中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (33): 5372-5377.doi: 10.3969/j.issn.2095-4344.1822

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

构建环状RNA mmu_circ_Rab11a质粒载体并转染293T细胞

元佩燕，陈  蕾，徐帅妹，童方丽，徐平平   

  1. 南方医科大学口腔医院，广东省广州市  510280
  • 修回日期:2019-05-22 出版日期:2019-11-28 发布日期:2019-11-28
  • 通讯作者: 徐平平，博士，主任医师，教授，南方医科大学口腔医院，广东省广州市 510280
  • 作者简介:元佩燕，女，1985年生，汉族，广东省揭阳市人，汉族，2012年南方医科大学毕业，硕士，主治医师，主要从事细胞压力和自噬方面的研究。
  • 基金资助:

    广东省省级科技计划基金项目(2017A020215050)，项目负责人：徐平平

Construction of circular RNA mmu_circ_Rab11a overexpression vector to transfect 293T cells

Yuan Peiyan, Chen Lei, Xu Shuaimei, Tong Fangli, Xu Pingping   

  1. Hospital of Stomatology, Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Revised:2019-05-22 Online:2019-11-28 Published:2019-11-28
  • Contact: Xu Pingping, MD, Chief physician, Professor, Hospital of Stomatology, Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • About author:Yuan Peiyan, Master, Attending physician, Hospital of Stomatology, Southern Medical University, Guangzhou 510280, Guangdong Province, China
  • Supported by:

    Guangdong Provincial Science and Technology Project, No. 2017A020215050 (to XPP) 

PDF

82

摘要：

文章快速阅读：

文题释义：
载体构建：是指将目的基因与运载体相互结合，形成重组载体。其构建目的是通过转导或转化的方式使目的基因被运送到受体细胞中并进行后续的复制与扩增，从而获得目的基因的扩增。目前理想的运载体是质粒，此次实验采用人工构建的质粒作为载体。
转染：是指将外源DNA片段导入真核细胞而获得新的表型的过程。伴随着基因和蛋白功能研究的快速发展，转染目前已经成为目前已成为研究真核细胞基因功能的基本方法。常规转染技术可分为瞬时转染和稳定转染两类。文章利用脂质体LipofectamineTM 3000转染试剂将mmu_circ_Rab11a过表达载体转染293T细胞和MC3T3细胞。
环状RNA：是一类特殊的非编码RNA分子。与传统的线性RNA不同，环状RNA呈封闭环状结构，不受RNA外切酶影响，表达更稳定，不易降解。环状RNA可解除miRNA对其靶基因的抑制作用，升高靶基因的表达水平。

 

摘要
背景：研究发现Rab11a在细胞自噬、增殖过程中发挥重要作用。由于circRNA可通过调控亲本基因的表达参与生物学过程的调控，因此，mmu_circ_Rab11a是否同样在细胞自噬、增殖过程中发挥重要作用值得关注。
目的：探讨环状RNA mmu_circ_Rab11a质粒载体构建并转染293T细胞的可行性。
方法：构建PLC5+mmu_circ_Rab11a过表达质粒载体，采用PCR、基因测序等方法对重组质粒进行验证鉴定。利用脂质体Lipofectamine 3000转染试剂将mmu_circ_Rab11a过表达载体瞬时转染至293T细胞中。利用 RT-PCR检测对照组、空载组(pLC5-ciR)及mmu_circ_Rab11a过表达组中 mmu_circ_Rab11a的基因表达情况并对PCR产物进行Sanger测序验证。mmu_circ_Rab11a过表达质粒载体转染MC3T3，CCK8检测细胞增殖能力。
结果与结论：实验成功构建了mmu_Circ_Rab11a过表达载体，可促使293T细胞高效转染mmu_circ_Rab11a。过表达mmu_circ_Rab11a可提高MC3T3细胞的增殖能力。提示可利用基因工程技术构建mmu_circ_Rab11a过表达载体，通过脂质体法转染法转染293T细胞，使其高效转录mmu_circ_Rab11a，为进一步研究mmu_circ_Rab11a的生物功能奠定实验基础。可通过调控mmu_circ_Rab11a的表达影响MC3T3细胞的增殖能力。


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: 0000-0002-5777-7609(徐平平)

关键词: 环状RNA, mmu_circ_Rab11a, 载体构建, 293T细胞, 脂质体转染, 过表达载体, MC3T3细胞, 细胞增殖

Abstract:

BACKGROUND: Rab11a plays an important role in the cell autophagy and proliferation, and circular RNA is involved in the regulation of biological processes by regulating the expression of parental genes. Therefore, whether mmu_circ_Rab11a also plays an important role in the process of cell autophagy and proliferation deserves further study.
OBJECTIVE: To investigate the feasibility of circular RNA (mmu_circ_Rab11a) plasmid vector construction and transfection of 293T cells.
METHODS: We constructed PLC5+mmu_circ_Rab11a overexpressed plasmid vectors, verified the recombinant plasmid by PCR and gene sequencing. mmu_circ_Rab11a overexpression vector was transiently transfected into 293T cells using Lipofectamine 3000 transfection reagent. Expression of mmu_circ_Rab11a gene in control, pLC5-ciR and mmu_circ_Rab11a overexpression groups was detected by RT-PCR, and PCR products were verified by Sanger sequencing. mmu_circ_Rab11a overexpression plasmid vector was transfected into MC3T3 cells and cell counting kit-8 was used to detect cell proliferation ability.
RESULTS AND CONCLUSION: mmu_circ_Rab11a overexpression vector was successfully constructed, which could efficiently promote mmu_circ_Rab11a transcribed into 293T cells. Over-expression of mmu_circ_Rab11a could enhance the proliferation of MC3T3 cells. Our findings indicate that gene engineering technology can be used to construct mmu_circ_Rab11a overexpression vector, which is then transfected into 293T cells by liposomal transfection method to make efficient transcription of mmu_circ_Rab11a, laying an experimental foundation for further investigation on the biological function of mmu_circ_Rab11a. The proliferation of MC3T3 cells could be affected by regulating the expression of mmu_circ_Rab11a.

Key words: circular RNA, mmu_circ_Rab11a, vector construction, 293T cells, liposome transfection, overexpression vector, MC3T3 cells, cell proliferation

中图分类号: