腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

中国生物工程杂志

2011, Vol. 31

Issue (11): 24-30

研究报告

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

张凤娟, 杨吉成, 盛伟华, 王家融, 缪竞诚

苏州大学基础医学与生物科学学院细胞生物学系苏州 215123

The Growth-inhibitory Effect of Recombinant OSM-Adenovirus on A375 Melanoma Cells

ZHANG Feng-juan, YANG Ji-cheng, SHENG Wei-hua, WANG Jia-rong, MIAO Jing-cheng

Department of Cell Biology, School of Biology & Basic Medical Sciences, Medical College of Soochow University, Suzhou 215123, China

全文: PDF(712 KB) HTML

摘要：

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。

关键词： 抑瘤素OSM; 腺病毒载体; 构建; 黑色素瘤; 凋亡

Abstract:

Objective: To construct recombinant adenovirus harboring OSM gene and to study its growth-inhibitory effects on A375 melanoma cells. Methods: In order to obtain the recombinant OSM Adenovirus vector, PCR technique was used and PEGZ-OSM was used as the template.Then the recombinant Ad-OSM was obtained by gene recombination and in vitro packaging technique. A375 cells were infected with Ad-OSM and RT-PCR was used to detect the transcription of OSM in the cells. Western blot was used to detect the expression of OSM in the cells. Cell growth inhibition was detected by MTT assay. Morphologic changes were observed by light microscope and fluorescence microscope. The cell cycle and apoptosis were detected by flow cytometry and RT-PCR was used to detect the transcription of Bax and Bcl-2. Results: Ad-OSM was proved in correct construction by gene sequence and RT-PCR results. Expression of OSM was identified by RT-PCR.By microscope and flow cytometry displayed the apoptosis of the melanoma cells positively expressing OSM. Additionally,the mRNA of Bax was up-regulated and mRNA of Bcl-2 was down-regulated in the cells by RT-PCR.Conclusion: Ad-OSM was proved in correct construction. Ad-OSM can inhibit the growth of A375 cells and induce cell apoptosis,which may result in changing Bax and Bcl-2 expression.

Key words: Apoptosis Oncostatin M Adenovirus vector Construction Melanoma

收稿日期: 2011-06-07 出版日期: 2011-11-25

ZTFLH:

Q81

基金资助:

国家自然科学基金资助项目(81001016)

通讯作者: 缪竞诚 E-mail: jcmiao@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

张凤娟, 杨吉成, 盛伟华, 王家融, 缪竞诚. 腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用[J]. 中国生物工程杂志, 2011, 31(11): 24-30.

ZHANG Feng-juan, YANG Ji-cheng, SHENG Wei-hua, WANG Jia-rong, MIAO Jing-cheng. The Growth-inhibitory Effect of Recombinant OSM-Adenovirus on A375 Melanoma Cells. China Biotechnology, 2011, 31(11): 24-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/24

[1] Glenn M,Frances P N. Modeling gene-environment interactions in malignant melanoma.TRENDS in Molecular Medicine,Review,2003,9(3):102-108.

[2] Thomas B,Meenlaard H. Cell-surface proteolysis,growth factor activation and intercellular communication in the progression of melanoma.Oncology Hematology,2002,44(1):1-15.

[3] Monica R,Maria D,Viviana V,et al.Genetic progression of metastatic melanoma.Cancer letters,2004,214(2):133-147.

[4] Catherine G,Wenbin M,Francesca S,et al.High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens. J Exp Med, 2004,201(2):241-248.

[5] 陈瑶,缪竞诚,韩亚丽,等.IRES/polyA-promoter介导的Ang-1与VEGF165双基因表达效率的对比分析.中国生物工程杂志,2010,30(12):10-19. Chen Y,Miao J C,Han Y L,et al. China Biotechnology,2010, 30(12):10-19.

[6] 金晓琳. 抑瘤素M 的研究进展. 细胞与分子免疫学杂志,1997,13 (增刊2):31-33. Jin X L. Chinese Journal of Cellular and Molecular Immunology,1997,13(supplementary issue 2):31-33.

[7] Robbins P D,Chivizzani S C. Viral vector for genetherapy.Pharmacology & Therapeutics,1998,80(1):35-47.

[8] Dougherty J P, Temin H M. High mutation rate of a spleen necrosis virus-based retrovirus vector. Mol Cell Biol, 1986, 6(12):4387-4395.

[9] Bruce A G,Linsley P S,Rose T M,et al.Oncostatin M.Prog Growth Factor Res,1992,4(2):157-170.

