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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(gold nanoparticles,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/mL 6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/mL 6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/mL 3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50μg/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10 000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;G1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。
OBJECTIVE: To construct a new type of trastuzumab nano-probe and investigate its effect on the inhibition of breast cancer cells in vitro. Methods: Gold nanoparticles (GNPs) were prepared by citrate reduction method, and Herceptin was adsorbed by electrostatic interaction to synthesize GNP @ Herceptin. Immunohistochemistry The expression of HER-2 protein was detected in MCF-7 and SK-BR-3 cells. The proliferation inhibition of GNPs was determined at concentrations of 12.5,25.0,50.0,100.0,200.0 and 400.0μg / mL, and blank cells (0mg / mL) , And the kit was used to detect the cytotoxicity of GNPs. The GNPs, Herceptin and GNP @ Herceptin groups were divided into 6 groups (6.25,12.5,25.0,50.0,100.0 and 200.0ng / mL), and blank control group The inhibitory effect of MCF-7 and SK-BR-3 cells was detected by ELISA kit. Three concentrations of Herceptin and GNP @ Herceptin at 0.25, 0.5 and 1.0 μg / mL were added and the blank cells (0 μg / mL ). The apoptosis and cycle arrest of SK-BR-3 cells were detected by kit. Corresponding experimental data were analyzed by ANOVA and independent sample t-test. Results: The gold nanoparticle was approximately circular with a particle size of (14.11 ± 1.134) nm. After adsorbed with trastuzumab, the solution was clear and bright. The binding rate of GNPs to Herceptin was 2.25:1. When the concentration of GNPs was 400 μg / mL, The survival rate of MCF-7 cells was reduced to 43.9% (t = 39.709, P = 0.001). The survival rate of SK-BR-3 cells was 65.1% (t = 6.796, P = 0.002) However, the survival rates of SK-BR-3 and MCF-7 cells were 82.8% (t = 1.979, P = 0.119) and 99.3% (t = 0.177, P = 0.868), and did not show the effect of cell proliferation inhibition, low concentrations of GNPs have good biocompatibility. Herceptin treatment of SK-BR-3 cells, the best dose of 7.143ng per 10,000 cells, GNP @ Herceptin cells after treatment the best dosage decreased from 50ng / mL to 25ng / mL, the difference was statistically significant, t = 14.774, P <0.001). When treated with the highest concentration of GNP @ Herceptin, the apoptotic rate increased from 9.37% to 16.87%, the difference was statistically significant, t = 6.537, P = 0.001; (T = 5.286, P = 0.006). The number of cells in G2 / M phase decreased from 14.14% to 6.22%, the difference was statistically significant, t = 6.732, P = 0.003. CONCLUSION: The stable GNP @ Herceptin tracer gold nanoparticles can reduce the dosage of Herceptin in a large scale and have a promising prospect for the treatment of HER-2 overexpression breast cancer.