论文部分内容阅读
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。50μmol/L和100μmol/L的NO联合Ad-UPⅡ-E1A 30MOI作用后能够促进肿瘤细胞的增殖,而200μmol/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。
Objective: To study the effects of nitric oxide (NO) on tumor-specific oncolytic virus transfection and the regulation of exogenous gene expression. Methods: Tissue-specific oncolytic adenovirus was constructed, and bladder tumor BIU-87 and 5637 were routinely cultured. Sodium nitroprusside was used as donor of exogenous NO. PTIO and L-NMMA were used as inhibitors of endogenous NO scavenger and inducible nitric oxide synthase (NOS) respectively. Nitrate / Nitrite Assay Kit was used to detect NO levels in bladder tumor cell culture medium before and after the action of NO and / or virus. The effects of NO on the proliferation of recombinant virus were detected by MTT assay. The changes of adenovirus particles entering cell and subcellular structure were observed by transmission electron microscopy. Results: The level of NO in bladder tumor cells BIU-87 and 5637 itself was very low. After exogenous NO donor, NO level increased with time. Recombinant virus Ad-UPⅡ-E1A can exert oncolytic effect through E1A gene. NO promoted the transfection of this virus into BIU-87, 5637 cells. The effects of 50μmol / L and 100μmol / L NO combined with Ad-UPⅡ-E1A 30MOI could promote the proliferation of tumor cells, while 200μmol / L NO combined with recombinant adenovirus could promote the death of tumor cells. The effect of NO on Ad-UPII-E1A is time-dependent. Transmission electron microscopy showed that recombinant adenovirus Ad-UPⅡ-E1A could enter and replicate in bladder tumor cells. However, NO could enhance the transfection efficiency of the virus and induce tumor cell autophagy and apoptosis. CONCLUSION: NO can promote the transfection efficiency of oncolytic adenovirus Ad-UPIIE1A into bladder tumor cells. However, NO can regulate the oncolytic effect of oncolytic adenovirus differently because of its different concentrations. Low concentration of NO can down-regulate the expression of E1A Expression and thus promote the proliferation of tumor cells, and high doses of NO play an oncolytic effect by up-regulating the expression of E1A.