| 肠上皮细胞RNA干扰Hsp70基因转染条件的研究 |
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| 引用本文： | 钟翔,李兴美,李伟,黄雪新,张莉莉,王恬.肠上皮细胞RNA干扰Hsp70基因转染条件的研究[J].生物技术通报,2011(9). |
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| 作者姓名： | 钟翔 李兴美 李伟 黄雪新 张莉莉 王恬 |
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| 作者单位： | 南京农业大学动物科技学院,南京,210095 |
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| 基金项目： | 国家自然科学基金项目(30771569,30972116) |
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| 摘 要： | 旨在研究肠上皮细胞(IEC-6)RNA干扰效率的特染条件,并对4对Hsp70干抚序列进行筛选,为进一步以RNA干扰(RNAi)技术研究Hsp70对胃肠道的保护功能以及介导信号通路的机理打下基础.以24孔培养板培养IEC-6细胞,利用阳高子脂质体特染FAM-siRNA片段进入IEC-6细胞,采用荧光倒置显微镜和流式细胞仪检测FAM-siRNA和特染试剂Lipofectamine 2000不同剂量比例对特染效率的影响,并进一步采用RT-PCR和Western blotting方法对Hsp70干扰序列进行筛选.试验结果表明,采用脂质体转染法可获得较高的转染效率,其中以80 nmol/L FAM-siRNA+1μL Lipofectamine 2000组的转染效率最高(85.77％).针对4个不同靶位点,进行Hsp70基因干涉,结果表明,4个组中Hsp70 mRNA的表达显著下降,其中oligo 4组的基因表达极显著降低.Western blotting的结果表明,oligo 2、oligo3和oligo4组中的Hap70蛋白表达极显著降低.最终确定80 nmol/L FAM-siRNA+1μL Lipofectamine 2000为最佳转染条件,以oligo 4(Hap70的基因位点为3672 -3692)为最佳RNA干扰靶基因.
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| 关 键 词： | 肠上皮细胞 RNA干扰 Hsp70 转染 |
Research on Transfection Condition of RNA Interference Targeting Hsp70 Gene in Intestinal Epithelial Cells |
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| Zhong Xiang,Li Xingmei,Li Wei,Huang Xuexin,Zhang Lili,Wang Tian.Research on Transfection Condition of RNA Interference Targeting Hsp70 Gene in Intestinal Epithelial Cells[J].Biotechnology Bulletin,2011(9). |
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| Authors: | Zhong Xiang Li Xingmei Li Wei Huang Xuexin Zhang Lili Wang Tian |
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| Institution: | Zhong Xiang Li Xingmei Li Wei Huang Xuexin Zhang Lili Wang Tian(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095) |
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| Abstract: | This study was conducted to investigate the transfection condition for affecting RNA interference efficiency on intestinal epithelial cells(IEC-6)cells,and determine the highest efficiency of RNA interference targeting Hsp70,therefore,it could lay a foundation for the further research on the protection of Hsp70 in gastro-intestinal tract and cell signal transduction of Hsp70 by the method of RNA interference.IEC-6 cells were cultured in 24-well plate.With the help of cation liposome,the transfection of FAM-... |
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| Keywords: | Intestinal epithelial cells RNA interference Hsp70 Transfection |
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