首页> 中文期刊> 《现代肿瘤医学》 >FeIT 对急性髓系白血病细胞 eL60影响的研究

FeIT 对急性髓系白血病细胞 eL60影响的研究

         

摘要

Objective:The eL60 of acute myelogenous leukemia cell lines lacked FeIT exPression,to determine the biological effects of FeIT gene exPression in eL60. Methods:We constructed a eukaryotic exPression vector PEG-FP - FeIT and transfected it into the eL60 cells by electroPoration transfect. Fluorescence microscoPe,RT - PCR and Western blot were used to examine the exPression of recombinant Plasmid at mRNA and Protein levels after transfec-tion. MTT assay and flow cytometry were used to determine the effects of FeIT exPression on the Proliferation and aP-oPtosis. Results:A correct eukaryotic exPression vector PEGFP - FeIT was identified by PCR,endoenzyme analysis and sequence of DNA. The GFP exPression was observed by fluorescence microscoPy,transfection efficiency was 40% . The FeIT mRNA and Protein could be detected within the transfected cells by RT - PCR and Western blot. Re-sults of MTT assay indicated that the exPression of FeIT significantly inhibited the Proliferation of eL60 cells in the exPerimental grouP(P ﹤ 0. 05). Flow cytometry Proved that the aPoPtosis index of FeIT - eL60 was higher than the control grouP. Conclusion:Re - exPression of FeIT gene using gene transfection technology effectively inhibits the growth and Proliferation of eL60 cell,and induces aPoPtosis of eL60 cell.%目的:进行外源 FeIT 基因转染人白血病细胞缺乏 FeIT 表达的 eL60,研究 FeIT 基因对转染细胞生长的生物学影响。方法:构建 PEGFP - FeIT 真核表达质粒(实验组)与质粒 PEGFP - N1(对照组)。分别电穿孔法转染 eL60细胞。应用荧光显微镜、RT - PCR、Western blot 检测转染基因的转录和表达情况。四甲基偶氮唑盐(MTT)法、流式细胞术研究转染基因对 eL60细胞体外增殖、凋亡情况的影响,并与转染对照质粒的细胞株进行比较。结果:经 PCR、酶切和 DNA 测序证实 PEGFP - FeIT 真核载体构建成功。荧光显微镜下实验组 eL60细胞可见绿色荧光,转染率为40%。RT - PCR 和 Western blot 分别从 mRNA 和蛋白水平检测到 FeIT的表达。MTT 检测结果显示:转染后实验组抑制率明显升高,细胞增殖受到抑制( P ﹤0.05)。流式细胞术结果显示:实验组细胞凋亡率明显增加(P ﹤0.05)。结论:转染 FeIT 基因对白血病细胞 eL60的生长有抑制作用,并能诱导其凋亡。

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