SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤
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国家自然科学基金资助项目(30670649).


Overexpressing SelS May Protect Human Umbilical Vein Endothelial Cells From Injuring by H2O2
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This work was supported by a grant from The National Natural Science Foundation of China (30670649).

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    摘要:

    采用以下方法探讨SelS在内皮细胞中的表达和作用:将SelS基因克隆到真核表达载体pLNCX2,RT-PCR、XhoⅠ/ClaⅠ双酶切以及DNA序列分析验证目的基因;利用脂质体转染技术将pLNCX2-SelS或pLNCX2转染至人脐静脉内皮细胞(ECV304细胞),RT-PCR检测重组基因SelS的表达;MTT方法检测转染后过氧化氢(H2O2)对内皮细胞增殖能力的影响;硫代巴比妥酸法测定暴露于H2O2中不同转染组细胞脂质过氧化产物丙二醛含量. 结果表明:成功构建真核表达载体pLNCX2-SelS;转染后重组SelS mRNA表达水平是内源性水平的1.76倍;H2O2对ECV304细胞损伤后,高表达SelS组细胞活性增强、H2O2诱导产生的丙二醛减少. 上述结果表明,高表达SelS可保护内皮细胞免于H2O2诱导的细胞损伤,其作用机制与抗氧化有关.

    Abstract:

    In order to investigate the expression and roles of SelS in human umbilical vein endothelial cells (HUVECs, ECV304 cells), total RNAs were extracted with TRIzol from adipose tissues of human, then the 1 102 bp fragment of SelS was amplified by RT-PCR. After the expected 1 102 bp PCR fragment was purified, SelS was cloned into pMD18-T vector. Purification, RT-PCR, restriction endonuclease analysis and DNA sequencing were performed to confirm the results. pMD18-SelS was incubated in presence of XhoⅠ/ClaⅠ and then ligated into pLNCX2 vector corresponding restriction sites. RT-PCR and DNA sequencing were performed to confirm a 1 102 bp SelS gene fragment was successfully inserted into pLNCX2. The ECV304 cells were stably infected with pLNCX2-SelS or pLNCX2 alone and positive clone was obtained by G418 selection. After transient transfection, ECV304 cells expressed pLNCX2 was verified by neor gene detection and the expression of recombinant SelS in the ECV304 cells was 1.76-fold higher than the endogenous level by RT-PCR. After the cultured ECV304 cells was treated with hydrogen peroxide (H2O2, 100, 200, 300, 400 μmol/L) for 24 h, cell viability was measured by MTT assay and the intracellular maleic dialdehyde(MDA)was detected by TBA method. The results showed that ECV304 cells were injured by H2O2, overexpressing SelS increased the cell viability apparently and inhibited the production of MDA induced by H2O2. So overexpressing SelS protects ECV304 cells from injuring by H2O2, and SelS may have the antioxidant protection effective on endothelial cells.

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杜建玲,安利佳,孙长凯,门莉莉,张秀娟,李昌臣. SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤[J].生物化学与生物物理进展,2007,34(4):425-430

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  • 收稿日期:2006-10-16
  • 最后修改日期:2007-01-04
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  • 在线发布日期: 2007-03-27
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