双特异性溶瘤腺病毒通过调节活性氧引起人肺癌细胞凋亡
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摘要
目的探讨双特异性溶瘤腺病毒ATV引起人肺癌A549细胞凋亡与活性氧含量之间的关系。方法应用本实验室构建的双特异性溶瘤腺病毒ATV,通过CCK-8法检测ATV对A549细胞增殖的抑制作用;通过Hoechst和Annexin V-FITC/PI流式检测法检测A549细胞的凋亡情况;通过人活性氧Elisa试剂盒测定A549细胞内活性氧水平。结果 CCK-8法检测结果表明,ATV感染剂量为10 MOI和50 MOI作用时间为48 h和72 h时,对A549细胞具有显著性的抑制作用。Hoechst染色结果显示,感染ATV的A549细胞呈现核碎裂典型的浓染和边集现象,具备细胞凋亡的典型特征;Annexin V-FITC/PI流式检测结果表明ATV在12、24、48、72 h均能诱导A549细胞不同程度的凋亡,并且凋亡率随时间增长而提高;胞浆活性氧检测结果表明,ATV感染A549细胞12、24、48、72 h,A549细胞活性氧水平随时间的增长而提高,具有时间效应。结论 ATV对A549细胞有抑制作用且该作用具有明确的时效与剂效关系;ATV通过使A549细胞内活性氧含量增加引起细胞凋亡进而发挥对A549细胞的抑制作用。
Objective To investigate the relationship between apoptosis and the level of reactive oxygen in ATVinfected A549 cells. Methods The dual cancer-specific oncolytic adenovirus ATV was constructed in our laboratory. The suppression effect of ATV-infected A549 cells was detected by CCK-8 cell proliferation assay. The apoptosis of ATV-infected A549 cells was analyzed by Hoechst staining and Annexin V-FITC/PI flow cytometer detection. The level of reactive oxygen in ATV-infected A549 cells was evaluated by human reactive oxygen Elisa kit. Results The results of CCK-8 cell proliferation assay showed that A549 cells were suppressed significantly by 10 MOI or 50 MOI ATV at 48 h and 72 h. The results of hoechst staining were showed that typical characteristics of apoptosis and typical nuclear fragmentation in ATV-infected A549 cells could be observed. The result of Annexin V-FITC/PI flow detection was showed that apoptosis of A549 cells was induced by ATV at 12 h, 24 h, 48 h and 72 h, and the apoptosis rate increased with time. The result of cytoplasm reactive oxygen detection was showed that the level of reactive oxygen in ATV-infected A549 cells increases with time at 12 h, 24 h, 48 h and 72 h, which has a significant time effect. Conclusion ATV has suppression effect on A549 cells, and this effect has a clear time-dependent and dose-dependent relationship. The apoptosis of A549 cells can be induced by ATV via causing the increase of reactive oxygen level in A549 cell.
Objective To investigate the relationship between apoptosis and the level of reactive oxygen in ATVinfected A549 cells. Methods The dual cancer-specific oncolytic adenovirus ATV was constructed in our laboratory. The suppression effect of ATV-infected A549 cells was detected by CCK-8 cell proliferation assay. The apoptosis of ATV-infected A549 cells was analyzed by Hoechst staining and Annexin V-FITC/PI flow cytometer detection. The level of reactive oxygen in ATV-infected A549 cells was evaluated by human reactive oxygen Elisa kit. Results The results of CCK-8 cell proliferation assay showed that A549 cells were suppressed significantly by 10 MOI or 50 MOI ATV at 48 h and 72 h. The results of hoechst staining were showed that typical characteristics of apoptosis and typical nuclear fragmentation in ATV-infected A549 cells could be observed. The result of Annexin V-FITC/PI flow detection was showed that apoptosis of A549 cells was induced by ATV at 12 h, 24 h, 48 h and 72 h, and the apoptosis rate increased with time. The result of cytoplasm reactive oxygen detection was showed that the level of reactive oxygen in ATV-infected A549 cells increases with time at 12 h, 24 h, 48 h and 72 h, which has a significant time effect. Conclusion ATV has suppression effect on A549 cells, and this effect has a clear time-dependent and dose-dependent relationship. The apoptosis of A549 cells can be induced by ATV via causing the increase of reactive oxygen level in A549 cell.
引文
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[9]尹逊哲,陈爽,李文杰,等.携带凋亡素基因的溶瘤腺病毒ATV对人宫颈癌HeLa细胞的抑制作用[J].中国肿瘤生物治疗杂志, 2017, 24(12):1356-1361.
[10]范园园.溶瘤腺病毒Ad-VT对荧光素酶标记人肺癌细胞的抑制作用研究[D].北京:中国人民解放军军事医学科学院, 2017.
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[12]朱文赫,沈楠,徐俊杰,等.微波辐射对大鼠能量代谢及心肌细胞凋亡的影响[J].中国病理生理杂志, 2015, 31(4):647-651.
[13] PRZEMYS?AW S, EWA S, MONIKA T, et al. A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus, L[J]. Tumour Biology the Journal of the International Society for Oncodevelopmental Biology&Medicine, 2016, 37(7):1-12.
