骨髓间充质干细胞介导NIS基因及金纳米颗粒治疗乳腺癌的实验研究  

Vitro experiment of bone marrow mesenchymal stem cells mediated NIS gene and gold nanoparticles in the treatment of breast cancer

作　　者：张璐[1] 郭睿[1] 郑本超 苗莹[1] 张春富[2] 李彪[1] Zhang Lu;Guo Rui;Zheng Benchao;Miao Ying;Zhang Chunfu;Li Biao(Department of Nuclear Medicine,Ruijin Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200025,China;School of Biomedical Engineering&Med-X Research Institute,Shanghai Jiao Tong University,Shanghai 200030,China)

机构地区：[1]上海交通大学医学院附属瑞金医院核医学科,200025 [2]上海交通大学Med-X研究院、生物医学工程学院,200030

出　　处：《中华核医学与分子影像杂志》2020年第5期275-280,共6页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家自然科学基金(81471686,81671715)。

摘　　要：目的制备转染治疗基因早期生长反应蛋白1(Egr1)-钠碘同向转运体(NIS)并携带金纳米颗粒(AuNPs)的骨髓间充质干细胞(BMSCs),探讨Egr1对NIS表达的促进和AuNPs的辐射增敏作用。方法采用慢病毒(Lv)-Egr1-NIS-巨细胞病毒(CMV)-绿色荧光蛋白(GFP)及Lv-Egr1-GFP颗粒对BMSCs进行基因转染,制备BMSCs-Egr1-NIS及BMSCs-Egr1-GFP(对照)。通过摄碘实验验证在不同放射性浓度碘诱导下NIS基因的表达;用激光共聚焦显微镜观察BMSCs吞噬AuNPs的最佳温育时间、质量浓度;进行细胞毒性实验,分析AuNPs对BMSCs-Egr1-NIS细胞活性的影响;通过摄碘实验研究BMSCs-Egr1-NIS吞噬和未吞噬AuNPs对基因表达的影响;用细胞迁移实验验证BMSCs-Egr1-NIS在吞噬和未吞噬AuNPs的情况下对乳腺癌细胞MDA-MB-231的体外靶向性;探索不同质量浓度AuNPs对131I杀伤乳腺癌细胞MDA-MB-231的辐射增敏作用。采用单因素方差分析及Dunnett t检验比较多组间数据差异。结果成功制备转染治疗基因Egr1-NIS的BMSCs(非稳转),即BMSCs-Egr1-NIS。在辐射诱导下Egr1可以增加NIS的表达,BMSCs-Egr1-NIS较BMSCs-Egr1-GFP摄碘能力提高2.5~5倍或更高;BMSCs吞噬AuNPs的最佳温育条件是AuNPs 0.20 g/L温育24 h或者0.10 g/L温育48 h;AuNPs的细胞毒性非常低,对细胞的摄碘能力和体外靶向性没有影响;BMSCs-Egr1-NIS对乳腺癌细胞MDA-MB-231具有体外靶向性;AuNPs对131I的辐射增敏实验示,各131I杀伤组与无131I的空白对照组活细胞染色吸光度(A)570差异有统计学意义(F=60.670,P<0.01),AuNPs质量浓度为0.20和0.40 g/L的实验组131I对MDA-MB-231细胞的杀伤作用高于0 g/L组,A570 nm值分别为0.87±0.05、0.41±0.07和1.39±0.11(均P<0.01)。结论BMSCs可转染治疗基因Egr1-NIS并携带AuNPs,作为靶向乳腺癌的载体,在放射性碘的作用下增强NIS基因表达,同时AuNPs可作为131I治疗的辐射增敏剂。Objective To prepare bone marrow mesenchymal stem cells(BMSCs)transfected with therapeutic gene early growth reactive protein 1(Egr1)-sodium/iodine symporter(NIS)and carrying gold nanoparticles(AuNPs),and to investigate the feasibility of Egr1 in promoting NIS expression and the radiasensitization effect of AuNPs.Methods BMSCs transfected with lentivirus(Lv)-Egr1-NIS-cytomegalovirus(CMV)-green fluorescent protein(GFP)in the experimental group and Lv-Egr1-GFP in the control group were prepared and the expression of NIS induced by radioiodine was verified by iodine uptake determination.The optimal incubation time and concentration of AuNPs were observed with laser confocal microscopy.The cytotoxicity of AuNPs suspension liquid was investigated using cytotoxicity test.Iodine uptake assay was performed to investigate NIS gene expression of BMSCs-Egr1-NIS incubated with AuNPs.In vitro chemotaxis of BMSCs-Egr1-NIS incubated with/without AuNPs to breast cancer cells were verified by cell migration experiment.The radiosensitization effect of AuNPs for 131I on killing breast cancer cells MDA-MB-231 were explored.The one-way analysis of variance and Dunnett t test were used for data analysis.Results BMSCs-Egr1-NIS(unstable transformation)was successfully prepared.Egr1 could promote NIS expression with the induction of radioiodine.The iodine uptake capacity in BMSCs-Egr1-NIS increased by 2.5-5 times or even higher compared with BMSCs-Egr1-GFP.The better incubation conditions of AuNPs for BMSCs phagocytosis were 0.20 g/L(24 h)or 0.10 g/L(48 h).The cytotoxicity of AuNPs was low in appropriate incubation time and concentration,and there was no effect on iodine uptake and chemotaxis.The chemotaxis to MDA-MB-231 of BMSCs-Egr1-NIS was identified.AuNPs radiasensitization assay showed that absorbance(A)570 nm of MDA-MB-231 cells were significantly deferent in 131I killing groups and blank control group without 131I(F=60.670,P<0.01),and the cytotoxicity of 131I to MDA-MB-231 cells in the 131I killing groups with 0.20 g/L AuNPs and 0

关 键 词：间质干细胞 骨髓 转染 早期生长反应蛋白质 钠碘转运体 金属纳米粒子 乳腺肿瘤 肿瘤细胞 培养的 

分 类 号：R737[医药卫生—肿瘤]