中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (50): 9433-9436.doi: 10.3969/j.issn.1673-8225.2011.50.030

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

稳定表达人热休克蛋白22内皮细胞株的构建和鉴定

鲍慧慧，陈  琦，王  玲，程晓曙   

  1. 南昌大学第二附属医院心内科，南昌大学心血管疾病研究所，江西省南昌市 330006
  • 收稿日期:2011-05-21 修回日期:2011-06-26 出版日期:2011-12-10 发布日期:2011-12-10
  • 通讯作者: 程晓曙，博士，教授，博士生导师，南昌大学第二附属医院心内科，南昌大学心血管疾病研究所，江西省南昌市 330006 xiaoshumenfan@126.com
  • 作者简介:鲍慧慧☆，女，1977年生，江西省九江市人，汉族， 2009年南昌大学医学院毕业，博士，主治医师，讲师，主要从事血管内皮保护研究。 huihui_bao@hotmail.com
  • 基金资助:

    国家自然基金青年科学基金资助项目(30800467)；江西省自然基金资助项目(0640089)。

Construction and identification of endothelial cell lines stably expressing human heat shock protein 22

Bao Hui-hui, Chen Qi, Wang Ling, Cheng Xiao-shu   

  1. Department of Cardiology, Second Affiliated Hospital of Nanchang University, Institute of Cardiovascular Disease of Nanchang University, Nanchang  330006, Jiangxi Province, China
  • Received:2011-05-21 Revised:2011-06-26 Online:2011-12-10 Published:2011-12-10
  • Contact: Cheng Xiao-shu, Doctor, Professor, Doctoral supervisor, Department of Cardiology, Second Affiliated Hospital of Nanchang University, Institute of Cardiovascular Disease of Nanchang University, Nanchang 330006, Jiangxi Province, China xiaoshumenfan@126.com
  • About author:Bao Hui-hui☆, Doctor, Attending physician, Lecturer, Department of Cardiology, Second Affiliated Hospital of Nanchang University, Institute of Cardiovascular Disease of Nanchang University, Nanchang 330006, Jiangxi Province, China huihui_bao@hotmail.com
  • Supported by:

    the National Natural Science Foundation for Distinguished Young Scholars, No. 30800467*; the Natural Science Foundation of Jiangxi Province, No. 0640089*

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摘要：

背景：热休克蛋白22的应激表达可能对缺氧复氧内皮具有一定的保护作用。
目的：建立稳定表达人热休克蛋白22基因的内皮细胞株。
方法：EcoRⅠ/kpnⅠ双酶切鉴定真核表达重组质粒pEGFP-N1-HSP22。脂质体法以重组质粒pEGFP-N1-HSP22转染人脐静脉内皮细胞，G418筛选，获得G418抗性单克隆细胞。用荧光显微镜、RT-PCR及Western-blot检测热休克蛋白22在人脐静脉内皮细胞中的表达。
结果与结论：经双酶切电泳检测重组质粒pEGFP-N1-HSP22符合预期大小片段。稳定转染的单克隆人脐静脉内皮细胞中，荧光显微镜观察到每个细胞均有绿色荧光蛋白表达，RT-PCR和Western-blot检测到热休克蛋白22mRNA和蛋白表达。说明成功获得稳定表达人热休克蛋白22基因的内皮细胞株。

关键词: 热休克蛋白22, 人脐静脉内皮细胞, 稳定表达, 转染, 质粒

Abstract:

BACKGROUND: Stress expression of heat shock protein 22 (HSP22) may protect endothelial cells against hypoxia/reoxygenation injury.
OBJECTIVE: To establish endothelial cell line stably expressing human HSP22 gene.
METHODS: The eukaryotic expression vector pEGFP-N1-HSP22 was identified by EcoRⅠ/kpnⅠdouble enzyme digestion. The recombinant plasmids were transfected into human umbilical vein endothelial cells (HUVECs) by lipofectamine. After screening culture by G418, a stably transfected cell line was established. The transcription and expression of the HSP22 gene were identified by fluorescence microscope, RT-PCR and western-blot assay.
RESULTS AND CONCLUSION: Agarose gel electrophoresis detection showed that HSP22 DNA segment was in accordance with the expected results after enzyme digestion. Green fluorescent protein expression was observed in each cell through the use of fluorescence microscope. HSP22 mRNA and protein expression were detected by RT-PCR and western blot analysis. The HUVEC monoclonal cells with a stable expression of HSP22 gene have been established successfully.

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