基于RNA测序的SHED体外连续扩增后差异表达基因的研究

doi:10.12439/kqhm.1005-4979.2023.04.002

《口腔颌面外科杂志》 ›› 2023, Vol. 33 ›› Issue (4): 211-218. doi: 10.12439/kqhm.1005-4979.2023.04.002

基于RNA测序的SHED体外连续扩增后差异表达基因的研究

王慧慧¹(), 吴情¹, 赵玉梅²()

¹ 复旦大学附属上海市第五人民医院口腔科，上海　200240
² 同济大学口腔医学院，同济大学附属口腔医院儿童口腔科，上海牙组织修复与再生工程技术中心，上海　200072

收稿日期:2022-11-07 接受日期:2022-12-09 出版日期:2023-08-28 发布日期:2023-08-29
通讯作者: 赵玉梅，主任医师. E-mail：yumeizhao@tongji.edu.cn
作者简介:
王慧慧，住院医师. E-mail：tjwanghuihui@126.com
基金资助:
上海市闵行区自然科学研究课题(2020MHZ030)

Differentially expressed genes of SHED based on RNA-seq analysis after long-term expansion in vitro

WANG Huihui¹(), WU Qing¹, ZHAO Yumei²()

¹ Department of Stomatology, Shanghai Fifth People's Hospital, Fudan University, Shanghai 200240
² Department of Pediatric Dentistry, Stomatological Hospital and Dental School of Tongji University,Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China

Received:2022-11-07 Accepted:2022-12-09 Online:2023-08-28 Published:2023-08-29

摘要/Abstract

摘要：

目的： 利用RNA测序（RNA sequencing，RNA-seq）技术研究人脱落乳牙干细胞（stem cells derived from human exfoliated deciduous teeth，SHED）体外长时期扩增至20代（P20）后基因表达的差异，初步探讨其体外扩增衰老相关的信号通路。方法： 从健康儿童脱落的乳牙中分离牙髓干细胞，在常规条件下将其扩增培养至20代，利用RNA-seq筛选出差异表达的基因，并对其进行相关生物学信息分析，寻找SHED体外连续扩增衰老相关的信号通路。结果： RNA-seq结果显示，SHED第4代（P4）和P20代差异表达的基因共有1 884个，其中上调表达的基因有575个，下调表达基因1 309个，这些差异表达的基因分布在生物过程（biological progress，BP）、细胞组成（cellular component，CC）和分子功能（molecular function，MF）等生物学过程中。早、晚期代次的SHED差异基因及相关蛋白之间的作用主要富集在剪接体、核糖体、细胞周期、p53通路相关的信号通路上。结论： 本研究揭示了SHED体外连续扩增培养至第20代后基因表达谱的改变，为后续进一步研究其细胞体外衰老机制指明了方向。

关键词: 人脱落乳牙干细胞, 体外连续扩增培养, RNA测序, 细胞衰老

Abstract:

Objective: To investigate the differences in gene expression of stem cells derived from human exfoliated deciduous teeth (SHED) after long-term continuous subculture to passage 20 (P20) in vitro by RNA sequencing (RNA-seq) analysis technology, and to preliminarily explore their signaling pathways related to in vitro expansion and aging.Methods: SHED were isolated from deciduous teeth of healthy children in mixed dentition stage and subculture to P20 under the standard condition. RNA-seq was performed to identify the differentially expressed genes among different cell passages, then the relevant biological information was analyzed to explore the senescence related signal pathway of continuous amplification of SHED in vitro.Results: RNA-seq results showed that a total of 1 884 genes were differentially expressed in SHED at passage 4 (P4) and P20, including 575 up-regulated genes and 1 309 down-regulated genes. Gene ontology analysis indicated that the differentially expressed genes were distributed in biological processes (BP), cell composition (CC) and molecular function (MF). Moreover, the pathways most enriched for differentiallygenes and related proteins were mainly concentrated in the spliceosome, ribosome, cell cycle and p53 pathway related signaling pathways.Conclusion: This study revealed that the changes of gene expression profile of SHED after continuous amplification culture to the 20th generation in vitro, which indicated the direction for the further study of in vitro senescence mechanism of SHED.

Key words: stem cells derived from human exfoliated deciduous teeth, long-term expansion in vitro, RNA sequencing, cell senescence

中图分类号:

R782
R788

王慧慧, 吴情, 赵玉梅. 基于RNA测序的SHED体外连续扩增后差异表达基因的研究[J]. 《口腔颌面外科杂志》, 2023, 33(4): 211-218.

WANG Huihui, WU Qing, ZHAO Yumei. Differentially expressed genes of SHED based on RNA-seq analysis after long-term expansion in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(4): 211-218.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I4/211

图/表 6

图1 SHED的形态特征和表面标志物 A.P4代 SHED的形态（×100）；B.P20代 SHED的形态（×100）；C.流式细胞仪检测P4代 SHED的表面标志物；D.流式细胞仪检测P20代 SHED的表面标志物。

Figure 1 The morphological characteristics and cell surface markers of SHED

表1 各样本转录组测序数据统计

Table 1 Transcriptome sequencing data of each sample

样本编号	原始数据序列数	有效序列数	有效序列/%	平均序列长度/bp	碱基≥Q30/%
SHED P4-1	42 477 430	40 504 097	95.35	150	90.70
SHED P4-2	46 054 152	44 034 078	95.61	150	91.25
SHED P4-3	42 583 272	40 407 759	94.89	150	89.53
SHED P20-1	45 591 452	43 509 359	95.43	150	90.62
SHED P20-2	43 272 988	41 310 853	95.47	150	91.08
SHED P20-3	53 988 110	51 611 769	95.60	150	90.90

图2 P4和P20代SHED PCA和系统树图聚类分析 A.转录组数据PCA；B.转录组数据系统树图聚类分析。

Figure 2 PCA and system tree cluster analysis of SHED at passage 4 and passage 20

图3 P4和P20代SHED差异表达的基因热图

Figure 3 Heatmap shows the different expression genes of SHED at P4 and P20

表2 P4和P20代SHED差异基因表达数目统计

Table 2 Statistics of the different expression genes of SHED at P4 and P20

样本	总差异基因数	上调基因数	下调基因数
SHED P20/SHED P4	1 884	575	1 309

图4 P4和P20代SHED差异表达基因功能富集分析 A.差异表达基因的GO分析；B.KEGG通路富集分析；C.PPI网络分析。

Figure 4 Functional enrichment analysis of different expression genes in SHED at P4 and P20

参考文献 19

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[2]	王慧慧, 赵玉梅. 体外连续扩增对乳牙源牙髓干细胞生物学特性的影响[J]. 《口腔颌面外科杂志》, 2017, 27(3): 172-178.

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