大的差异,并且不存在基因表达模式完全相同的两个单细胞。以基因表达模式相似的48个单细胞作为对照,进一步分析了单细胞D11_1、D12_1、DW61_2和DW99_2的差异基因表达模式:首先利用R语言筛选4个单细胞的差异表达基因,并对前50差异表达基因进行汇总;然后对差异表达基因进行GO富集分析和KEGG通路分析。富集分析发现差异表达基因的主要分子功能包括能量代谢、蛋白质代谢和细胞对刺激的反应等;主要通路包括KEGG中富集的与细胞周期、细胞代谢、DNA复制相关的通路。根据以上研究结果并结合SCNT研究进展讨论了4个单细胞的差异基因表达模式对核移植胚胎发育效率的潜在影响。本研究揭示了猪耳组织成纤维细胞的转录组异质性,并提供了分析精英供体细胞的一种有效方法,为提高克隆效率带来新的思路。

There is heterogeneity among donor cells of the same source.Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer(SCNT).However,the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level.In this study,single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns.The results showed that 48 cells had similar gene expression patterns,whereas 4 cells(D11_1,D12_1,DW61_2,DW99_2)had significantly different gene expression patterns from those of other cells.There were no two cells with identical gene expression patterns.The gene expression patterns of D11_1,D12_1,DW61_2 and DW99_2 were analyzed,using the 48 cells with similar gene expression patterns as controls.Firstly,we used the R language statistics to select the differentially expressed genes in the 4 single cells,and identified the top 50 most significant differentially expressed genes.Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes.Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism,protein metabolism and cell response to stimulation.The main pathways from KEGG enrichment were related to cell cycle,cell metabolism,and DNA replication.Finally,based on the above results and in consideration with the SCNT research progress,we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation.This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells,thereby providing new ideas on improving the cloning efficiency of SCNT.