| GCF低表达宫颈癌HeLa细胞系的构建及辐射对其调控IER5基因表达的初步探究 |
| 投稿时间：2014-10-05 修订日期：2014-10-20 点此下载全文 |
| 引用本文：史海敏,丁库克,周平坤,陈丹,郭冬梅,赵春丽,张新.GCF低表达宫颈癌HeLa细胞系的构建及辐射对其调控IER5基因表达的初步探究[J].医学研究杂志,2015,44(4):37-41 |
| DOI：
10.11969/j.issn.1673-548X.2015.04.011 |
| 摘要点击次数: 1583 |
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| 基金项目:国家自然科学基金资助项目(30770573/31170806);北京市自然科学基金资助项目(7092013);沈阳市科技攻关项目(F12193948) |
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| 中文摘要:目的 构建GCF低表达的宫颈癌HeLa细胞系,在60Co γ射线照射下初步探究其细胞增殖以及调控宫颈癌放射敏感基因IER5的表达。 方法 经带有荧光标记的GCF低表达的质粒稳定转染HeLa细胞, G418筛选,蛋白印迹法、实时定量PCR法鉴定得到GCF-shRNA-HeLa单克隆细胞系。倒置显微镜观察细胞形态变化,CCK-8法探究不同剂量照射后的细胞增殖情况,在不同照射剂量下用蛋白印迹法检测GCF-shRNA-HeLa及对照细胞中IER5蛋白的表达情况。 结果 成功构建稳定表达的GCF-shRNA-HeLa单克隆细胞系,显微镜下观察GCF-shRNA-HeLa细胞比正常HeLa细胞体积小,细胞间隙较大,其中梭形细胞较多。蛋白印迹法及实时定量PCR验证了GCF-shRNA-HeLa的蛋白表达及mRNA转录水平均低于对照组(P<0.05)。辐射情况下实验结果显示在同一照射剂量、同一时间下GCF-shRNA-HeLa细胞比control对照细胞增殖缓慢(P=0.014)。不同剂量照射情况下蛋白印迹法检测HeLa细胞、control-shRNA-HeLa与GCF-shRNA-HeLa细胞IER5蛋白表达情况,结果同一辐照剂量下GCF沉默的细胞系中IER5的表达量比对照细胞高, 4Gy照射的GCF-shRNA-HeLa的IER5表达量最高(P<0.05)。 结论 本实验构建了低表达GCF的HeLa细胞系,初步探讨GCF作为IER5基因抑制性转录因子的作用。结合临床基因靶向治疗,可寻求可以降低GCF表达的方式,通过上调IER5表达这一双效途径来加速宫颈癌细胞凋亡。 |
| 中文关键词:GCF IER5 HeLa细胞 细胞增殖 |
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| Constructing the Cervical Cancer HeLa Cell Line with Lower Expression of GCF Gene and Gxplore the Radiational Effects of it's Regulating on IER5 Gene |
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| Abstract:Objective To construct and identify the HeLa cell line which express low level of GCF gene, investigate the effects on its proliferation and cell-cycle changes and IER5 gene after 60Co γ radiation. Methods GCF low expression vector was stably transfected into the HeLa cells and then we got GCF-shRNA-HeLa monoclonal cells line by G418 screening. Western blot and real-time PCR were used to identificated it. The morphological characteristics of cells was observed with light microscope.Cells' proliferation was counted by CCK-8. IER5 protein expression in GCF-shRNA-HeLa cell line and control cells were detected by Western blot. Results Constructing the HeLa cell line which express low level of GCF gene was successfully and the conclusion was that this cell line had smaller size,bigger intercellular space and more fusiform cells than normal HeLa cells observed by microscope. The results of Western blot and real-time PCR showed the level of mRNA of GCF-shRNA-HeLa lines was lower than control groups(P<0.05).At the same time and radiation dose, the proliferation of GCF-shRNA-HeLa lines were slower than control cells(P<0.05). At the same radition dose,the expression of IER5 in GCF-shRNA-HeLa was higher than control groups, and the 4Gy was the highest point. The results had significant difference P<0.05. Conslusion We confirmed that the GCF could effectively knock down in HeLa cells, established the stable GCF-shRNA-HeLa cell line and validated GCF's influences as IER5 gene's inhibitive transcription factor. Combined with clinical gene targeted therapy,we could explore the way to reduce the expression of GCF to accelerate the apoptosis of cervical cancer cells by up-regulating IER5. |
| keywords:GCF IER5 HeLa Cells cell proliferation |
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