免疫金纳米笼子介导热疗诱导人乳腺癌细胞凋亡研究

目的：研究抗体修饰的金纳米笼子对乳腺癌细胞的光热杀伤作用。方法：由银（Ag）立方纳米晶与次氯金酸（HAuCl4）置换反应得到金纳米笼子,经Anti-CK19抗体修饰后制成具有生物靶向性的免疫金纳米笼子。将其链接细胞后,由激光照射,在荧光显微镜下观察免疫金纳米笼子光热杀伤乳腺癌细胞的效果,通过流式细胞仪检测其对乳腺癌细胞株MCF-7、MDA-MB-231和MCF-7/TMA热疗效果的差异,用免疫组化方法检测热疗后细胞内Caspase-9蛋白的表达量,MMT比色法观察其细胞毒性。结果：免疫金纳米笼子可以通过光热作用来杀伤乳腺癌细胞;MCF-7细胞株对照组凋亡率为（0.056±0.007 8）%,实验组为（50.2±4.56）%,MCF-7/TAM细胞株对照组凋亡率为（0.006 3±0.005 4）%,实验组为（52.6±5.07）%,MDA-MB-231细胞株对照组凋亡率为（0.054±0.006 4）%,实验组为（49.0±4.81）%,差异无统计学意义,F=0.84,P=0.456;Caspase-9蛋白在37℃时表达水平为0.06±0.02,41℃表达水平为0.15±0.04,43℃表达水平为0.34±0.04,对照组光照后温度为34℃,表达水平为0.05±0.01,〈43℃时,随着温度的升高Caspase-9的表达量明显升高,F=14.914,P〈0.001;MMT法结果显示,高剂量免疫金纳米笼子可抑制乳腺癌细胞的增殖（F=5.774,P〈0.001）,且存在时间-效应（F=42.378,P〈0.001）和剂量-效应（F=76.440,P〈0.001）关系。结论：免疫金纳米笼子介导的光热疗法可以靶向杀伤乳腺癌细胞,上调Caspase-9的表达,是免疫金纳米笼子介导的热疗诱导乳腺癌细胞凋亡的机制之一。

Investigation on hyperthermia mediated by immuno gold nanocages induces apoptosis of human breast cancer

OBJECTIVE： To observe the photothermal effect of antibody of immune gold nanocages on human breast cancer ceil. METHODS： The gold nanocages were obtained through the replacement reaction between silver（Ag） cubic nanoerystalline and chloroauric acid（ HAuC14 ）, then it was modified with Anti-CK19 antibody so as to have biological targeting characteristics. In this experiment,we attached the gold nanocage-antibody probe to the cell and exposured it to the laser. The photothermal killing effect of immune gold nanocage was detected by fluorescence microscope. The difference of photothermal killing effect was observed by flow cytometry on breast cancer cell MCF-7, MDA-MB-231, MCF-7/TMA. Caspase-9 protein expression index in heated cells was tested by immunohistochemical method. The cytotoxicity of the im- mune gold nanoparticle cage was estimated by colorimetric method of MTT. RESULTS： Immuno Gold Nanocages can kill breast cancer celle by photothermy. The apoptosis fete MCF-7 in control groups was （0. 056± 0. 007 8）% ,in experiment groups it was （50.2±4.56）%. The apoptosis rate of MCF 7/TAM in control groups was （0. 006 3 ±0. 005 4）%,in experiment groups it was（52.6±5.07）%. The apoptosis rate of MDA-MB-231 in control groups was （0. 054±0. 006 4）%, in experiment groups it was （49.0±4.81）%. There was no significant difference among the apoptosis rates of three kinds of cells （F=0.84,P=0. 456）. Caspase-9 expression quantity in 37℃ was 0. 06 ± 0. 02 , the expression quantity in 41℃ were 0.15±0. 04,the expression quantity in 43 ℃ was 0. 34 ±0. 04. The temperature in control groups after light exposure was 34℃ ,its Caspase-9 expression quantity was 0.05 ±0.01. Caspase-9 expression quantity was increased with increasing of temperature when the temperature below 43℃ （F= 14. 914,P〈0. 001）. MMT method showed that high doses of immune gold nanoparticle cage could inhibit the cell proliferation of breast cancer cell （F= 5. 774, P〈0. 001） and there were time-