磁性纳米颗粒Fe_3O_4@PEI介导靶向自杀基因联合磁流体热疗对肝癌移植瘤的抑制作用
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摘要
目的:探讨磁性纳米颗粒Fe_3O_4@PEI介导靶向自杀基因联合磁流体热疗对肝癌移植瘤的特异性杀伤作用。方法:亚克隆基因重组法构建靶向肝癌的自杀基因p[HRE]AFP-HSVTK和肿瘤细胞成像报告基因载体p[HRE]AFP-Luc,并用限制性内切酶凝胶电泳法检测重组质粒是否构建成功。共沉淀法制备Fe_3O_4纳米粒并以聚乙烯亚胺(PEI)修饰后得到磁性纳米颗粒Fe_3O_4@PEI,将其作为肿瘤基因治疗的载体和磁流体热疗的介质,并利用透射电镜、粒径仪、傅里叶转换红外光谱等对Fe_3O_4@PEI进行表征鉴定。利用小动物活体成像仪检测Fe_3O_4@PEI系统运送报告基因p[HRE]AFP-Luc至荷瘤裸鼠后的生物发光信号。p[HRE]AFP-HSVTK/Fe_3O_4@PEI作用于肝癌细胞后,以MTT法检测细胞增殖抑制率,流式细胞仪检测凋亡细胞比例,动物实验验证体内肿瘤生长速度及瘤体质量抑制效果,并用透射电镜分析肿瘤组织的亚细胞结构。结果:成功构建磁性纳米颗粒Fe_3O_4@PEI以及重组载体p[HRE]AFP-HSVTK和p[HRE]AFP-Luc,经尾静脉注射Fe_3O_4@PEI运送的p[HRE]AFP-Luc后,活体成像系统显示仅能在裸鼠肿瘤组织检测到明显的成像信号,其主要器官病理组织分析无明显病理损伤。在体外肿瘤细胞杀伤试验中,联合治疗组细胞增殖抑制率分别高于磁流体热疗组和基因治疗组([76.02±7.33)%vs (42.31±4.28)%、(47.76±4.81)%,均P<0.05],联合治疗组瘤细胞的凋亡率高于单独热疗组和基因治疗组([34.05±3.41)%vs (14.41±1.55)%、(11.64±1.20)%,均P<0.01]。体内治疗实验显示,联合治疗组移植瘤体积增长明显减慢甚至下降,瘤块质量显著小于其他单独治疗组(P<0.05);瘤块细胞亚结构出现明显的凋亡形态。结论:自杀基因p[HRE]AFP-HSVTK对肝癌细胞具有选择性杀伤作用,Fe_3O_4@PEI可以作为有效的基因治疗载体和磁流体热疗的介质,其介导的肝癌靶向治疗联合磁流体热疗能特异地抑制肝癌移植瘤。
Objective: To investigate the specific killing effect of magnetic nanoparticles Fe_3O_4@PEI induced targeted suicide gene therapy combined with magnetic fluid hyperthermia on hepatoma xenograft. Methods: The suicide gene targeting hepatoma p[HRE]AFP-HSVTK and tumor cell imaging reporter gene vector p[HRE]AFP-Luc were constructed by sub-cloning gene recombination method, and tested by restriction endonuclease gel electrophoresis. Fe_3O_4 nano-particles were prepared by co-precipitation method and modified by Polyethyleneimine(PEI) to obtain the magnetic nano-particles Fe_3O_4@PEI,which could be used as carrier for tumor gene therapy and a medium for magnetic fluid hyperthermia treatment; and the characterization of Fe_3O_4@PEI was identified by transmission electron microscopy, particle size analyzer and Fourier transform infrared spectroscopy. The reporter genes p[HRE]AFP-Luc were delivered into the nude mice bearing xenografts via tail vein by Fe_3O_4@PEI, then the bioluminescence signals of mice were observed in an IVIS system. After the treatment of p[HRE]AFP-HSVTK/Fe_3O_4@PEI, the tumor cell inhibition rate was examined by MTT assay, the cell apoptosis was tested by Flow cytometry, the in vivo tumor development rate and tumor inhibition rate was tested by animal experiment, and the sub-cellular construction of tumor cells was observed by Transmission electron microscopy. Results: Nano-particles Fe_3O_4@PEI and recombinant vectors p[HRE]AFP-HSVTK and p[HRE]AFP-Luc were successfully constructed; after tale vein injection, image signals were detected only in tumor tissues via IVIS system, but no obvious pathologic damage in other major organs. In the in vitro cell killing test, the cell proliferation inhibition rate and the cell apoptosis rate in combination group was higher than that in hyperthermia treatment group andgene treatment group [inhibition rate:(76.02±7.33)% vs(42.31±4.28)%,(47.76±4.81)%, all P<0.05; apoptosis rate:(34.05±3.41)% vs(14.41±1.55)%,(11.64±1.20)%,all P<0.01]. The in vivo treatment showed that tumor volume development significantly slowed-down and even decreased in combination treatment group, and the tumor mass were significantly smaller than those of the single treatment groups(all P<0.05); and the tumor cell sub-cellular structure showed obvious apoptotic morphology. Conclusion: the suicide gene p[HRE]AFP-HSVTK has specific killing effect on hepatoma cells, Fe_3O_4@PEI can be used as effective gene treatment carrier and media of magnetic heperthermia treatment; Fe_3O_4@PEI mediated target treatment combined with magnetic fluid hyperthermia treatment could specifically inhibit the hepatoma xenograft.
