构建稳定表达病毒相关RNA的真核细胞株及其对抗肿瘤免疫促凋亡分子的影响  

Construction of stably expressing virus-associated RNA cell and the influence on anti-tumor immunoproapoptotin expression

作　　者：李昂[1] 伦新新 陈园坤 杨甜 杨安钢[1] 阎博 Li Ang;Lun Xinxin;Chen Yuankun;Yang Tian;Yang Angang;Yan Bo(Department of Immunology,Air Force Medical University,Shaanxi Xi'an 710032,China;School of Labratory Medicine,Xinxiang Medical University,Henan Xinxiang 453000,China;Department of Biochemistry and Molecular Biology,Air Force Medical University,Shaanxi Xi'an 710032,China)

机构地区：[1]中国人民解放军空军军医大学免疫学教研室,陕西西安710032 [2]新乡医学院检验学教研室,河南新乡453000 [3]中国人民解放军空军军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《现代肿瘤医学》2019年第3期367-371,共5页Journal of Modern Oncology

基　　金：国家自然科学基金重点项目(编号:81630069)

摘　　要：目的:构建稳定表达病毒相关RNA(virus-associated RNA,VA RNA)的293F-VA细胞株,并探讨其对靶向HER2阳性肿瘤的免疫促凋亡分子表达水平的影响。方法:采用PCR方法,扩增含VA RNA的功能序列,克隆入Piggy Bac转座系统质粒,将VA RNA编码序列转入293F细胞中,经过抗性筛选和流式细胞仪鉴定,得到293F-VA细胞株。将萤光素酶、红色荧光蛋白及免疫促凋亡分子编码质粒分别转入293F-VA细胞株或亲本293F细胞株,通过萤光素酶活力检测和Western Blot实验,比较两种细胞株对外源目的蛋白表达水平的影响。结果:成功构建了Piggy Bac-VA质粒。共转染Piggy Bac-VA质粒可以明显提高细胞内Fluc蛋白的表达水平(P <0. 05)。VA RNA编码序列通过Piggy Bac转座系统转入293F细胞中,获得293F-VA细胞株。与293F亲本细胞相比,293F-VA细胞株将萤火虫萤光素酶Fluc蛋白的表达水平提高363. 9%(P<0. 05);对海肾萤光素酶Rluc蛋白及红色荧光蛋白m Cherry的表达均表现出3倍左右的提高(P <0. 05)。293F-VA细胞株不仅能够提高免疫促凋亡分子e23-Fc-Fdt-t Bid在细胞内的表达水平,并且显著增加目的蛋白向细胞外分泌的水平,较293F亲本细胞提高4. 2倍(P <0. 05)。结论:建立了稳定表达VA RNA的细胞株293F-VA,该细胞株能够显著提高抗肿瘤免疫促凋亡分子的表达水平。Objective:To discuss the influence on anti-tumor immunoproapoptotin in 293F cell line by constructing a 293F-VA cell line stably expressing virus-associated RNA(VA RNA).Methods:The functional sequence encoding VA RNA was amplified by PCR and cloned into the plasmid of PiggyBac transposon system.The VA RNA coding sequence was transfected into 293F cells using the PiggyBac transposon system,and was identified by resistance screening and flow cytometry to obtain 293F-VA cell line.The plasmids encoding luciferase,red fluorescent protein and immunoproapoptotin were transfected into 293F-VA cell line or parental 293F cell line respectively,and the two cell lines were compared for expression level of exogenous protein by luciferase activity assay and Western Blot.Results:The coding sequence of VA RNA was amplified by PCR,cloned into PiggyBac transposon vector,and constructed into PiggyBac-VA plasmid.PiggyBac-VA plasmid and pcDNA3.1-Fluc plasmid were transiently transfected into 293F cells.The intracellular protein expression of Fluc was significantly increased compared with the control group(P<0.05).The VA RNA coding sequence was transfected into 293F cells by the PiggyBac transposon system.After puromycin resistance screening and validation by fluorescence microscopy and flow cytometry,almost 100%of the 293F-VA cells were EGFP-positive.The intracellular protein expression capacity of pcDNA3.1-Fluc plasmid was increased by 363.9%in the 293F-VA cell line(P<0.05).The intracellular protein expression capacity of pcDNA3.1-Rluc plasmid was increased by 302.2%(P<0.05).The intracellular protein expression capacity of plasmid pcDNA3.1-mCherry was increased by 3 fold(P<0.05).The intracellular expression level of the immunoproapoptotin plasmid pcDNA3.1-e23-Fc-Fdt-tBid was increased by 1.2-fold(P<0.05),and the secretion level was increased by 4.2-fold(P<0.05).Conclusion:A stable cell line 293F-VA expressing VA RNA was established.The cell line could significantly improve the expression of anti-tumor immunoproapoptotin.

关 键 词：病毒相关RNA 哺乳动物细胞蛋白表达系统 293F细胞 肿瘤靶向治疗 

分 类 号：R730.54[医药卫生—肿瘤]