[14] QIN F. Nucleophosmin mutations induce chemosensitivity in THP-1 leukemia cells by suppressing NF-κB activity and regulating Bax/Bcl-2 expression[J]. Journal of Cancer, 2016,7(15):2270-2279.
[15]陈国柱,徐元基,杜芝燕,等.雷公藤红素对非小细胞肺癌细胞株H1299增殖与凋亡的影响[J].生物技术通讯,2008, 19(6):826-829.
[16]张志强,高体云,李暐,等.雷公藤红素通过ROS诱导结肠癌SW620细胞凋亡[J].中国医院药学杂志, 2015,35(17):1558-1563.
[17]丁成国.雷公藤红素通过ROS-JNK信号通路诱导T98G细胞发生凋亡[J].浙江实用医学, 2015, 20(5):325-328.
[18]李恒元.雷公藤红素通过ROS/JNK通路诱导骨肉瘤细胞凋亡和自噬的研究[D].杭州:浙江大学, 2015.
[19] MAITI A K. Overcoming drug resistance through elevation of ROS in cancer[J]. Resistance to Targeted Anti-Cancer Therapeutics, 2013(1):135-149.
[20]刘艳霞,张璐玉,崔艳艳,等.β-胡萝卜素对食管鳞癌EC1细胞内活性氧和细胞凋亡的影响[J].郑州大学学报,2018, 53(3):272-276.
[2] LIU T C, GALANIS E, KIRN D. Clinical trial results with oncolytic virotherapy:a century of promise, a decade of progress[J]. Nature Clinical Practice Oncology, 2007, 4(2):101-117.
[3]李文杰,陈智飞,郭丽娇,等.溶瘤腺病毒ATV联合顺铂协同抑制A549细胞的研究[J].中国病原生物学杂志,2017(3):252-257.
[4]王华,王宗平.膀胱癌溶瘤腺病毒治疗研究进展[J].中国肿瘤, 2014, 23(2):148-152.
[5]徐一鹏,王华.溶瘤腺病毒治疗前列腺癌研究进展[J].中国肿瘤, 2015, 24(7):581-588.
[6]兰添.双特异性溶瘤腺病毒Ad--Apoptin--hTERTp--E1A对乳腺癌的体内外抑制作用研究[D].延吉:延边大学, 2016.
[7] LIU L, WU W, ZHU G, et al. Therapeutic efficacy of an hTert promoter-driven oncolytic adenovirus that expresses apoptin in gastric carcinoma[J]. International Journal of Molecular Medicine, 2012, 30(4):747-754.
[8]李霄.双特异性抗肿瘤重组腺病毒的构建、鉴定及实验免疫[D].长春:吉林大学, 2009.
[9]尹逊哲,陈爽,李文杰,等.携带凋亡素基因的溶瘤腺病毒ATV对人宫颈癌HeLa细胞的抑制作用[J].中国肿瘤生物治疗杂志, 2017, 24(12):1356-1361.
[10]范园园.溶瘤腺病毒Ad-VT对荧光素酶标记人肺癌细胞的抑制作用研究[D].北京:中国人民解放军军事医学科学院, 2017.
[11]JAYAVELU A K, M?LLER J P, BAUER R, et al. NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells[J].Experimental Hematology, 2016, 44(12):1113-1122.
[12]朱文赫,沈楠,徐俊杰,等.微波辐射对大鼠能量代谢及心肌细胞凋亡的影响[J].中国病理生理杂志, 2015, 31(4):647-651.
[13] PRZEMYS?AW S, EWA S, MONIKA T, et al. A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus, L[J]. Tumour Biology the Journal of the International Society for Oncodevelopmental Biology&Medicine, 2016, 37(7):1-12.
[14] QIN F. Nucleophosmin mutations induce chemosensitivity in THP-1 leukemia cells by suppressing NF-κB activity and regulating Bax/Bcl-2 expression[J]. Journal of Cancer, 2016,7(15):2270-2279.
[15]陈国柱,徐元基,杜芝燕,等.雷公藤红素对非小细胞肺癌细胞株H1299增殖与凋亡的影响[J].生物技术通讯,2008, 19(6):826-829.
[16]张志强,高体云,李暐,等.雷公藤红素通过ROS诱导结肠癌SW620细胞凋亡[J].中国医院药学杂志, 2015,35(17):1558-1563.
[17]丁成国.雷公藤红素通过ROS-JNK信号通路诱导T98G细胞发生凋亡[J].浙江实用医学, 2015, 20(5):325-328.
[18]李恒元.雷公藤红素通过ROS/JNK通路诱导骨肉瘤细胞凋亡和自噬的研究[D].杭州:浙江大学, 2015.
[19] MAITI A K. Overcoming drug resistance through elevation of ROS in cancer[J]. Resistance to Targeted Anti-Cancer Therapeutics, 2013(1):135-149.
[20]刘艳霞,张璐玉,崔艳艳,等.β-胡萝卜素对食管鳞癌EC1细胞内活性氧和细胞凋亡的影响[J].郑州大学学报,2018, 53(3):272-276.
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