摘要
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 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.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2014年第11期816-821,835,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
江苏省基础研究计划(BK2011036)
