沉默Krüppel样因子5对电离辐射后小肠上皮细胞生物学功能的影响
首发时间:2018-06-20
摘要:目的:观察电离辐射后下调Krüppel样因子5(KLF5)表达对小肠上皮细胞生长、周期分布、凋亡及DNA双链断裂的影响。方法:采用Western Blot法检测不同照射剂量及照射后不同时间大鼠小肠隐窝上皮细胞IEC-6中KLF5的表达。设计并合成特异性针对大鼠KLF5基因的shRNA靶序列,构建到慢病毒载体中,通过感染293T细胞对慢病毒进行包装和滴度测定。使用包装好的慢病毒感染IEC-6细胞,荧光显微镜下观察转染效率,分别采用Real time-PCR和Western Blot方法检测感染后细胞中KLF5 mRNA及蛋白的表达情况。采用CCK-8法观察电离辐射后KLF5沉默细胞的生长增殖活性,流式细胞术检测电离辐射后KLF5沉默细胞的周期分布和凋亡情况,免疫荧光染色观察电离辐射后KLF5沉默细胞中γ-H2AX foci数量。结果:电离辐射可以诱导体外培养的IEC-6细胞表达KLF5,呈现明显的时间和剂量依赖关系。KLF5 shRNA慢病毒载体构建成功,感染的IEC-6细胞中KLF5 mRNA及蛋白水平均明显降低。KLF5敲低组照射后细胞阻滞在G2/M期现象极为显著,细胞增殖明显受到抑制,细胞凋亡率高于对照组,同一时间点细胞核中γ-H2AX foci数量明显多于对照组。结论:本研究成功构建了KLF5 shRNA慢病毒载体,建立了KLF5敲低小肠上皮细胞株,下调细胞内KLF5表达能够使细胞周期阻滞在G2/M期,明显抑制照射后细胞的增殖,促进细胞的凋亡,DNA双链断裂水平增加,修复延迟。
关键词: 电离辐射 Krüppel样因子5 放射性肠损伤 DNA损伤
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Effects of Krüppel-like factor 5 gene silencing on biological functions of intestinal epithelial cells post radiation
Abstract:Objective: To investigate the effects of downregulation of Krüppel-like factor 5 (KLF5) on cell proliferation, cell cycle distribution, apoptosis and DNA double strand break (DSB) in intestinal epithelial cells in response to ionizing radiation. Methods: The expression of KLF5 in rat intestinal crypt epithelial cells IEC-6 in response to different doses of radiation at indicated time points post radiation was detected by Western Blot. shRNA sequences targeting rat KLF5 gene were designed and synthesized and inserted into the lentiviral vector. The recombinantlentiviral vectors were packaged in 293T cells, and the lentivirus titers were determined. IEC-6 cells were infected with the recombinantlentivirus, and the transfection efficiency was observed under fluorescence microscope. Realtime-PCR and Western Blot were used to detect theexpressions of KLF5 mRNA and protein in the transfected cells. The cell viability was observed in KLF5 silencing cells post radiation using CCK-8 assay. The cell cycle distribution and apoptosis were detected in KLF5 silencing cells post radiation by flow cytometry. Immunofluorescence staining was applied to visualize the γ-H2AX foci in nucleus of KLF5 silencing cells post radiation. Results: KLF5 was induced in response to ionizing radiation in a time- and dose-dependent manner. KLF5 shRNA lentiviral vectors were successfully constructed. The mRNA and protein level of KLF5 were downregulated in recombinantlentivirus transfected IEC-6 cells. Knockdown of KLF5 markedly inducedG2/M phase arrest, proliferation inhibition, more apoptosis rate, and more γ-H2AX foci in nucleus post exposure than negative control. Conclusion: We have successfully established a KLF5 knockdown intestinal epithelial cell line, which shows suppressed cell proliferation, increased cell apoptosis, enhanced DNA DSB and prolonged DNA damage repair.
Keywords: ionizing radiation Krüppel-like factor 5 radiation induced intestinal injury DNA damage
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沉默Krüppel样因子5对电离辐射后小肠上皮细胞生物学功能的影响
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