曲妥珠金纳米探针对乳腺癌细胞抑制影响体外实验

目的：构建新型曲妥珠纳米生物探针，探讨其对乳腺癌细胞抑制影响的体外实验。方法：以柠檬酸盐还原法制备金纳米颗粒（goldnanoparticles，GNPs），通过静电作用吸附曲妥珠单抗（Herceptin），进而合成曲妥珠金纳米探针（GNP@Herceptin）；免疫组化检测MCF-7与SK-BR-3细胞HER-2蛋白表达；GNPs增殖抑制检测设12．5、25．0、50．0、100．0、200．0和400．0μg／mL6个浓度，并设置空白细胞组（0mg／mL），试剂盒检测GNPs细胞毒性；综合细胞增殖抑制检测设GNPs、Herceptin和GNP@Herceptin组，每组设6．25、12．5、25．0、50．0、100．0和200．0ng／mL6个浓度，并设置空白细胞组（0ng／mL），试剂盒检测MCF-7与SK-BR-3细胞增殖抑制效果；设0．25、0．5和1．0μg／mL3个浓度的Herceptin、GNP@Herceptin，并设置空白细胞组（0μg／mL），试剂盒检测SK-BR-3细胞凋亡与周期阻滞；利用方差分析与独立样本t检验处理相应实验数据。结果：金纳米颗粒近似圆形，粒径大小（14．11±1．134）nm，与曲妥珠单抗吸附后，溶液澄清透亮，GNPs与Herceptin结合率为2．25：1；GNPs浓度在400μg／mL时，MCF-7细胞存活率减少至43．9％（t=39．709，P=0．001），在100μg／mL时，SK-BR-3细胞存活率为65．1％（t=6．796，P=0．002），均有明显细胞毒性效果，但金纳米颗粒浓度下降至50ug／mL时，SK-BR-3与MCF-7细胞生存率分别为82．8％（t=1．979，P：0．119）、99．3％（t=0．177，P=0．868），且未显示出细胞增殖抑制效果，低浓度GNPs有良好的生物相容性。Herceptin处理SK-BR-3细胞后，最佳用药剂量为每10000个细胞7．143ng，GNP@Herceptin处理细胞后最佳用药量从50ng／mL降低至25ng／mL，差异有统计学意义，t=14．774，P〈0．001；GNP@Herceptin最高浓度处理细胞时，细胞凋亡率从9．37％增大至16．87％，差异有统计学意义，t=6．537，P=0．001；Gl期阻滞从74．70％增大到83．12％，差异有统计意义，t=5．286，P=0．006；G2／M期细胞数从14．14％减少至6．22％，差异有统计学意义，t=6．732，P=0．003。结论：构建的曲妥珠金纳米探针GNP@Herceptin性状稳定，可大比例降低Herceptin用药剂量，对HER-2过表达乳腺癌治疗具有实用研发前景。

Inhibitory effect of breast cancer cells by gold nanoparticles probe that absorbing trastuzumab in vitro

OBJECTIVE： To explore the cytotoxicity of GNPs and the application of tumor cells of probe by con- structing a new nano-bio-probe in the fields of nano-medicine. METHODS： Synthesizing the gold nanoparticles （GNPs） by the citrate reduction method and the probe of GNP@ Herceptin was obtained using the method of electrostatic dsorption; The HER-2 protein of MCF-7 and SK-BR-3 was detected by immunohistochemical;Designing 6 kinds of concentrations of GNPs from 12.5 mg/mL to 400 mg/mL and the control group （0 mg/mL） and detecting the cytotoxicity of GNPs by a kitDesigning 6 kinds of concentrations of GNPs, Herceptin and GNP@ Herceptin from 6.25 ng/mL to 200 ng/mL and the control group（0 ng/mL） and investigating the proliferation inhibition of MCF-7 and SK-BR-3 by a kit~ Designing 3 kinds of concentrations of Herceptin and GNP@ Herceptin from 0. 25μg/mL to 1. 0 /lg/mL and the control group （0μg/mL） and studying the apoptosis and cell cycle of SK-BR-3 by a kit. The data was analyzed by analysis of variance and independent two-sample t-test. RESULTS： The gold nanoparticles was approximately round in the diameter of （14.11±1. 134） nm and claried after absorbing Herceptin. The GNPs and Herceptin had a perfect coupling with the com- bing rate of 2.25 ： 1;The survival of MCF-7 was 43. 9% （t=39. 709,P=0. 001） when the concentration of GNPs was 400μg/mL,and when that was 100 μg/mL the survival of SK-BR-3 was 65.1%（t=6. 796,P〈0. 002） ,it had the toxicity to breast cancer cells. When the concentration of GNPs was 50μg/mL the survival of SK-BR-3 and MCF-7 were 82.80/00 （t=l. 979,P〈0. 119） and 99.3% （t=0. 177 ,P=0. 868） respectively,they didn＇t show any toxicity to breast cancer. The low concentration of GNPs had a good biocompatihility. The optimal dose of Herceptin was 7. 143 ng for ten thousand cells if it treated with SK-BR-3 cells and GNP@ Herceptin could decrease the optimal dose from 50 ng/mL to 25 ng/mL, and the difference had statisticall significance （t= 14. 774,P〈0. 001）. When the SK-BR-3 was dealt with the highest con- centration of GNP@ Herceptin, the probe could led the effect of apoptosis from 9.37 % to 16.87 % （t = 6.537, P = 0. 001 ）, and block the G1 phase of cell cycle from 74.70% to 83.12% （t=5. 286,P- 0. 006） ,as well as reduce the cell number of the G2/M phase of cell cycle from 14.14% to 6.22%（t=6. 732,P=0. 003） while comparing with Herceptin. CONCLUSION： The probe of GNP@ Herceptin has the stahle traits and reduces the dosage of Herceptin at a large proportion, it would have a well medical prospect for the breast cancer patients who has the positive expression of HER-2.