| 闵凤玲,张红,李文建,高清祥,刘兵,周清明,段昕.辐射联合外源性野生型p53基因对不同p53状态的黑色素瘤细胞系的生物学效应[J].中华放射医学与防护杂志,2007,27(2):113-116 |
| 辐射联合外源性野生型p53基因对不同p53状态的黑色素瘤细胞系的生物学效应 |
| Biologic effect of exogenous wild p53 combined with irradiation on human melanoma cell lines with different p53 status |
| 投稿时间：2006-04-17 |
| DOI： |
| 中文关键词: p53 腺病毒载体 辐射 基因治疗 黑色素瘤 |
| 英文关键词:p53 gene Adenovirus vector Irradiation Gene therapy Melanoma |
| 基金项目:中国科学院2002 年百人计划基金资助项目; 科技部重大基础研究前期研究专项(2003CCB00200) |
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| 全文下载次数: 2013 |
| 中文摘要: |
| 目的 研究辐射结合腺病毒(Ad CMV)载体介导的p53基因转导对不同p53状态的人黑色素瘤细胞系基因转移效率、凋亡和辐射敏感性的影响。方法 用复制缺陷的重组腺病毒载体(AdCMV-p53)介导人p53基因转导1 Gy X射线预照射的黑色素瘤细胞系A375(wt p53)和WM983a(mu p53),RT-PCR检测mRNA水平,流式细胞仪测定细胞周期阻滞及外源性P53蛋白表达情况,Tunel法检测细胞凋亡,克隆形成率测定辐射后细胞存活率。用携带报道基因的复制缺陷重组腺病毒载体AdCMV-GFP作为对照。结果 1 Gy X射线照射可较高地增加AdCMV-p53对A375和WM983a细胞系的基因转导效率,转导的外源性野生型p53可在两种细胞中高效表达,并诱导细胞周期G1期阻滞;单纯转导p53对A375(wt p53)细胞无明显诱导凋亡和生长抑制效应,但可部分诱导WM983a(mu p53)细胞凋亡;而转导p53基因48 h后给予X射线辐射,两种细胞的克隆存活率较其对照组均明显减低,外源性p53基因对WM983a(mu p53)细胞的辐射增敏作用较A375(wt p53)细胞明显。结论 外源性野生型p53基因过表达可增加黑色素瘤细胞系A375和WM983a的辐射敏感性,但对WM983a细胞系的辐射增敏作用高于A375细胞系。表明p53是基因治疗黑色素瘤较好的候选基因。 |
| 英文摘要: |
| Objective To investigate the effect of low dose irradiation on gene transfer efficiency and the effect of adenoviral-mediated exogenous P53 overexpression on apoptosis and radiosensitivity of radioresistant human melanoma cell lines A375(wild type p53)and WM983a(mutant type p53). Methods Control vector, a replication deficient recombinant adenoviral vector containing a CMV promoter and green fluorescent protein (AdCMV-GFP), was used to transfect A375 cells and WM983a cells preirradiated with or without 1 Gy X-ray. The transduction efficiency of GFP gene was determined with fluorescence microscope directly. These two types of cells irradiated by 1 Gy X-ray were transfected with a replication deficient recombinant adenoviral vector carrying human wild p53 (AdCMV-p53), and mRNA level was detected by RT-PCR. The cell cycle delay and the expression of exogenous P53 were detected using flow cytometry (FCM) at different times after transfection. Tunel technique was used to detect cell apoptosis. The radiosensivity of A375 and WM983a cells after p53 transduction was analyzed by colony formation. Results It is found that 1 Gy irradiation increased the gene transfection efficiency of A375 and WM983a cells. The expression of exogenous P53 was found to range from 60% to 80% among transfected cells during the first three days after transduction and then declined continuously down to the control level on day 10. G1 cell cycle arrest was also observed after p53 gene transduction. WM983a cells transfected with p53 showed higher sensitivity to X-ray-induced cell killing than A375 cells. Conclusions It is indicated that low dose of ionizing radiation can improve gene transfection efficiency of A375 and WM983a cells mediated by adenovirus vector. Althrough the overexpresion of exogenous p53 may not inhibit cell growth and induce apoptosis of melanoma cell line A375 and WM983a in vitro, the two cell lines are much more sensitive to cell death induced by irradiation. It is suggested that p53 might be a potential gene for melanoma. |
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