纳米微粒偶联抗OX40/抗AFP抗体在细胞毒性T细胞抗肝癌中的作用  

Effect of PLGA nanoparticles conjugated with anti-OX40/anti-AFP mAbs on cytotoxicity of CTL cells against hepatocellular carcinoma

作　　者：陈明水[1,2] 欧阳海超 周山勇[3] 李洁羽[1,2] 叶韵斌[1,2] 

机构地区：[1]福建医科大学教学医院,福建省肿瘤医院肿瘤免疫学研究室,福建福州350014 [2]福建省肿瘤转化医学重点实验室,福建福州350014 [3]中国科学院福建物质结构研究所

出　　处：《细胞与分子免疫学杂志》2014年第4期337-341,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：福建省自然科学基金(2011J01140)

摘　　要：目的探讨乳酸-羧基乙酸共聚物(PLGA)制备的纳米微粒偶联抗OX40及抗AFP抗体(抗OX40/抗AFP-NP)对甲胎蛋白AFP158-166抗原肽特异性细胞毒性T细胞(CTL/AFP158-166)体外杀伤肝癌细胞的影响。方法用溶剂蒸发法制备纳米微粒(NP),并与抗OX40和抗AFP共价偶联。用扫描电镜观察所制得PLGA-NP纳米颗粒的形态;ZetaPlusTM粒度测定仪检测纳米微粒的粒径及电荷;用BCA蛋白定量方法检测纳米微粒偶联抗体的效率;用人外周血单核细胞诱导形成树突状细胞(DC),用AFP158-166抗原肽负载DC诱导AFP特异性的CTL/AFP158-166;分别用2-(4-碘苯)-3-(4硝基苯)-5-(2,4-磺苯基四氮唑)-2H-四唑单钠盐-1法(WST-1)法、ELISA及乳酸脱氢酶(LDH)法分别检测抗OX40/抗AFP-NP对CTL/AFP158-166细胞增殖能力、分泌IL-2和IFN-γ及杀瘤活性的影响。结果所制得的PLGA-NP为圆形,大小较均一,粒径约为(300±42)nm,带负电荷,约为-(25.12±5.34)mV。蛋白定量显示每mg的PLGA-NP偶联约100μg抗体,偶联效率为25%;增殖和活化实验显示偶联有抗OX40的纳米微粒能显著刺激CTL的增殖、IL-2和IFN-γ的分泌;杀伤实验显示,抗OX40/抗AFP-NP能显著增强AFP特异性CTL/AFP158-166细胞对HepG2细胞特异性杀伤作用,而对SMMC-7721杀伤作用无明显效果。结论抗OX40/抗AFP-NP能刺激CTL/AFP158-166细胞的增殖、细胞因子的分泌并增强CTL/AFP158-166细胞对AFP阳性肝癌细胞的特异性杀伤作用。Objective To evaluate the effect of anti-OX40 and anti-AFP antibodies conjugated onto poly （DL-lactide-co- glycolide）-nanoparticles （PLGA-NPs） on the cytotoxic activity of AFP158-166 -specific cytotoxic T lymphocyte （CTL） against hepatocellular carcinoma cells in vitro. Methods PLGA-NPs were prepared by oil-in-water single emulsion solvent evaporation method and covalently conjugated with anti-OX40 and anti-AFP monoclonal antibodies. Scanning electron microscopy （SEM） was utilized for the characterization of the surface morphology and estimation of the size of the PLGA-NPs. The mean diameter and zeta potential of the nanoparticles were measured by dynamic light scattering （DLS） performed in a Zetasiser Nano Series ZEN3600. Antibody conjugation efficiency was determined using bicinchoninic acid （BCA） protein assay. Dendritic cells （DCs） were induced from human peripheral blood mononuclear cells （PBMCs） in the presence of GM-CSF and IL-4, and loaded with AFP158-166 peptide to generate AFP-specific CTL （CTL/AFP158-166 ）. WST-1, ELISA and lactate dehydrogenase （LDH） methods were respectively used to examine the effects of the anti-OX40/anti-AFP-NPs on CTL/AFP158-166 proliferation, IL-2 and IFN-y production, and cytotoxicity against the tumor cells. Results The obtained nanoparUcles were found to be of regular spherical shape and the smooth surface with an average diameter of （300 ± 42） nm and a negative zeta potential of -（25.12 ±5.34） inV. Approximately 100 IJg antibodies were conjugated to every milligram of the nanoparticles with a conjugation efficiency of about 25% as estimated by BCA protein assay. Proliferation and activation analysis revealed that anti-OX40/anti-AFP mAb-NPs significantly induced CTL proliferation and the secretion of IL-2 and IFN-y. The cytotoxicity assay showed that anti-OX40/anti-AFP-NPs markedly enhanced CTL/AFP158-166 specific killing on HepG2 cells but had no obvious effect on SMMC-7721 cells. Conclusion Anti-OX40 mAb and anti-AFP rnAb co

关 键 词：纳米微粒 肝癌 细胞毒性T细胞 HepG2 OX40 

分 类 号：R735.7[医药卫生—肿瘤] R392.11[医药卫生—临床医学]