中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (17): 2659-2664.doi: 10.3969/j.issn.2095-4344.1704

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

慢病毒介导Notch1基因shRNA沉默大鼠骨髓间充质干细胞Notch信号通路的表达

闫锦玉，邢万红   

  1. 山西医科大学，山西省太原市 030001
  • 修回日期:2019-01-10 出版日期:2019-06-18 发布日期:2019-06-18
  • 通讯作者: 邢万红，博士，副主任医师，山西医科大学，山西省太原市 030001
  • 作者简介:闫锦玉，男，1990年生，山西省交城县人，汉族，2019年山西医科大学毕业，硕士研究生，主要从事心肌组织工程研究。
  • 基金资助:

    山西省自然科学基金(201601D011094)，项目负责人：邢万红

Lentiviral-mediated shRNA silencing of Notch-1 inhibits expression of Notch signaling pathway in rat bone marrow mesenchymal stem cells

Yan Jinyu, Xing Wanhong   

  1. Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Revised:2019-01-10 Online:2019-06-18 Published:2019-06-18
  • Contact: Xing Wanhong, MD, Associate chief physician, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • About author:Yan Jinyu, Master candidate, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China
  • Supported by:

    the Natural Science Foundation of Shanxi Province, No. 201601D011094 (to XWH)

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摘要：

文章快速阅读：

文题释义：
慢病毒载体：是基于人免疫缺陷Ⅰ型病毒工程改造的病毒载体系统。它区别于具有分裂和非分裂细胞转染能力的一般反转录病毒载体，能有效地将目的基因(或RNAi)引入动物和人的原代细胞或细胞系中，具有高效率、高稳定性，而且很少诱发宿主免疫反应的优点。因此，慢病毒载体介导的RNAi已经成为多数研究者的首选。
增强型绿色荧光蛋白基因：作为一种新型的报告基因，增强型绿色荧光蛋白已在许多研究领域得到应用。利用绿色荧光蛋白独特的发光机制，可将增强型绿色荧光蛋白作为蛋白质标签，即利用DNA重组技术，将目的基因与增强型绿色荧光蛋白基因构成融合基因，转染合适的细胞进行表达，然后借助荧光显微镜进行观察。由于增强型绿色荧光蛋白相对较小，只有238个氨基酸，将其与其他蛋白融合后不影响自身的发光功能。

 

摘要
背景：对于骨髓间充质干细胞向心肌细胞分化中Notch信号通路的机制研究较多，但是沉默Notch信号通路在工程化心肌样组织血管网络中的机制研究还比较少。
目的：构建慢病毒载体介导Notch1基因shRNA表达体系，观察骨髓间充质干细胞中rNotch基因沉默效果。
方法：构建pLV[shRNA]-EGFP: T2A: Puro- U6]{ rNotch1[ shRNA]_19 nt}(PLV-Notch)和pLV[shRNA]-EGFP: T2A:Puro-U6>Scramble_shRNA(PLV-Scramble)拼装慢病毒表达载体，并将该慢病毒载体包装质粒形成混合物共转染到HEK-293T细胞中。收集慢病毒悬液，通过定量PCR检测重组病毒滴度。将构建的PLV-Notch与PLV-Scramble分别感染骨髓间充质干细胞，经荧光显微镜观察和RT-qPCR、Western blot鉴定转染后两组骨髓间充质干细胞中增强型绿色荧光蛋白基因和目的基因rNotch1表达情况。
结果与结论：慢病毒载体滴度大于1×108 TU⁄mL，稳定转染筛选得到成熟的细胞株。病毒转导效果良好，荧光率达80%，rNotch1基因在干扰组中的表达量是scrambled对照组的75.2%。由此可见，慢病毒介导转染rNotch1基因的骨髓间充质干细胞稳转株构建成功，并对Notch信号通路的表达有沉默作用。


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
ORCID: 0000-0003-0395-542X(闫锦玉)

关键词: 骨髓间充质干细胞, Notch基因, 慢病毒载体, RNA干扰, Notch信号通路, 山西省自然科学基金

Abstract:

BACKGROUND: Number studies have insight into the mechanism of Notch signaling pathway in the differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, but the mechanism of silencing Notch signaling pathway in the engineered myocardial-like tissue vascular network is less elucidated.
OBJECTIVE: To investigate the role of Notch signaling pathway in bone marrow mesenchymal stem cells in the construction of engineered myocardial-like tissue vascular network.
METHODS: The lentivirus expression vector was constructed by pLV[shRNA]-EGFP: T2A: Puro-U6] {rNotch1 [shRNA]_19 nt}(PLV-Notch) and pLV[shRNA]-EGFP: T2A: Puro-U6>Scramble_shRNA(PLV-Scramble) and packaged with the lentivirus vector. The mixture was co-transfected into HEK-293 T cells. The lentivirus suspension was collected and the titer of recombinant virus was detected by quantitative PCR. The constructed PLV-Notch and PLV-Scramble were infected with bone marrow mesenchymal stem cells respectively. The expression of enhanced green fluorescent protein gene and target gene rNotch1 in the transfected cells was identified by fluorescence microscopy, RT-qPCR and western blot.
RESULTS AND CONCLUSION: The titer of lentivirus vector was more than 1×108 TU/mL. The mature cell lines were screened. The fluorescence rate of the two transfection groups was 80%, and the expression of rNotch1 gene in the interference group was 75.2% of that in the scrambled group. Therefore, lentiviral-mediated rNotch1 gene transfection of bone marrow mesenchymal stem cells can be successfully constructed and silence the expression of Notch signal pathway.

Key words: bone marrow mesenchymal stem cells, Notch gene, lentiviral vector, RNA interference, Notch signaling pathway, Natural Science Foundation of Shanxi Province

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