摘要： 目的：考察介孔二氧化硅纳米微粒(mesoporous silica nanoparticle，MSNP)共转运体系逆转肿瘤多药耐药、杀伤肿瘤细胞的效果，证实阿霉素（doxorubicin，DOX）与MDR1shRNA共同应用具有协同抗肿瘤作用。方法：首先采用溶胶-凝胶法制备MSNP，通过表面修饰阳离子聚合物聚乙烯亚胺（polyethylenimine，PEI）使其带有正电荷，能够与带负电的MDR1shRNA相结合，同时将抗肿瘤药物DOX吸附于介孔内部，形成载药、载基因的共转运体系。体外采用紫外可见光分光光度法测定载药量；通过MTT法测定DOX对口腔鳞癌细胞株KB及耐药株KBV的半抑制浓度IC50；使用荧光显微镜、流式细胞仪测定转染效率；应用Real-Time PCR技术检测MDR1基因的表达水平；AnnexinV-FITC/7-AAD双染法经流式细胞仪检测细胞凋亡情况。结果：纳米粒度仪、透射电镜测得所合成的MSNP尺寸为100~200 nm，表面介孔直径约3~5 nm，分散性较好，尺寸较均一；紫外分光光度计法测得IC50(KB-DOX)=182.9 ng/mL、IC50(KBV-DOX)=9 233.5 ng/mL，计算耐药株KBV的耐药倍数为50.483 871倍；荧光显微镜、流式细胞仪测得转染效率为6.73%；Real-Time PCR结果显示：与单纯载药、载基因组相比，双载组MDR1基因的表达水平显著下调；AnnexinV-FITC/7-AAD双染法经流式细胞仪测得单纯载药、单纯载基因组细胞凋亡率分别为12.74%、10.08%，而双载组细胞凋亡率增加到25.68%。结论：介孔二氧化硅纳米微粒共转运体系能够有效逆转肿瘤多药耐药，与单纯载药、载基因组相比，同时应用DOX与MDR1shRNA具有协同抗肿瘤作用。

关键词:  , 介孔二氧化硅纳米微粒；共转运；DOX；MDR1shRNA

Abstract: Objective: To investigate the effect of reversing multi-drug resistance and killing tumor cells of mesoporous silica nanoparticles(MSNP) co-delivery system, and to confirm whether combined application of Doxovubicin(DOX) and MDR1 shRNA can exert a synergistic antitumor effect. Methods: Firstly, mesoporous silica nanoparticles were prepared by sol-gel method under room temperature and coated by cationic polymerpolyethylenimine(PEI) on the surface to stay positive charge, which could facilitate its combination with negatively charged MDR1 shRNA in the next step. With the absorption of DOX, an antitumor drug, the drug and gene co-delivery system was then formed. In vitro, drug loading was evaluated by UV-Vis spectrophotometer; antitumor drugs were co-cultured respectively with oral squamous cells carcinoma cells lines KB and resistant cell lines KBV. Then MTT assay was conducted to measure the half maximal inhibitory concentration (IC50); Fluorescence microscope and flow cytometry instrument were used to measure the transfection efficiency of the co-delivery system; Real-Time PCR technique was applied to detect the expression of MDR1 gene; AnnexinV-FITC/7-AAD double staining by flow cytometry was conducted to detect cell apoptosis. Results: The MSNP appeared to have a high dispersibility and homogeneous size by particle size analyzer and transmission electron microscopy (TEM). The formed nanoparticles have a diameter distribution of 100-200 nm, and the surface mesoporous diameter is 3-5 nm. IC50 (KB-DOX) was 182.9 ng/mL, and IC50 (KBV-DOX) was 9 233.5 ng/mL by ultraviolet spectrophotometer. The resistance index of KBV was 50.483 871. The transfection efficiency is 6.73% measured by fluorescence microscope and flow cytometry . The Real-Time PCR test indicated that MDR1 mRNA expression in the co-delivery system decreased dramatically, compared with co-delivery nanoparticles. The results of AnnexinV-FITC/7-AAD double staining showed that the cell apoptosis rates were 12.74% and 10.08% for drug-loaded and gene-loaded nanoparticles respectively, while cell apoptosis rate increased to 25.68% for co-delivery nanoparticles. Conclusion: The results demonstrate that comparing with the pure medicine or genome, the mesoporous silica nanoparticles co-delivery system can effectively reverse multi-drug resistance of tumor and inhibited the growth of tumor dramatically. At the same time, DOX and the MDR1 shRNA can exert synergistic antitumor effect when applied in combination.

Key words: mesoporous silica nanoparticles, co-delivery, DOX, MDR1 shRNA

中图分类号: 

• R782

• R739.8