一氧化氮对组织特异性溶瘤腺病毒转染膀胱肿瘤细胞的影响

田俊强; 李仁举; 王志平; 卢建中; 付生军; 史庭凯; 马宝良; 熊彬; 陶燕

一氧化氮对组织特异性溶瘤腺病毒转染膀胱肿瘤细胞的影响

- 1 兰州大学第二医院泌尿外科;兰州大学第二医院泌尿外科研究所;甘肃省泌尿系疾病重点实验室(兰州, 730030);
- 2 兰州大学第二医院血液科
基金项目:
国家自然科学基金项目(No:30800206/C050605)

兰州大学中央高校基本科研业务费专项资金资助(No:lzujbky-2009-103)

详细信息

通讯作者: 田俊强,E-mail:tjq007263@sina.com

中图分类号: R737.14

收稿日期: 2011-09-15

The effect of nitric oxide on the tissue-specific oncolytic adenovirustransfection of bladder tumor cells

- 1. Department of Urology, Institute of Urology, the Second Hospital of Lanzhou University;
- 2. the Key Laboratory of Urinary Diseases of Gansu Province, Lanzhou, 730030, China;

More Information

Corresponding author: TIAN Junqiang, E-mail: tjq007263@sina.com

Received Date: 15 September 2011

摘要

摘要: 目的:研究一氧化氮(NO)对肿瘤组织特异性溶瘤病毒转染过程的影响及对外源基因表达的调节作用。方法:构建组织特异性溶瘤腺病毒,常规培养膀胱肿瘤BIU-87和5637细胞株,以硝普钠作为外源性NO的供体。应用PTIO和L-NMMA分别作为内源性NO的清除剂和诱导型一氧化氮合酶(NOS)的抑制剂。采用Nitrate/Nitrite Assay Kit检测NO和(或)病毒作用前后膀胱肿瘤细胞培养液中的NO水平。应用四甲基偶氮唑盐(MTT)法检测NO对重组病毒抗肿瘤细胞增殖的影响;透射电镜观察腺病毒颗粒进入细胞情况和亚细胞结构变化。结果:膀胱肿瘤细胞BIU-87和5637本身培养液中NO水平很低,给予外源性NO供体后NO水平随时间延长而升高。重组病毒Ad-UPⅡ-E₁A 能通过E1A基因发挥溶瘤作用。NO能够促进该病毒转染入BIU-87、 5637细胞。50 μmol/L和100 μmol/L的NO联合Ad-UPⅡ-E₁A 30 MOI作用后能够促进肿瘤细胞的增殖,而200 μmol/L的NO联合重组腺病毒作用后则促进肿瘤细胞的死亡。NO对Ad-UPⅡ-E₁A 的作用具有时间依赖性。透射电镜观察发现,重组病毒Ad-UPⅡ-E₁A 能够进入并在膀胱肿瘤细胞内复制,而NO能够提高病毒的转染效率并引起肿瘤细胞自吞噬和凋亡。结论:NO能够促进溶瘤腺病毒Ad-UPIIE₁A转染膀胱肿瘤细胞的效率,但NO因其浓度不同对溶瘤腺病毒的溶瘤效果具有双向调节作用,低剂量的NO能够下调重组病毒E1A的表达从而促进肿瘤细胞增殖,而高剂量的NO通过上调E1A的表达因而发挥溶瘤效应。
- 膀胱肿瘤 /
- 一氧化氮 /
- 溶瘤腺病毒 /
- 基因治疗
Abstract: Objective:To study the effect of nitric oxide(NO) on tumor-specific oncolytic adenovirus transfection process and the impact of exogenous gene expression regulation. Methods:A tissue-specific oncolytic adenovirus was constructed previously. BIU-87 and 5637 bladder cancer cell lines were cultured. Sodium nitroprusside (SNP), Carboxy-PTIO (PTIO) and NG-Momomethethy-L-arginine (L-NMMA) were applied respectively as exogenous NO donor, endogenous NO scavenger and nitric oxide synthase(NOS) inhibitor. NO level in cell culture medium before and after SNP and/or virus intervention was detected by Nitrate/Nitrite Assay. The proliferation of bladder cancer cells before and after intervention by recombinant viruses and / or SNP detected by MTT assay. Transmission electron microscopy(TEM) was used to observe adenovirus particles within cells and the changes of subcellular structure.Results:Low levels of NO were detected in BIU-87 or 5637 cell culture medium itself, but NO concentration increased with time when cells exposed to exogenous NO donor SNP. Recombinant virus Ad-UPⅡ-E₁A has high purity and titer, and plays the oncolytic effect through E1A gene. NO can promote the virus transfer into BIU-87, 5637 cells, 50 μmol/L or 100 μmol/L NO combined with Ad-UPⅡ-E₁A 30 MOI can enhance
- bladder cancer /
- nitric oxide /
- oncolytic adenovirus /
- gene therapy

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