Objective: To investigate the specific killing effect of magnetic nanoparticles Fe_3O_4@PEI induced targeted suicide gene therapy combined with magnetic fluid hyperthermia on hepatoma xenograft. Methods: The suicide gene targeting hepatoma p[HRE]AFP-HSVTK and tumor cell imaging reporter gene vector p[HRE]AFP-Luc were constructed by sub-cloning gene recombination method, and tested by restriction endonuclease gel electrophoresis. Fe_3O_4 nano-particles were prepared by co-precipitation method and modified by Polyethyleneimine(PEI) to obtain the magnetic nano-particles Fe_3O_4@PEI,which could be used as carrier for tumor gene therapy and a medium for magnetic fluid hyperthermia treatment; and the characterization of Fe_3O_4@PEI was identified by transmission electron microscopy, particle size analyzer and Fourier transform infrared spectroscopy. The reporter genes p[HRE]AFP-Luc were delivered into the nude mice bearing xenografts via tail vein by Fe_3O_4@PEI, then the bioluminescence signals of mice were observed in an IVIS system. After the treatment of p[HRE]AFP-HSVTK/Fe_3O_4@PEI, the tumor cell inhibition rate was examined by MTT assay, the cell apoptosis was tested by Flow cytometry, the in vivo tumor development rate and tumor inhibition rate was tested by animal experiment, and the sub-cellular construction of tumor cells was observed by Transmission electron microscopy. Results: Nano-particles Fe_3O_4@PEI and recombinant vectors p[HRE]AFP-HSVTK and p[HRE]AFP-Luc were successfully constructed; after tale vein injection, image signals were detected only in tumor tissues via IVIS system, but no obvious pathologic damage in other major organs. In the in vitro cell killing test, the cell proliferation inhibition rate and the cell apoptosis rate in combination group was higher than that in hyperthermia treatment group andgene treatment group [inhibition rate:(76.02±7.33)% vs(42.31±4.28)%,(47.76±4.81)%, all P<0.05; apoptosis rate:(34.05±3.41)% vs(14.41±1.55)%,(11.64±1.20)%,all P<0.01]. The in vivo treatment showed that tumor volume development significantly slowed-down and even decreased in combination treatment group, and the tumor mass were significantly smaller than those of the single treatment groups(all P<0.05); and the tumor cell sub-cellular structure showed obvious apoptotic morphology. Conclusion: the suicide gene p[HRE]AFP-HSVTK has specific killing effect on hepatoma cells, Fe_3O_4@PEI can be used as effective gene treatment carrier and media of magnetic heperthermia treatment; Fe_3O_4@PEI mediated target treatment combined with magnetic fluid hyperthermia treatment could specifically inhibit the hepatoma xenograft.
引文
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[17]BRüNINGK S,POWATHIL G,ZIEGENHEIN P,et al.Combining radiation with hyperthermia:a multiscale model informed by in vitro experiments[J].J Royal Soc Interfact,2018,15(138):20170681.DOI:10.1098/rsif.2017.0681.
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[19]赵建夫,赵凤芝,陈文慧,等.CIK细胞联合贝伐单抗对肝癌Hep G2细胞的体内外抗瘤活性[J].中国肿瘤生物治疗杂志,2017,24(4):404-410.DOI:10.3872/j.issn.1007-385X.2017.04.012.
[2]MAZZANTI R,GRAMANTIERI L,BOLONDI L.Hepatocellular carcinoma:epidemiology and clinical aspects[J].Mol Aspects Med,2008,29(1/2):130-143.DOI:10.1016/j.mam.2007.09.008.
