| 引用本文: | 祁雨心,张秉君,张晓玲.人脐带间充质干细胞的单细胞RNA测序及功能亚群分析[J].中国临床新医学,0,():-. |
| qi yu xin,zhang bing jun.人脐带间充质干细胞的单细胞RNA测序及功能亚群分析[J].中国临床新医学,0,():-. |
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| 摘要: |
| 目的 比较不同人的脐带间充质干细胞（hUCMSCs）在干性、成骨、成软骨和减轻炎症方面的不同。对hUCMSCs进行亚群分类，分析亚群之间功能的异质性。方法 由郑州奥博细胞医学实验室提供两株hUCMSCs，通过实时荧光定量聚合酶链反应（qPCR）对两株hUCMSCs进行干性基因（Sox2、Nanog）的检测。对两株hUCMSCs进行成骨诱导，三天后检测成骨基因（Alp）的表达，7天后进行碱性磷酸酶染色，14天后进行茜素红染色。对两株hUCMSCs进行成软骨诱导，7天后检测成软骨基因（Col2、Acan）的表达。用10ng/ml白介素-1β（IL-1β）刺激大鼠软骨细胞24小时，与两株hUCMSCs共培养后，检测对软骨细胞合成（Acan）和分解代谢(Mmp3)基因表达的影响。通过单细胞转录组测序技术对hUCMSCs进行亚群分群，系统性地描绘各个亚群标志基因热图、富集的信号通路。结果 两株hUCMSCs的干性基因的表达(Sox2、Nanog）有极显著性差异（P<0.01），成骨基因（Alp）表达有极显著性差异（P<0.01），碱性磷酸酶和茜素红染色显示出不同，成软骨基因（Col2、Acan）表达有显著性差异（P<0.05）。在炎症情况下，两株hUCMSCs对软骨细胞合成基因（Acan）表达没有显著性差异(P>0.05)，对分解代谢基因（Mmp3）表达有极显著性差异（P<0.01）。通过单细胞测序技术可以将hUCMSCs分为3个亚群，C1亚群主要表达成骨和减轻炎症方面的基因，C2亚群主要表达干性基因，C3亚群主要表达细胞周期相关基因。结论 不同人来源的hUCMSCs成骨、成软骨和抗炎能力有差异，可能是由于hUCMSCs的异质性所造成的。通过单细胞测序等技术透彻了解MSCs的特性及作用机制，并对其进行精准操作，才能科学造福病患。 |
| 关键词: 人脐带间充质干细胞 干性 成骨 成软骨 减轻炎症 亚群 |
| DOI： |
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| 基金项目:国家重点研发计划项目（编号：2020YFC2002800），上海市科委项目（编号：23141901200） |
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| Single cell RNA sequencing and functional subset analysis of human umbilical cord mesenchymal stem cells |
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qi yu xin,zhang bing jun
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| Abstract: |
| Objective To compare the stemness, osteogenesis, chondrogenesis and inflammation reduction of human umbilical cord mesenchymal stem cells (hUCMSCs) from different people. Classify hUCMSCs into subpopulations and find functional heterogeneity between subpopulations.Methods Two kinds of hUCMSCs were obtained from Zhengzhou Aobo Cell Medicine Laboratory, and the stemness genes (Sox2, Nanog) of the two kinds of hUCMSCs were detected by real-time quantitative polymerase chain reaction (qPCR). Osteogenic induction was performed on two kinds of hUCMSCs. The expression of osteogenic gene (Alp) was detected three days later, alkaline phosphatase staining was performed 7 days later, and alizarin red staining was performed 14 days later. Two kinds of hUCMSCs were induced into cartilage, and the expression of chondrogenic genes (Col2, Acan) was detected 7 days later. Rat chondrocytes were stimulated with 10ng/ml interleukin-1 β (IL-1 β) for 24 hours. After co-culture with two kinds of hUCMSCs, the effects on chondrocyte synthesis (Acan) and catabolism (Mmp3) gene expression were detected. hUCMSCs were subgrouped by single cell transcriptome sequencing technique, and the heat map and enrichment signal pathways of marker genes in each subgroup were systematically described.Results There was a highly significant difference in the expression of stemness genes (Sox2, Nanog) between the two kinds of hUCMSCs (P<0.01), there was a highly significant difference in the expression of the osteogenic gene (Alp) (P<0.01), alkaline phosphatase and Alizarin red staining showed differences, and the expression of chondrogenic genes (Col2, Acan) was significantly different (P<0.05). Under inflammation, there was no significant difference in the expression of chondrocyte synthesis gene (Acan) between the two kinds of hUCMSCs (P>0.05), but there was a highly significant difference in the expression of catabolic gene (Mmp3) (P<0.01). hUCMSCs can be divided into three subgroups by single cell sequencing technology, the C1 subpopulation mainly expresses osteogenesis and inflammation-reducing genes, the C2 subpopulation mainly expresses stemness genes, and the C3 subpopulation mainly expresses cell cycle-related genes.Conclusion The osteogenic, chondrogenic and anti-inflammatory abilities of hUCMSCs from different human sources are different, which may be due to the heterogeneity of hUCMSCs. Through single cell sequencing and other techniques to thoroughly understand the characteristics and mechanism of MSCs, and to accurately operate it in order to benefit patients scientifically. |
| Key words: human umbilical cord mesenchymal stem cells stemness osteogenesis chondrogenesis inflammation reduction |
