| 刘建香,苏旭.辐射诱导表达载体pEgr-p16的构建及其体外蛋白表达的研究[J].中华放射医学与防护杂志,2005,25(5):416-419 |
| 辐射诱导表达载体pEgr-p16的构建及其体外蛋白表达的研究 |
| Construction of expression vector pEgr-p16 and determination of p16 in vitro expression induced by ionizing radiation |
| 投稿时间：2005-01-18 |
| DOI： |
| 中文关键词: 人抑癌基因p16 pEgr-p16 辐射诱导表达载体 基因放射联合治疗 |
| 英文关键词:Human suppressor gene p16 pEgr-16 gene Expression vector induced by ionization radiation Gene combined with radiation therapy |
| 基金项目:中国科学院近代物理研究所重离子加速器国家实验室课题基金资助 |
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| 中文摘要: |
| 目的 构建辐射诱导表达载体pEgrp16并研究其体外稳定转染联合60Coγ射线照射对人宫颈癌HeLa细胞p16蛋白表达变化的影响。方法 利用双酶切、粘端连接的方法构建了含有辐射诱导特性的早期生长反应因子Egr1和p16的pEgrp16的质粒载体,以脂质体介导的方法,将重组载体导入人HeLa细胞,采用免疫细胞化学和流式细胞术的方法检测照射转染后的HeLa细胞p16蛋白表达的变化。结果 经全自动测序证明辐射诱导表达载体pEgrp16构建正确;稳定转染可见有明显的p16表达增强;5Gy以内p16蛋白表达呈剂量依赖性的增加,在2Gy照射后2hp16蛋白表达水平即开始增高,4h达到最高,12h趋于正常水平。结论 本研究成功构建了辐射诱导表达载体pEgrp16,在HeLa细胞中蛋白表达明显增强。 |
| 英文摘要: |
| Objective In this study, a recombinant plasmid pEgr-p16 was constructed, and the protein expression of p16 in HeLa cells was determined after the cells were transfected in combination with 60Co γ-rays irradiation. Methods The pEgr-p16 recombinant plasmid containing radio-sensitive promoter Egr-1 and p16 encoding sequence was constructed by double-enzyme cleavage and sticky end ligation techniques. pEgr-p16 plasmid was packed with lipofectamine to transfect human cervix epithelial carcinoma HeLa cell line, and p16 protein level was detected by immunocytochemistry and FCM. Results The plasmid pEgr-p16 was constructed correctly as confirmed by sequencing in both directions. The expression level of p16 protein was increased significantly. The level of p16 expression was dose-dependently increased below 5 Gy irradiation. The p16 level began to increase at 2 h after 2 Gy irradiation and there was a significant increase at 4 h, then gradually decreased to the control level at 12 h. Conclusion The expression vector pEgr-p16 which can be induced by ionizing radiation has been constructed successfully in this study. The expression level of p16 protein in HeLa cells increases markedly. |
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