Anti-cMet抗体耦合空心纳米金球对宫颈癌光热治疗增敏作用研究

Anti-c Met antibody conjugated hollow gold nanospheres as a new nano-material for enhancing the effect of photothermal therapy

目的:探讨偶联anti-cMet抗体的靶向空心纳米金球(anti-cMet/HGNs)对肿瘤光热治疗的增敏作用和诱导凋亡的分子机制。方法:透射电子显微镜(TEM)和分光光度计对纳米金球进行表征。共聚焦荧光显微镜观察人宫颈癌细胞He La和CaSki荧光染色后cMet的表达情况。电感耦合等离子体原子发射光谱法(ICP-AES)测定两种细胞对HGNs和anti-cMet/HGNs的摄取量。CCK-8法检测不同浓度HGNs和anti-cMet/HGNs对细胞的抑制率。使用近红外光(NIR)进行光热实验,细胞分组:对照、NIR、HGNs+NIR、anti-cMet/HGNs+NIR,酶标仪测定吸光度值。Western blot法测定凋亡相关蛋白。结果:HGNs吸收峰为788nm,聚乙二醇(PEG)修饰结合抗体后右移为808nm。He La和CaSki均高表达cMet,两种细胞对anti-cMet/HGNs的摄取量均多于HGNs。HGNs半数抑制浓度(IC_(50))为1.1nmol/L(He La)和0.6nmol/L(CaSki)。NIR照射后,anti-cMet/HGNs+NIR组的细胞抑制率为34.8%(He La)和53.6%(CaSki),其余3组无明显细胞死亡(P<0.05);Bax、Caspase-3和Cyclin E2表达均高于对照,Bcl-2表达与之相反,anti-cMet/HGNs+NIR组趋势最明显。结论:Anti-cMet抗体耦合空心纳米金球对宫颈癌光热治疗有增敏作用,通过破坏Bcl-2/Bax平衡和激活Fas通路来诱导肿瘤细胞凋亡。

Objective： To investigate the molecular mechanism of anti-cMet/HGNs in mediating targeted cancer treatment and enhancing treatment sensitivity in photothermal therapy. Methods： The successful synthesis of HGNs was observed and photographed by transmission electron microscope（ TEM）. The absorbance of naked HGNs and anti-cMet/HGNs was examined by UV-Visible-NIR spectrophotometry. Over-representation of cMet in cancer cells was verified by immunofluorescence. Uptake of HGNs and anti-cMet/HGNs was tested by inductively coupled plasma atomic emission spectroscopy（ ICP-AES）. The in vitro photothermal therapy was conducted in four groups-control,laser alone,naked HGNs with laser and anti-cMet/HGNs with laser. To test the toxicity of functionalized gold nanospheres using cell counting kit-8（ CCK-8）. To investigate these four groups＇ inhibitory rate of cell proliferation by CCK-8. The expression of apoptosis-related proteins was examined by western blot. Result： Modification with PEG and conjunction with targeting antibody made an approximately 20 nm right shift of the largest infrared absorption peak（ from 788 nm to 808nm） of gold nanomaterials. Both two human cervical cancer cell lines- He La and CaSki-were detected a layer of Fluorescent green marker. Correspondingly,ICP-AES confirmed the distinct uptake of anti-cMet/HGNs by both two cell lines.IC_（50） was 1. 1nmol/L in He La and 0. 6nmol/L in CaSki. Compared with other three groups,anticMet/HGNs with laser（ 1. 7J/cm^2） possessed a higher inhibitory rate of cell proliferation by34. 8% in He La and 53. 6% in CaSki. Meanwhile this group was indicated obviously overexpression of Caspase-3,Bax and Cyclin E2 and a low expression of Bcl-2 in both two cell lines.Conclusions： Anti-cMet/HGNs have the potentials to breaking Bcl-2/Bax balance to induce apoptosis and to enhancing the effect of photothermal ablation therapy on cervical cancer.