[10] 聂晶,范永星,赵洪礼. 抑瘤素M 对照射后小鼠造血系统的影响.沈阳部队医药,2004,17:267-268. Nie J,Fan Y X,Zhao H L. Shenyang Budui Yiyao,2004,17:267-268.

[11] Minehata K,Takeuchi M,Hirabayashi Y,et al. Oncostatin m maintains the hematopoietic microenvironment and retains hematopoietic progenitors in the bone marrow. Int J Hematol,2006,84(4):319-327.

[12] Gearing D P,Comeau M R,Griend D J,et al. The IL-6 signal transducer gp130:An oncostatin M receptor and affinity converter for the LIF receptor. Science, 1992, 255(5050): 1434-1437.

[1]	黄蕾,万常青,刘美琴,赵敏,郑妍鹏,彭向雷,虞结梅,付远辉,何金生. 利用DNA Assembly方法构建重组腺病毒载体[J]. 中国生物工程杂志, 2021, 41(6): 23-26.
[2]	陶守松,任广明,尹荣华,杨晓明,马文兵,葛志强. 敲低去泛素化酶USP13抑制K562细胞的增殖^*[J]. 中国生物工程杂志, 2021, 41(5): 1-7.
[3]	段阳阳,张凤亭,成江,石瑾,杨娟,李海宁. SIRT2抑制对MPP⁺诱导的帕金森病细胞模型凋亡和线粒体动态平衡的影响^*[J]. 中国生物工程杂志, 2021, 41(4): 1-8.
[4]	郭利成,曹雪玮,傅龙云,王富军,赵健. 一种用于药物蛋白亲和纯化和跨膜转运的双功能标签的开发 ^*[J]. 中国生物工程杂志, 2020, 40(6): 40-52.
[5]	何询,张鹏,张俊祥. 类器官的构建与应用进展[J]. 中国生物工程杂志, 2020, 40(12): 82-87.
[6]	朱永朝,陶金,任萌萌,熊燃,何亚琴,周瑜,卢震辉,杜勇,杨芝红. 自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 ^*[J]. 中国生物工程杂志, 2019, 39(9): 62-67.
[7]	刘叶,潘玥,郑魏,胡晶. miR-186-5p在酒精诱导的心肌细胞中高表达并通过靶基因XIAP调控细胞凋亡水平 ^*[J]. 中国生物工程杂志, 2019, 39(5): 53-62.
[8]	马占兵,党洁,杨继辉,霍正浩,徐广贤. 基于慢病毒系统的双荧光标记多功能自噬流监测系统建立与应用 ^*[J]. 中国生物工程杂志, 2019, 39(5): 88-95.
[9]	汪路,杨丽媛,唐雨婷,陶瑶,雷力,敬一佩,蒋雪坷,张伶. 干扰PKM2对人白血病细胞增殖和凋亡的影响及潜在机制 ^*[J]. 中国生物工程杂志, 2019, 39(3): 13-20.
[10]	黄翔,杨杰,何佩彦,吴志慧,曾慧兰,王新宁,蒋建伟. 白花地胆草单体EM-12诱导2774-C10细胞G₁/S期阻滞及细胞凋亡的分子机制研究[J]. 中国生物工程杂志, 2018, 38(4): 17-23.
[11]	代立婷, 吴忠南, 黄翔, 杨杰, 曾慧兰, 王国才, 蒋建伟. 卤地菊乙醇提取物W40单体诱导GLC-82细胞凋亡的分子机制研究[J]. 中国生物工程杂志, 2017, 37(8): 1-7.
[12]	徐安健, 李艳萌, 李斯文, 乌姗娜, 张蓓, 黄坚. PHP14沉默对肺癌细胞凋亡的影响及其机制[J]. 中国生物工程杂志, 2017, 37(7): 12-17.
[13]	田聪慧, 谢雪梅, 李英, 尹晓东, 韩军, 李军. 基于IRES序列的多基因共表达载体构建[J]. 中国生物工程杂志, 2017, 37(7): 97-104.
[14]	李艳伟, 马义, 韩磊, 肖兴, 党诗莹, 文涛, 王得华, 范志勇. Fas凋亡抑制分子FAIM 1表达缺失诱发单纯性肥胖的初步研究[J]. 中国生物工程杂志, 2017, 37(6): 37-42.
[15]	白欣艳, 温丽敏, 王玉晶, 王海龙, 解军, 郭睿. ANKRD49通过上调Bcl-xL的表达抑制UV诱导GC-1细胞的凋亡[J]. 中国生物工程杂志, 2017, 37(4): 40-47.

Viewed

Full text

Abstract

Cited

Shared

Discussed