| 赵文玉,杨斯茗,杨志敏,等.沉默Krüppel样因子5对电离辐射后大鼠小肠上皮IEC-6细胞生物学功能的影响[J].中华放射医学与防护杂志,2020,40(7):500-506.Zhao Wenyu,Yang Siming,Yang Zhimin,et al.Effects of Krüppel-like factor 5 gene silencing on biological functions of rat intestinal epithelial cells IEC-6 after radiation[J].Chin J Radiol Med Prot,2020,40(7):500-506 |
| 沉默Krüppel样因子5对电离辐射后大鼠小肠上皮IEC-6细胞生物学功能的影响 |
| Effects of Krüppel-like factor 5 gene silencing on biological functions of rat intestinal epithelial cells IEC-6 after radiation |
| 投稿时间：2019-08-08 |
| DOI：10.3760/cma.j.issn.0254-5098.2020.07.002 |
| 中文关键词: 电离辐射 Krüppel样因子5 放射性肠损伤 细胞凋亡 |
| 英文关键词:Ionizing radiation Krüppel-like factor 5 Radiation induced intestinal injury Apoptosis |
| 基金项目:国家自然科学基金（31300694，81673091） |
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| 摘要点击次数: 3426 |
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| 中文摘要: |
| 目的 探讨沉默Krüppel样因子5（KLF5）对电离辐射后体外培养的大鼠小肠上皮IEC-6细胞生物学功能的影响。方法 给予大鼠小肠上皮IEC-6细胞12 Gy照射，分别在照后0、0.5、1、2、3、5、7、24 h，检测KLF5的表达。给予IEC-6细胞0、2、4、8、12和16 Gy X射线照射，照后3 h收集细胞蛋白，采用Western blot法检测IEC-6细胞中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，通过感染人胚肾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，转染72 h后分别采用实时-PCR和Western blot方法检测感染后细胞中KLF5 mRNA及蛋白的表达。后续实验分为阴性对照组、shKLF5组、单纯照射组、照射+shKLF5组4组。采用CCK-8法观察8 Gy照射后KLF5沉默细胞的增殖活性，流式细胞术检测8 Gy照射后KLF5沉默细胞的周期分布和凋亡，免疫荧光染色观察2 Gy照射后KLF5沉默细胞中γ-H2AX焦点数量。结果 不同剂量射线照射后KLF5表达逐渐增加，呈现明显的剂量效应关系。12 Gy照射后KLF5表达呈现先升高后降低的趋势，照后5 h表达量最高。KLF5 shRNA慢病毒载体构建成功，感染的IEC-6细胞中KLF5 mRNA及蛋白水平均在转染72 h明显降低。照射+shKLF5组细胞在照射后24 h阻滞在G2/M期现象显著（t=11.56，P<0.05），细胞增殖明显受到抑制，其细胞凋亡率（12.49±0.63）%，明显高于单纯照射组（7.42±0.49）%，两组比较差异有统计学意义（t=10.98，P<0.05），照射+shkLF5组细胞核中各时间点的γ-H2AX焦点数量明显多于同一时间点阴性对照组（t=22.07、23.89、11.24、59.97、20.85，P<0.05）。结论 成功构建KLF5 shRNA慢病毒载体，并建立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，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。 |
| 英文摘要: |
| Objective To investigate the effects of down-regulation of Krüppel-like factor 5 (KLF5) on biological functions of rat intestinal epithelial cells IEC-6 in response to ionizing radiation. Methods Rat intestinal epithelial IEC-6 cells were irradiated with 0, 2, 4, 8, 12, 16 Gy of X-rays and 3 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. IEC-6 cells were irradiated to 12 Gy and 0, 0.5, 1, 2, 3, 5, 7 and 24 h later the expression of KLF5 in IEC-6 cells was detected by Western blot. shRNA sequences targeting rat KLF5 gene were designed, synthesized and inserted into the lentiviral vector. The recombinant lentiviral vectors were packaged in human embryonic kidney 293T cells, and the lentivirus titers were determined. IEC-6 cells were infected with the recombinant lentivirus, and the transfection efficiency was observed under fluorescence microscope. Real-time PCR and Western blot were used to detect the expressions of KLF5 mRNA and protein in the transfected cells 72 h post transfection. The consequent experiments included four groups:negative control group, shKLF5 group, radiation group and radiation + shKLF5 group. The cell viability was observed in KLF5 silencing cells irradiated with 8 Gy by using CCK-8 assay. The cell cycle distribution and apoptosis were detected in KLF5 silencing cells irradiated with 8 Gy by flow cytometry. Immunofluorescence staining was applied to visualize the γ-H2AX foci in nucleus of KLF5 silencing cells irradiated with 2 Gy. Results The expression of KLF5 increased with the different doses. The expression of KLF5 increased first, then decreased and peaked at 5 h post-irradiation with 12 Gy. KLF5 shRNA lentiviral vectors were successfully constructed. The mRNA and protein level of KLF5 were down-regulated in recombinant lentivirus transfected IEC-6 cells 72 h after transfection. Knockdown of KLF5 markedly induced G2/M phase arrest (t=11.56, P<0.05), proliferation inhibition, more apoptosis rate[radiation group:(7.42±0.49)%, radiation + shKLF5 group:(12.49±0.63)%, t=10.98, P<0.05], and more γ-H2AX foci in nucleus post-irradiation than negative control (t=22.07, 23.89, 11.24, 59.97, 20.85, P<0.05). Conclusions The KLF5 knockdown intestinal epithelial cell line was successfully established. The down-regulation of KLF5 expression could induce cell arrest at G2/M, suppress the proliferation of irradiated cells and improve the cell apoptosis, enhance DNA double strand breaks and prolong DNA damage repair. |
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