中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3670-3674.doi: 10.3969/j.issn.1673-8225.2011.20.015

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

构建内皮脂肪酶慢病毒表达载体及在HepG2细胞中的表达

徐  波，邓宇斌   

  1. 中山大学中山医学院病理学与病理生理学教研室，广东省广州市  510080 
  • 收稿日期:2010-12-29 修回日期:2011-03-23 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 邓宇斌，博士，教授，中山大学中山医学院病理学与病理生理学教研室，广东省广州市 510080 dengyub@mail. sysu.edu.cn
  • 作者简介:徐波★，男，1985年生，山西省太原市人，汉族，中山大学在读硕士，主要从事干细胞移植及心脑血管疾病方面的研究。 aquila851@ yahoo.com.cn
  • 基金资助:

    国家教育部博士点基金(20090171110048)，课题名称：中枢神经损伤模型中MSCs向神经元样细胞分化的在体MRI功能显像研究。

Construction of lentiviral vector of endothelial lipase and expression in infected HepG2 cells

Xu Bo, Deng Yu-bin   

  1. Department of Pathology and Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
  • Received:2010-12-29 Revised:2011-03-23 Online:2011-05-14 Published:2011-05-14
  • Contact: Deng Yu-bin, Doctor, Professor, Department of Pathology and Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China dengyub@mail.sysu.edu.cn
  • About author:Xu Bo★, Studying for master’s degree, Department of Pathology and Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China aquila851@yahoo. com.cn
  • Supported by:

    the Doctoral Foundation of Ministry of Education of China, No.20090171110048*

PDF

312

被引次数

3

摘要：

背景：内皮脂肪酶主要由血管内皮细胞分泌并作用于血管内皮，内皮脂肪酶可能在动脉粥样硬化等疾病的发生发展中起重要作用，但目前机制尚不明确。
目的：构建内皮脂肪酶慢病毒表达载体，产毒感染HepG2细胞，评价重组载体提高内皮脂肪酶表达的效果。
方法：RT-PCR调取大鼠内皮脂肪酶基因，SpeⅠ、Eco RⅠ双酶切后，将内皮脂肪酶基因插入慢病毒载体PRRL.sin.CMV.eGFP中构建内皮脂肪酶过表达慢病毒表达载体PRRL.sin.CMV.EL-eGFP；使用PRRL.sin.CMV.EL-eGFP质粒及病毒包装系统于293FT细胞中包装产毒，用于感染HepG2细胞，RT-PCR及Western blot检测感染后HepG2细胞内皮脂肪酶mRNA及蛋白的表达。
结果与结论：酶切及测序鉴定证实内皮脂肪酶基因成功插入PRRL.sin.CMV.EL-eGFP慢病毒表达质粒；转染PRRL.sin.CMV.EL-eGFP慢病毒表达质粒后，HepG2细胞中可见内皮脂肪酶mRNA及蛋白的表达。说明重组PRRL.sin.CMV.EL-eGFP慢病毒可感染HepG2细胞，并使其表达内皮脂肪酶mRNA和蛋白。

关键词: 内皮脂肪酶, 慢病毒载体, 构建, 表达, HepG2细胞

Abstract:

BACKGROUND: Endothelial lipase expresses mainly in endothelial cells, and plays an important role in atherosclerosis and other diseases. But the mechanism is not very clear.
OBJECTIVE: To construct a lentiviral vector carrying rat endothelial lipase (EL) gene to transfect HepG2 cells.
METHODS: 1 482 bp DNA fragment of rat EL was amplified by PCR, cleaved with SpeⅠ, EcoRⅠand cloned into the lentiviral vector PRRL.sin.CMV.eGFP to construct PRRL.sin.CMV.EL-eGFP vector. 293FT cells were co-transfected with recombined PRRL.sin.CMV.EL-eGFP vector and packaging plasmids to produce PRRL.sin.CMV.EL-eGFP lentiviral viruses. Viruses were used to infect HepG2 cells. RT-PCR assay and western blot were used respectively to evaluate the mRNA and protein expressions of EL in the transfected HepG2 cells.
RESULTS AND CONCLUSION: The recombinant inducible lentiviral vector containing EL gene was successfully constructed. The lentiviruses produced by 293FT were efficient to transfect HepG2 cells. The mRNA and protein expressions of EL in the infected HepG2 were obviously up-regulated. It is indicated that EL expression in HepG2 cells can be effectively and stably up-regulated by infecting with the recombined PRRL.sin.CMV.EL-eGFP lentivirus.

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