中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (36): 6779-6784.doi: 10.3969/j.issn.2095-4344.2012.36.023

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

超极化激活环化核苷酸门控通道2基因转染后293T细胞中目的基因的表达

李先花，李建美，陶四明，张新金   

  1. 昆明医学院第四附属医院心内科，云南省昆明市 650021
  • 收稿日期:2012-01-20 修回日期:2012-02-26 出版日期:2012-09-02 发布日期:2012-09-02
  • 通讯作者: 李建美，教授，昆明医学院第四附属医院心内科，云南省昆明市 650021 lijianmei090@sina.com
  • 作者简介:李先花★，女，1985年生，山西省大同市人，汉族，2010年昆明医学院毕业，硕士，医师，主要从事心血管内科研究。 lixianhua0223@163.com

Expression of target gene in 293T cells transfected by hyperpolarization-activated cyclic nucleotide-gated channel 2 gene

Li Xian-hua, Li Jian-mei, Tao Si-ming, Zhang Xin-jin   

  1. Department of Cardiology, the Fourth Affiliated Hospital of Kunming Medical College, Kunming 650021, Yunnan Province, China
  • Received:2012-01-20 Revised:2012-02-26 Online:2012-09-02 Published:2012-09-02
  • Contact: Li Jian-mei, Professor, Department of Cardiology, the Fourth Affiliated Hospital of Kunming Medical College, Kunming 650021, Yunnan Province, China lijianmei090@sina.com
  • About author:Li Xian-hua★, Master, Physician, Department of Cardiology, the Fourth Affiliated Hospital of Kunming Medical College, Kunming 650021, Yunnan Province, China lixianhua0223@163.com

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摘要：

背景：研究证实超极化激活环化核苷酸门控通道电流在调控心脏的自发搏动中起着非常重要的作用。
目的：观察超极化激活环化核苷酸门控通道2基因重组质粒转染后293T细胞中目的基因在核酸和蛋白质水平的表达。
方法：采用脂质体转染试剂Lipofectamine2000将含全长超极化激活环化核苷酸门控通道2基因的质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。
结果与结论：用脂质体转染试剂Lipofectamine2000成功地将质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。反转录PCR定性地检测到在100-250 bp处可见高度特异DNA的条带，与携带超极化激活环化核苷酸门控通道2基因的质粒扩增出的片段位置相同。经过RT-PCR、Western-blot方法检测提示超极化激活环化核苷酸门控通道2基因在mRNA和蛋白水平都有高表达。表明质粒CMV-hHCN2-3xHA-IRES-EGFP成功转染后的293T细胞中超极化激活环化核苷酸门控通道2基因可以在核酸和蛋白水平表达。

关键词: 超极化激活环化核苷酸门控通道2, 293T细胞, 生物心脏起搏器, 基因, 基因重组质粒, 起搏电流

Abstract:

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) currently plays an important role in regulating heart spontaneous pulsation.
OBJECTIVE: Target gene expression was observed at mRNA and protein level in 293T cells transfected with human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) gene recombinant plasmid.
METHODS: Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length hHCN2 gene was transfected into 293T cells by Liposome Lipofectamine 2000. Expression of hHCN2 mRNA was detected by reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction respectively. Expression of HCN2 protein was examined by western blot method.
RESULTS AND CONCLUSION: Plasmid CMV-hHCN2-3xHA-IRES-EGFP including full length HCN2 gene was successfully transfected into 293T cells by Liposome Lipofectamine 2000. Reverse transcription polymerase chain reaction detected the highly visible specific DNA bands at 100-250 bp, which was the same as hHCN2 gene carried by plasmid. Reverse transcription-polymerase chain reaction and western blot method results showed that hHCN2 gene was highly expressed at mRNA and protein levels. These findings suggest that hHCN2 gene was expressed at the nucleic acid and protein levels in 293T cells transfected by plasmid of CMV-hHCN2-3xHA-the IRES-EGFP.

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