中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (30): 4847-4851.doi: 10.12307/2022.765

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

人脐带间充质干细胞向肝细胞分化过程中长链非编码RNA核富集转录本1的作用

王丹丹1,禹亚彬2,刘世奇1,严雨楼1,张建淮2   

  1. 1南京医科大学,江苏省南京市   210000;2南京医科大学附属淮安第一医院肝胆外科,江苏省淮安市   223300
  • 收稿日期:2021-08-02 接受日期:2021-10-11 出版日期:2022-10-28 发布日期:2022-03-29
  • 通讯作者: 张建淮,副教授,主任医师,南京医科大学附属淮安第一医院肝胆外科,江苏省淮安市 223300
  • 作者简介:王丹丹,女,1994年生,江苏省淮安市人,汉族,在读硕士。
  • 基金资助:
    淮安市自然科学项目(HAB202016),项目负责人:禹亚彬;南京医科大学附属淮安第一医院高层次人才科研项目(YGRX201902),项目负责人:禹亚彬

Role of long non-coding RNA nuclear-enriched abundant transcript 1 in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes

Wang Dandan1, Yu Yabin2, Liu Shiqi1, Yan Yulou1, Zhang Jianhuai2   

  1. 1Nanjing Medical University, Nanjing 210000, Jiangsu Province, China; 2Department of Hepatobiliary Surgery, Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, Huaian 223300, Jiangsu Province, China
  • Received:2021-08-02 Accepted:2021-10-11 Online:2022-10-28 Published:2022-03-29
  • Contact: Zhang Jianhuai, Associate professor, Chief physician, Department of Hepatobiliary Surgery, Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, Huaian 223300, Jiangsu Province, China
  • About author:Wang Dandan, Master candidate, Nanjing Medical University, Nanjing 210000, Jiangsu Province, China
  • Supported by:
    Huaian Municipal Natural Science Program, No. HAB202016 (to YYB); the Research Program for Advanced Talents of the Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, No. YGRX201902 (to YYB)

摘要:

文题释义:
长链非编码RNA:是一类超过200个核苷酸的非编码RNA分子,位于细胞核或胞质部分。在大多数情况下,由于缺乏开放的阅读框架,它们几乎从不参与蛋白质编码;相反,它们在RNA水平上调节基因表达;此外,长链非编码RNA还可与转录因子蛋白、染色体空间构象或mRNA结合,通过激活或抑制转录因子来调节下游相关基因的表达。
转染:是真核细胞在一定条件下主动或被动导入外源DNA片段而获得新的表型的过程。从本质上讲,和转化区别不大,因为无论是转染还是转化,都是用氯化钙处理大肠杆菌细胞来提高细胞膜的通透性,再将重组噬菌体DNA直接导入受体细胞,进入感受态细菌的噬菌体DNA可以同样复制和繁殖,这种方式称为转染。广义上说,转染也称为转化。常规转染技术包括瞬时转染和稳定转染两类。

背景:人脐带间充质干细胞在体外借助细胞因子可以诱导分化为肝样细胞,但是形成的肝样细胞功能上尚不够成熟。研究发现长链非编码RNA与细胞分化关系密切,并通过干预某些长链非编码RNA的表达达到了促进细胞分化的目的。
目的:探讨长链非编码RNA核富集转录本1对人脐带间充质干细胞向肝细胞分化的影响。
方法:建立人脐带间充质干细胞向肝细胞分化体系,用糖原染色和吲哚菁绿摄取实验评价肝细胞功能特性,qRT-PCR检测肝细胞特异性基因及核富集转录本1的mRNA表达。然后通过构建慢病毒干扰核富集转录本1表达,转染7 d后得到稳定转染的细胞株,然后进行肝向诱导分化4周,设立转染空质粒的阴性对照组,未进行病毒转染的对照组,qRT-PCR、Western blot检测肝细胞特异性基因和蛋白表达。
结果与结论:①经糖原染色后可见细胞胞质内有红、紫色糖原颗粒,吲哚菁绿摄取后可见大量深绿色吲哚菁绿阳性细胞;②随着诱导时间延长,白蛋白、细胞色素酶P450 3A4 mRNA表达量逐渐增加,核富集转录本1的mRNA表达量逐渐降低;③与阴性对照组和未转染对照组比较,在干扰核富集转录本1表达后,白蛋白、细胞色素酶P450 3A4 mRNA和蛋白表达均明显增加;④结果表明,长链非编码RNA核富集转录本1可能参与了人脐带间充质干细胞向肝细胞的分化。
缩略语:核富集转录本1:nuclear-enriched autosomal transcript 1,NEAT1

https://orcid.org/0000-0001-7527-7918 (王丹丹) 

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 长链非编码RNA, NEAT1, 间充质干细胞, 肝细胞, 分化, 肝细胞特异性基因, 转染

Abstract: BACKGROUND: Human umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocytes with the help of cytokines in vitro, but the resulting hepatoid cells are not yet functionally mature. It has been gradually found that long non-coding RNA (lncRNA) is closely related to cell differentiation and the purpose of promoting cell differentiation is achieved by interfering with the expression of some lncRNAs.  
OBJECTIVE: To explore the role of long non-coding RNA nuclear-enriched abundant transcript 1 (lncRNA NEAT1) in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.
METHODS:  A differentiational cell line system of human umbilical cord mesenchymal stem cells into hepatocytes was established. Periodic Acid-Schiff staining and indocyanine green intake test were used to assess hepatocellular functional characteristics of the cells. Quantitative real-time reverse transcription polymerase chain reaction was used to detect the expression of hepatocyte specific genes and lncRNA NEAT1. The lentivirus was constructed to interfere with NEAT1 expression, and stably transfected cell lines were obtained 7 days after transfection, and hepatotropic differentiation was induced for 4 weeks. A negative control group for transfection with empty plasmids and a control group without virus transfection were set up. The expression levels of hepatocyte specific genes and proteins were detected by quantitative real-time reverse transcription polymerase chain reaction and western blot assay.  
RESULTS AND CONCLUSION:  (1) After Periodic Acid-Schiff staining, there were red and purple glycogen particles in the cytoplasm of cells, and a large number of indocyanine green positive cells were observed after indocyanine green ingestion. (2) With the extension of induction time, the mRNA expression levels of albumin and cytochrome enzyme P450 3A4 mRNA increased gradually, while the mRNA expression levels of NEAT1 decreased gradually. (3) Compared with the negative control group and the control group, the mRNA and protein expression levels of albumin and cytochrome enzyme P450 3A4 were significantly increased after the interference of NEAT1. (4) The results confirm that lncRNA NEAT1 may be involved in the differentiation of human umbilical cord mesenchymal stem cells into hepatocytes.

Key words: long non-coding RNA NEAT1, mesenchymal stem cell, hepatocyte, differentiation, hepatocyte specific genes, transfection

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