[3]SMERDOU C,MENNE S,HERNANDEZ-ALCOCEBA R,et al.Gene therapy for HCV/HBV-induced hepatocellular carcinoma[J].Curr Opin Investig Drugs,2010,11(12):1368-1377.
[4]JOHANNSEN M,THIESEN B,WUST P,et al.Magnetic nanoparticle hyperthermia for prostate cancer[J].Int J Hyperthermia,2010,26(8):790-795.DOI:10.3109/02656731003745740.
[5]KOLOSNJAJ-TABI J,DI CORATO R,LARTIGUE L,et al.Heatgenerating iron oxide nanocubes:subtle“destructurators”of the tumoral microenvironment[J].ACS Nano,2014,8(5):4268-4283.DOI:10.1021/nn405356r.
[6]EL-BOUBBOU K.Magnetic iron oxide nanoparticles as drug carriers:preparation,conjugation and delivery[J].Nanomedicine(Lond),2018,13(8):929-952.DOI:10.2217/nnm-2017-0320.
[7]赵成桂,袁晨燕,吴国球.p[5HRE]AFPp-p53/PEI-Fe3O4磁性纳米颗粒联合磁流体热疗对肝癌细胞的抑制作用[J].中国肿瘤生物治疗杂志,2016,23(1):44-51.DOI:10.3872/j.issn.1007-385X.2016.01.007.
[8]WANG Y,CANINE B F,HATEFI A.HSV-TK/GCV cancer suicide gene therapy by a designed recombinant multifunctional vector[J].Nanomedicine,2011,7(2):193-200.DOI:10.1016/j.nano.2010.08.003.
[9]KIM K I,CHUNG H K,PARK J H,et al.Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma[J].World J Gastroenterol,2016,22(27):6127-6134.DOI:10.3748/wjg.v22.i27.6127.
[10]YAN C,YANG M,LI Z,et al.Suppression of orthotopically implanted hepatocarcinoma in mice by umbilical cord-derived mesenchymal stem cells with s TRAIL gene expression driven by AFP promoter[J].Biomaterials,2014,35(9):3035-3043.DOI:10.1016/j.biomaterials.2013.12.037.
[11]DANDA R,KRISHNAN G,GANAPATHY K,et al.Targeted expression of suicide gene by tissue-specific promoter and micro RNAregulation for cancer gene therapy[J/OL].PLo S One,2013,8(12):e83398[2018-05-03].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877029/.DOI:10.1371/journal.pone.0083398.
[12]O'NEILL K,LYONS S K,GALLAGHER W M,et al.Bioluminescent imaging:a critical tool in pre-clinical oncology research[J].JPathol,2010,220(3):317-327.DOI:10.1002/path.2656.
[13]PARK J H,KIM K I,LEE Y J,et al.Non-invasive monitoring of hepatocellular carcinoma in transgenic mouse with bioluminescent imaging[J].Cancer Lett,2011,310(1):53-60.DOI:10.1016/j.canlet.2011.06.013.
[14]KE B,MA L,KANG T,et al.In vivo bioluminescence imaging of cobalt accumulation in a mouse model[J].Anal Chem,2018,90(8):4946-4950.DOI:10.1021/acs.analchem.8b00391.
[15]RAO W,DENG Z S,LIU J.A review of hyperthermia combined with radiotherapy/chemotherapy on malignant tumors[J].Crit Rev Biomed Eng,2010,38(1):101-116.DOI:10.1615/critrevbiomedeng.v38.i1.80.
[16]LI M,BU W,REN J,et al.Enhanced synergism of thermo-chemotherapy for liver cancer with magnetothermally responsive nanocarriers[J].Theranostics,2018,8(3):693-709.DOI:10.7150/thno.21297.
[17]BRüNINGK S,POWATHIL G,ZIEGENHEIN P,et al.Combining radiation with hyperthermia:a multiscale model informed by in vitro experiments[J].J Royal Soc Interfact,2018,15(138):20170681.DOI:10.1098/rsif.2017.0681.
[18]CERESOLI M,FRIGERIO L,ANSALONI L.Hyperthermic intraperitoneal chemotherapy in ovarian cancer[J].N Engl J Med,2018,378(14):1363-1368.DOI:10.1056/NEJMc1802033.
[19]赵建夫,赵凤芝,陈文慧,等.CIK细胞联合贝伐单抗对肝癌Hep G2细胞的体内外抗瘤活性[J].中国肿瘤生物治疗杂志,2017,24(4):404-410.DOI:10.3872/j.issn.1007-385X.2017.04.012.